STUDIES ON THE EFFECTS OF SIDEROPHORES OF SOME SOIL ...

STUDIES ON THE EFFECTS OF SIDEROPHORES OFSOME SOIL BACTERIA ONALTERNARU HELWVTHZ(Hanf.) Tubaki and Vishihar CAUSING FOLIAR DISEASEIN HELUNTHUS ANNUUS L .THESIS SUBMITTED TOWh'DICHERRY UNIVERSITY FOR TIIE AWARDOF THE DEGREE OFDOCTOR OF PHILOSOPHYIN LIFE SCIENCES (BOTANY)RAJATHILACAM. RDEPARTMENT OF BIOCHEMISTRY. AND . MOLECULAR BIOLOGYSCHOOL OF LIFE SCIENCESPONDICHERRY UNIVERSITYPONDICHERRY-605 014INDIAFEBRUARY- 2006

Dr. B. KannabiranReaderPONDICHERRY UNIVERSITYDEPARTMENT OF BIOCHEMISTRY ANDMOLECULAR BIOLOGYSCHOOL OF LIFE SCIENCESPONDICHERRY-605 014, INDIACERTIFICATEI hereby certify that this thesis entitled "STUDIES ON THE EFFECTSOF SIDEROPHORES OF SOME SOIL BACTERIA ON ALTERNARIAHEUANTHl (Hanf.) Tubaki and Vishiha CAUSING FOLIAR DISEASE INHELIANTHUS ANNUUS L." submitted for the award of degree of DOCTOROF PHILOSOPHY IN LIFE SCIENCES (BOTANY) is a bonaflde research workdone by the candidate MRS. RAJA1RPAGAM , R, durlng the period of herstudy under my guidance in the Department of Blochernlsby and MolecularBlology School of Life Scirncrs, Pondlcherry Unlvenity and the thesis hasnot previously formed the basis for the award of any Degrw or Diploma orother similar titles.candidate.I further certify that the thesis represents the independent work of the1 4 ,. [LLULL ILb;Dr. B. KannabiranResearch SupervisorProfessor S. JayachandranDeanHead of the Deparhnent

RAJATILAGAME. RDepartment Of Biochemistry AndMolecular BiologySchool Of Life SciencesPondicheny UniversithyPondicherry -605 0 14, INDIADECLARATION1 hereby declare that this thesis entitled "STUDIES ON THEEFFECTS OF SIDEROPHORES OF SOME SOILBACTERIA ON ALTERNARIA HELIANTHI (Hanf,) Tubakiand vishihar CAUSING FOLIAR DISEASE IN HELIANTHUSANNUUS L." submitted for the award of Degree of Doctor ofPhilosophy in Life Sciences (Botany) is a record of research workdone by me under the guidance of Dr. B. Kannabiran, Departmentof Biochemistry and Molecular Biology, School of Life Sciences,Pondicherry University and the thesis has not previously formed thebasis for the award of any Degree or Diploma or other similar titles.Date: 13/%/0bTqXRA ATILAGAM'

ACKNOWLEDGMENTI wish to place on record with immense pleasure andprc~found gratitude, m,y deep sense ofindehledness to Dr. B. Kannabiran. Ph. D., Rcadcr, Department of Riochemisty andMolecular BiOlOfl, School of LiJe science.^, Pondicherry University for his uniqui,,admirable, mcticlrlous and invaluahle guidance, constant encouragement, precioussirggestions and cortstrtrctir'e criticism during the course of the project in most effkctii~eand .~,v.stemutic munncr and ,for having provided me an idealistic methodologicalorientation lhrougholtt I/I(. course ofrhis study.I tvi.rh to express m.v heorfy thanks to Dr. P.P. Mathur, Ifcad, and Dr. E. V(ja,va~i, FormerHeud, ~eporlrnf~~il of Biochemi.sfy and Mo/rculur Biolo~y und Former Deun, Sc8hoo/ ufLifc Scie~iccs ,for hai'itrg provided me with rhc lahorarory and infrasrructltrc ,facilitiesduring thc period of my research work. Mv .sincere thanks crre due to Dr. S. ,ja,vachundran.Dean, School ofL@ Scic~nccsfur hi., encouragment. My siricere thank arc, dua IU Dr. K.Srikirmur, Reader and Dr A. Ra~nachandra Reddy. Former Reader, Deportment ofBiochemistw arid Molccular Biology, Pondicherty llni~~erui(v/or their help..I am e.rfrcme!v grurc:firl to the Doctoral Committee members, Dr. V. Rurnas.ro~nv. Rcader.Depurtmoit of Botanv, Kanchi Mamuni~jar Cerltrc for Post Graduate Stndie,~, and Dr. N.Parthasaruth,v, Reuder. Uepurtmt>nt of Ecologv and Eril~ironmental Sciences. PoridicherryU~liversip,for thcir ~~uluuhlc suggesrio~is and c*onstant encouragonmt.I express m,v gratitudi, to Dr. Puneerseh~rrn, Reader, Dc~purtmcnt uf French. Dr.T,Ganesun, Lecturer, Tagore Arts and Science College, Pondichern, and Dr.(Mrs) V.Gomathi. Rescurch Associate, Center fir Adl'anced Studie.s in Boran,v, Uniiirersip ofMadras, Chennai, for their untiring support, apt advice and needed inspiration whichenabled me to completc this task successfully.

I wish to express my sincere thanks to Profe,,r.ror Dr. J. Muthumary, Profes.ror Dr.Murugesan, Center ,for Advanced Studies in Botany. [lnil~er.rity of Madrus, Chennai fortheir help in the idontificution und confirmation of the,fungus und I OM.V un ohligution tothe Institute of MMiohiology Technique, (IMTECH). Chandigurh fir having prol~ided methe bacterial culture.I owe an ohligation to M,: H. Ra~ikumar, Physical education teacher and Mr. S.Huriharun, Field supervisor, Government o~f Pondichertyfor their continual helpI thunk profu.se1v und sincere!^ [hunks to Dr. (Mrs) K.C. Chitra, Dr. R. Soureche, Dr. H.Chitru und Dr,lMr~) Ashu, A.N, Mr. B. Nubi. B., Mr. J. P. Pavun, for their incessant hclp,constont sr~pport and encolrrugement.I cxpresx \~ulrruhle thu~~kv to thc Rcreurch Scholar.r, Mi.s.s S.N. Ruj!icsn,uri. Miss ShyamalaMr. Rai,ikumur, Mr. Muthrr. Depurtmr~rit of tliochemistty and Molcculur Biology andMr.Kumulukar~nu~i, Departmerit oj Chemisttyfor their rimelv help and limitless support.I express n{v gratitlrdr to [kc Research Scholurs, Departments oJ'Plunt Parl~ologv, PlantPhysiolo~ und Plorit Biochemistw, Tamil Nadu Agricult~rre ullil~n:~i(V, Coimhatorc fortheir valuable suggrsrion and ,for hu~dng prol'idcd me nmith needed informution withconcon.I wish to curn7e.y my siticere thank to Distributed Infirmation Center arid Ccrltrul Libra?.Pondichery Unii~ersity, Lihruries of Cmter,for udi~unced stirdies in Botany, tini\'ersiry of'Madras. Department of Plant Pathology, Plant Physiology and Plant Biochemistry,TamilNadu Agricultrrrc Uni~~ersiry, Coimhatore for ha~sing provided me with the neededupdute infirmation thut kept me abreast ofudvuncc in thcfield tilldate.

My heartfelt thankr are due to Mrs. Sivakami, Mr. Veeraragavan oflce stafi Depar~mentof Biochemistty and Molecular Biologv, Pondicherry Universiy for their help.1 profirsely thank my husband Mr. G. K. Jayasudhahar, Parents-in-law, Mrs. Pangajamand Mr. Gothandnpani for having provided me the needed source of inspiration during thelast phase of my work.And most of all, I owe immensely to my Beloved Dad and Mom, for their keen interest inme, initiative and inspiration but for which I would not have joined this Ph. D. programmeand completrdit success&lly. Equally my deepest thanks arc due to my sister Ranjeetha,brothers Dhanaraja and Barathiraja for everything that cannot be put down in words soeasily. Their limitless and unstintcd support backed me at all the stages and kept me goingahead in spite of all troubles. Let my hearlfelt thanks reach them for their love andaffeecrion tuwards me rhur made my research effort a memorable and succes.yfi1 one.

1. INTRODUCTION

lndia is considered as one of the largest producers of grains and oilseeds amongthe countries of the world. It is also the world's second largest consumer of vegoils.In recent years, India's production of oil has met or exceeded its domesticconsumption needs. (Anonymous, 2004).The main oilseed crops grown in India aregroundnuts, rapeseed, mustard, sesame, sunflower, safflower, niger, cotton andsoybeans. Since the late 1980s, lndia has focussed its efforts on increasing oilseedproduction to meet its growing demand for vegoils. Increased production has beenaccomplished by expanding cultivation area, increasing irrigation, improving cropproduction techniques and developing higher yielding varieties of oil seeds. Amongthe Kharif oilseed crops introduced for boosting edible oil production (Jiskani.,2005)Sunflower has gained higher popularity and acrcagc in recent times.Hclianrhus annuus L. (Sunflower) belongs to the familyAsteraceae(Compositae). 'Helianthus' is taken from the peek words: 'helios' (sun)and 'amthos' (flower). It is the world's fourth largest oil-seed crop. World seedproduction was 25.2 million tonnes during 199511996 from approximately 50million ha of cultivated land (Rodriguez et al., 2002). The important features of thiscrop are short growing pcriod, high yield potential and wide range of growingseason viz. autumn, spring and winter. It fits well in different cropping patterns, lowirrigation water requirements, wide adaptability to soil and moisture conditions. Itsseed contains high oil (over 40%) of good edible quality and meal of good qualityfree from toxic compounds.

The wild sunflower is native to North America but commercialization ofsunflower was developed first as an important commercial oilseed crop in theFormer Soviet Union (FSU). It was only recently that the sunflower plant returned toNorth America to become a cultivated crop. The American Indian may be the first todomesticate the plant into a single headed plant with a variety of seed colorsincluding black, white, red, and black /white stripe. The oil has found widespreadacceptance as a high quality, edible oil throughout most of the world. Majorproducing countries are: Argentina, Eastern Europe, USA, China, France, and Spain.These seven countries of the world produce about 84 percent of the world'sproduction of both oilseed and non oilseed sunflower. Historically, the Soviet Unionhas been the number one producer of sunflower, producing about 27 percent of theworld's production in 1991-92. During 1970% the United Statcs was the world'ssecond largest producer, but in the 1980's Argentina became firmly entrenched insecond place. Work on sunflower began in Manitoba in earnest about 20 yearsearlier than in the United States. Mennonite immigrants from Russia to the prairiesof Canada late in the nineteenth century pew suntlowers in farm gardens to roastand eat the seeds.MorphologyIt is an erect, often unbranched, fast-growing and annual herb; taproot strong,penetrating to the depth of 3 m with large lateral spread of surface roots; stem 0.7-3.5 m tall, hirsute; leaves alternate, ovate, long-petioled; lamina with 3 main veins.12-30 cm long, 5-20 cm wide; apex acute or acuminate; lower leaves opposite andcordate; flowering head terminal on main stem, 10-40 cm in diameter, rotating to

face the sun, sometimes drooping; heads on lateral branches smaller; outer rayflowers neuter with yellow ligulate corolla; disc florets numerous, spirally arranged,perfect; ovary inferior with a single basal ovule; achenes obovoid, compressed,slightly 4-angled, variable in size, usually 1-1.5 cm long, fill-colored or striped.Climate and soilSunflower thrives at medium and high elevation in the tropics. It requires awarm climate with moderate rainfall and shows a somewhat wide range of toleranceto wet and dry conditions. It is drought-resistant and can withstand several degreesof frost. It is adapted to a variety of soils and does well on acid soils, water-loggedlands or steep slopes. In highly fertile soils it tends to grow vcry tall,mature late.Economic usesSunflower is an important oilseed crop cultivated throughout the world and isalso valued for its oil which is used in paint industry and for cooking (domesticmarket). If ground, the seeds form an excellent flour which is more nutritious anddigestible. Like hemp, the plant tissue renders an excellent fiber which the Chineseuse along with silk. An excellent paper can be made from the stem and yellow dyecan be prepared from the petals. The dried stems provide a good fuel for burning.Sunflower seed oil is classified as a semi-drying oil and is employedparticularly in blends with linseed and other drying oils in paints and varnishes. It isvalued for the non-yellowing and the heat-resisting propenies of films produced. Ablend containing 30-40% sunflower seed oil and 60-70% linseed oil possessessatisfactory drying properties and gives films which are softer, glossier and more

elastic than those of linseed oil alone. Sunflower seed oil is used as lubricant, forlighting purposes and treatment of shoddy in woolen manufacture. It has potentialvalue as fuel in diesel motors. It is a fairly efficient foam destroyer and possessesbactericidal action against many bacteria. Sulphonated sunflower seed oil is suitablefor the manufacture of high co-efficient liquid disinfectants.The cake or meal left after the extraction of oil is used as a high grade proteinsupplement for livestock, specially dairy cows and poultry. When fed to dairy cowsin large amounts, the butter obtained tends to be soft. Pigs fed on the seed cake givesoft pork. It is an excellent source of calcium. thiamine and niacin. The protein ofthe meal is of high biological value (64%) and digestibility (94%). The amino acidspresent in the defatted meal are: arginine.5.46Yu; histidine. 1,43%;leucine, 3.71%;isoleucine, 1.14% ; methionine, 1.61% phenylalaninc, 2.39% ; threonine 1.64% ;tryptophan, 1.14% and valine, 2.7%.Seeds as Poultry and Cattle Food: Sunflower seeds have a high feeding value - theanalysis in round figures is I6 per cent albumin and 21 per cent fat. Fed with oats inequal quantities, they make a perfectly balanced ration. Since both of these articlescontain a big proportion of indigestible matter. particularly in the husks, grit must onno account be withheld, if the birds are to derive full benefit. Sunflower-seed oilcakeis a valuable article for bringing up the feeding value of some of the poultryfoods. It was specially in demand for this purpose in war-time, when the supply ofgood cereals ran short. It is more fattening to cattle than Linseed cake, being richerin nitrogenous substances, containing 34 per cent albumen. It is an excellent food forpoultry and also for rabbits. It keeps both horses and cattle in good condition. It is

said that cows, fed on sunflower-seed oil-cake, mixed with bran, will have anincreased flow of good, rich milk.Plants as Green Food: The green leaves, when gathered young, make a goodsucculent green food for poultry stock of all ages. They can be finely minced up andadded - raw - to the mash for young or adult stock, or they can be boiled and put inthe soft food.Littcr: Even the stems and seedless heads need not be wasted where fowls are kept.Many may prefer to use them as fire-kindlers, but they will, when thoroughly dry,come in useful as litter for the laying-houses. When dry, they can be passed through achaff cutting machine and be added to the other litter - peat-moss or dried leaves.They need to be made into o scratchable material for hens, but for ducks, the matenalcan be placed deeply in the house as a bedding. Ducks need litter to 'squat' on ratherthan to scratch in.Source of Fuel and Potash: Sunflowers, when the stalks are dry, are as hard aswood and make an excellent fire. The ash, obtained from the plants after the seedhas been harvested is known for its richness in potash, a manure of considerablevalue. At the time of cutting the leaves are stripped off and fed them to rabbits orpoultry. When the stems are dry and after the seed crop has been gathercd, bothstems and empty seed-heads serve as fuel. Of the ash obtained from burning theSuriflower stems and heads (apart from seeds) 62 per cent consists of potash and asan acre of Sunflower produces from 2,500 to 4,000 Ib. of top, the total yield ofpotash is considerable. Allowing 3,000 Ib. of top, there would be produced 160 Ib.of ash per acre of crop, which should contain upwards of 50 Ib. of potash.

SOU Improver: The growing herb is extremely useful for drying damp soils,because of its remarkable ability to absorb quantities of water. Swampy districts inHolland have been made habitable by an extensive culture of the sunflower, themalarial fog being absorbed and nullified, whilst abundant oxygen is emitted.Textile Use: The Chinese grow this plant extensively and it is believed that a largeportion of its fibre is mixed with their silks.Bee Plant: The Sunflower is a good bee plant, as it furnishes hive bees with largequantities of wax and nectar.Vegetable: The unexpanded buds boiled and served like Artichokes form a pleasantdish.Medicinal Uses: The seeds have diuretic and expectorant properties and have beenemployed with success in the treatment of bronchial, laygeal and pulmonaryaffliction, cough and cold.A tincture of the flowers and leaves has been recommended in combination withbalsamics in the treatment of hronchiectasis.The seeds, if browned in the oven and then made into an infusion are admirable forthe relief of whooping cough.Tincture ofHelianthtrs has been used in Russia for malarial fever.A tincture prepared from the seed with rectified spirit of wine is useful forintermittent fevers, instcad of quinine. It has been employed thus in Turkey andPersia, where quinine and arsenic have failed.Medicinally, seeds are diuretic, expectorant and used for colds, coughs, throatand lung ailments. According to Hartwell (1971), the flowers and seeds are used infolk remedies for cancer in Venezuela, often incorporated in white wine. It is

eported to be anodyne, antrseptlc, aphrodrs~ac, bactenc~dal, deobstruent, druretlc,emoll~ent, expectorant, rnsectlc~dal and malana preventative Sunflower 1s a folkremedy for bltndness, bronchrtls, carbuncles, catarrh, cold, wlrc, cough, drarrhoea,dysentery, dysuna, fever, flu, fractures, ~nflammat~ons, laryngtls, menorrhagra,pleunt~s, rheumatism, scorplon stlngs, snakcbrtc, splenetrs, umgenrtal aliments,wh~tlow and wounds (Duke and Waln, 1981)Chemical ConstituentsThe black-seeded vanety yelds between 50 and 60 per cent of the best gradeof 011 The or1 IS slrghtly yellowrsh, Irmprd. of a sweet~sh taste and odourless It has aspec~fic gravity from 0 924 to 0 926 and sol~d~fies at 5'F It dnes slowly and formsone of the best bumlng 011s known, bumrng longer than any other vegetable or1 Atannrn called hellanth~tanl~ acrd was obtained and its formula 1s CuHvOa On bo~llngwrth moderately drluted hydrochlonc acrd, a fermentable sugar and a vroletcolounng matter w~th ~nulrn, large quant~tles of levulrn and d dextro-rotatory sugarwere obtarned Sunflower oil IS a healthy chorce for cookrng 011 needs It is hrgh Invrtamrn E and ~t has a light clean taste Sunflower 011 found on grocery store shelvestoday IS hrgh In llnolelc acld, an essential fatty acrd It IS an excellent home cooklng011 and salad 0x1 w~th a hght, clean taste, hrgh smoke polnt and low level of saturatedfatHigh oleic sunflower 011 IS premrum sunflower or1 w~th monounsaturated fatty ac~dlevel of 80% and aboveIt 1s used m food and lndustnes where hrghmonounsaturated fatty acrd levels are requlred Saturated fat levels IS 20% lowerthan lrnolerc sunflower 011 The balance of ~ ts wmposltron 1s l~nolerc ac~d

ChemistryThe seed is reported to contain 560 calories, 4.8 g H20, 24.0 g protein, 47.3 gfat, 19.4 g total carbohydrate, 3.8 g fiber, 4.0 g ash, 120 mg Ca, 837 mg P, 7.1 mgFe, 30g Na, 920 mg of K, 30 mg of carotene equivalent, 1.96 mg thiamine, 0.23 mgriboflavin, 5.4 mg niacin per I00 seeds. Seeds contain 25-35% of oil, but cultivarshave been bred in Russia with upto 50% oil. Oil contains 4&72% linoleic acid, and13-20% protein of high biological value and digestibility. The forage contains 8.8%protein, 2.9% fat, 77.2% total carbohydrate, 30.3g fiber, and 11.1 g ash. Anotheranalysis showed that young shoots contain 13.0% protein, 1.9% fat, 70.3% totalcarbohydrate, 20.4 g fiber, 14.8 g ash, 1,670 mg Ca, and 370 mg PI100 g. Thetlowers contain 12.7% protein, 13.7% fat, 64.3% total carbohydrate, 32.9 g fiber, 9.3g ash, 630 mg Ca, and 80 mg PllOO g. Sunflower oil has a high concentration oflinoleic acid (polyunsaturates), intermediate level of oleic acid(monounsaturated)and very low levels of linolenic acid. The saturated acids, palmitic and stearic, rarelyexceed 12% and the minor acids viz, lauric, arachidic, behenic, lignoceric,eicosenoic, etc. rarely add upto as much as 2%. Tocopherol, or vitamln E, is animportant vitamin and natural antioxidant. Sunflower oil is somewhat unique in thatthe alpha form predominates(608 mg of alpha kg of oil) (Dorrell, 198 I).GermplasmIt is reported from the North American (and secondarily, the Eurosiberian)Center of Diversity. Sunflower is reported to tolerate disease, drought, frost, fungi,high pH, laterite, limestone, low pH, mycobacteria, photoperiod, poor soil, rust, salt,sand, smog, virus, weeds, and waterlogging (Duke, 1978). Botanically, the

sunflower is treated as the following subspecies: ssp. lenticularis in the wildsunflower; ssp, annuus is the weedy wild sunflower; and ssp. macrocarpus iscultivated for edible seeds. Cultivars are divided into several types: Giant types:1.84.2 m tall, late maturing; heads 30-50 cm diam.; seeds large, white or grey, orwith black stripes; oil content rather low: ex. 'Mammoth Russian'. Semi-dwarftypes: 1.3-1.8 m tall, early maturing; heads 17-23 cm diam.; seeds smaller, black,grey or striped; oil content higher: ex. 'Pole Star' and 'Jupiter'. Dwarf types: 0.6-1.4m tall, early maturing; heads 14-16 cm diam.; seeds small, oil content highest; ex.,'Advance' and 'Sunset'. Gene centers are in America with genuine resources forresistance in southern United States and Mexico. Although sunchoke is the namegiven to the hybrid with the jerusalem artichoke, much of what is sold as sunchokein the United States is, in fact, straight Jerusalem artichoke.EcologySunflowers are gown from the Equator to 55" N Lat. In the tropics, theygrow better at medium to high elevations. but tolerate the drier lowlands. Theythrive wherevcr good crops of corn are gown. Young plants withstand mildfreezing. As sunflowers havc highly efficient root systems, they can bc grown inareas which are too dry for many crops. Plants are quite drought-resistant exceptdu.-ing flowering. In South Africa, reasonable yields have been obtained with 25 cmof rainfall by dwarf cultivars. Giant types require more moist conditions. Crop maybe grown on a wide range of soils, including poor soils, provided they are deep andwell-drained. Plants are intolerant of shade and acid or waterlogged soils. Rangingfrom Boteal Moist through Tropical Thorn to Wet Forest Life Zones, sunflower

tolerates annual precipitation of 2-40 dm (mean of 195 cases =11.4), annualtemperature of 6-28'C (mean of 194 cases = 19.6), and pH of 4.5-8.7 (mean of 121cases = 6.6) (Duke 1978, 1979).CultivationSeeds harvested at 12% moisture content and stored, will retain viability forseveral years. Sunflower production may be adapted to mechanized orunmechanized societies. Propagation is always by seed. It is planted with corn orbeet planter, 2.5-7.5 cm deep, spaced 0.2 m apart in 0.W.9 m rows; seed rate of 5.6kgha. giving about 62,500 plants per ha. It may be planted earlier in spring thancorn sincc plants are more tolerant to frost. Early weed control is an important factorin yield. Sunflowers respond well to a balanced fertilizer based on soil test, usually a1-2-3 NPK ratio is best, with a need for boron and other trace elemcnts on lightersoils. Foliar fertilizers of liquid NPK on plants increase yield upto 62% with oneapplication and 97% with two applications. Sunflowers should not occur in rotationmore than once in every 4 years and should not bc in rotation with potatoes.Yields and EconomicsAverage yields range from 900-1,575 kg of seed per ha; however, yields ofover 3,375 kg/ha have been reported. Heads may contain 1,00&4,000 florets. Yieldsfrom dried seeds are 40% oil, 35% protein meal and 20-.25% hulls. In 1979, theworld's lowest production yield was 308 kgha in Algeria; the internationalproduction yield was 1,266 kgha and the world highest production yield was 2,420kgha in Austria (Anonymous, 1980). Pryde and Doty (1981) suggested average oil

yield of 589 kgha from 1,469 kgha seed. Telek and Martin (1981) suggest oilyields of 450 kgiha. In India, rainfed sunflower gave seed yields of 1,120 kgha inpure stands, 1,050-1,070 kgha and 1,010-1,070 kgha when intercropped withwwpea and peanuts respectively (Chandrasekar and Morachan, 1979).DiseasesIncrease in sunflower production has led to the increase in incidence of severediseases (Chattopadhyay,l999).Sunflower suffers on account of a number ofdiseases. The following fungi are known to cause diseases in sunflowers: Albugotragopogonis, Allernaria tcnuis. Alternaria zinniae, Armillaria mellea, Ascochytahelianthi. Bottytis cinerca. Cercospora bidmtis. Cercospora helianrhi, Cercosporahelianthicola. Cercospova pachypus, Corticiltm rolfiii. Uyslopus cubicus. Cystopustragopogonis, Diaporthe arctii, Diplodina helianthi. Entyloma po!vsporum,Erysiphe chicuracearum. Fusarium acuminatum, Fusarium conglutinans. Fusariumculmorum. Fusarium equiseti. Fusarium javanicurn, Fusariurn oxysporum,Fusarium sambucinum, Fusarium scirpi. Fusarium semitecrtm, Fusarium solani,Hclminthosporium helianthi.(Allernaria helianlhi) Leptosphaeria helianthi.Leveillula cornpositarum. Leveillula taurica, Macrophomina phaseoli, Oidiumhelianthi, Ophiobolus helianthi, Phialea cynrhoides, l'homa oleracca,Phymatotrichurn omnivorum. Plasmopara halstedii, Puccinia helianthi, Pythiumdebaryanum. Pythium irreguhre, Pyfhium splendens, Pvthium ultirnum,Rhabdospora helianthicola, Rhizacronia rocorum. Rhizoctonia solani. Rhizoctoniabataticola. Rhizopus nodosus. Sclerotinia ,fuckcliana, Sclerotinia libertiana,Sclerofinia minor, Sclerotinia sclerotiarum. Sclerotium rolfsii. Septoria helianthi,

Sphaerotheca fulginea, Sphaerotheca humuli, Uromyces junci, Verticillium alboatrum.Vcrticillium dahliae. Following bacteria are reported to infect sunflower:Agrobacterium tumefaciens, Bacterium mclleum, Erwinia aroides, Pseudomonascichorii, Pseudomonas helianthi, and Pseudomonas solanacearum. Virus diseasesreported from sunflower are: Apple mosaic, Argentine sunflower, Aster yellows,Brazilian tobacco streak, Cucumber mosaic, Tomato spotted wilt, Peach ringspot,Peach yellow-bud mosaic, Pelargonium leaf-curl, Tobacco necrosis, Tobaccoringspot, and Yellows. Sunflowers are parasitized by the following flowering plants:Cuscuta pentagona, Cuscuta arvensis. Orobanche aegyptiaca. Orobanche cumana,Orobanche muteli, Orobanche ramosa, Striga hermonthica, Striga asiatica. Strigaiutcu und Striga senegalcnsisFoliar blight due to Alternaria helianthi is one the most destructive diseasesand causes seed and oil-yield losses upto 80%(Balasubramaniyam and Kolte,1980;Agawathi et a/. ,1989). It is caused by Alternaria helianthi(Hansf)Tubaki andNishihara (Chattopadhyay and Appaiji,l999). Alternaria helianthi was firstdescribed by Hansford (1943) as Helminlhosporium helianthi Hansf. from diseasedsunflower leaves collected near Kampala in Uganda. Following a carefulexamination of diseased material in Japan in 1964 and a comparison withWansford's original type specimen, Tubaki and Nishihara (1969) renamed thepathogen as Alternaria helianthi. The disease has been reported on sunflower inTanzania in 1950 (Wallace and Wallace,1950), Japan in 1963(Takano, l963),Yugoslavia in 1964 (Acimovic,l969), India in 1971 (Narain andSaksena, 1973), Brazil in 1971 (De Aquino el a1.,1971),Iran in 1971 (Acimovic,

1975), Australia in 1971 (Alcorn and Pont ,1972 )and North America in 1980(Shane etal.,l981).A. helianthi belongs to the family Dematiaceae, under the class Hyromycetesof the sub division Deuteromycotina(Mehrotra and Aneja, 1998)Fungi Imperfecti. Itreproduces by wnidia borne on conidiophores. The conidiophores of A. helianthi arecylindrical, scattered, pale grey yellow, straight or curved, geniculate, simple orbranched, upto heptate, 25-80 X 8-10 pm. The conidia are cylindrical to longellipsoid,straight or slightly curved, pale grey yellow to pale brown, Ito 11 septa,40-1 l Ox 13-28 pm, round at both ends. The conidia are rarely produced in chains.Alternaria helianthi blight disease is epidemic (Mayee, 1991). Van der Westhuizenand Holtzhausen (1980) reported that occasional secondary conidia were producedon lateral conidiophores growing directly from the parent conidium.Altcrnaria helianlhi is now regarded as a major pathogen of sunflower,particularly in Yugoslavia (Acimovic, 1969; Islam e! a/., 1976; Islam and Maric,1978) and India (Abraham ct a/., 1976; Kolte and Tewari,1977). It is a seriouspathogen of sunflower that is being grown in tropical and subtropical regions of thcworld and causes rapid epidemic conditions. .Disease development is highlydependent on environmental condition and growth stage of the crop, location andyear (Allen ct a/., 1981, 1983). Symptom of the disease includes necrotic spots onfoliage which coalesce on leaves causing blight and premature senescence.Susceptibility of sunflower tissue increases with age; the older leaves are moresusceptible than the young and expanding leaves (Allen et al.. 1983). The funguscauses brown spots mostly on the leaves, but the spot may also appear on thecotyledons, stem, sepals and petals. The lesions on the leaves are dark brown with

pale margin and yellow halo, 2 to 3cm in dia. and are usually irregularly circular inshape. Foliar blight symptoms appear as circular, dark brown to black lesions withconcentric rings that resemble a target pattern. Some lesions have distinct yellowhalos on young plants. Lesions generally do not cross major leaf veins and becomeangular in shape as they age.Under disease favorable conditions lesions cancoalesce, leading to necrosis and withering of entlre leaves. Stem lesions begin asdark flecks that enlarge to form large elliptical to diamond-shaped sunken lesions.Large, blackened stem lesions can girdle plants and cause stem breakage. Highhumidity and moderate to warm temperatures favor foliar blight.Plants are highly susceptible at anthesis, when leaf assimilates are mobilizedby the plant for seed and oil production. Lcaf loss at this growth stagc results in anyield loss upto 80% (Carson, 1985). A very few sources of resistance to A. helianlhihave been identified and quantitat~ve differences existing among A. helianrhi havebeen reported (Carson, 1981).Epidemics of Altemaria foliar blight [Allernaria helianthi (Hansf.) Tubakiand Nishihara; A. alrcrnata (Fr.) Keissler] at different plant growth stages resulted inyield losses as high as 61.9% in 'Mordcn' and 49.7% in 'APSH 11' cultivars ofsunflower. The number of seeds per head and their test weight were alsosignificantly reduced. Infection at the late vegetative to budding stage causedgreatest losses in yield.Yield losses of 20 to 80%, with corresponding oil losses have been reportedfrom tropical and subttopical sunflower production regions. In the high plains, thedisease is only of importance during wet years or when sunflowers are grown underabundant irrigation.

Management ApproachesCultural ControlCrop rotation and strict sanitation of crop debris effectively manageAltemaria foliar blight in the High Plains.Chemical ControlFungicides generally are not necessary for Altemaria foliar blightmanagement in the High Plains. Chemical controls are most effective when appliedat or just before the first appearance of lesions and used in combination with culturalcontrol strategies. Addition of carbendazim, azadirachtin and bulb extract of Alliumsativum were inhibitory to mycelial growth of A. helianlhi (Chattopadhyay, 1999).Biological ControlA few reports on biological control strategy are available for Altcrnariaalternala and A. porri causing leaf spot but not for A. helianthi(Hussain,1960).Extensive use of chemicals to control plant diseases has disturbed the delicateecological balance of the soil, leading to groundwater contamination, developmentof resistant races of pathogen and health risks to humans. The sensitive issueregarding the demerits as well as the harmful effects of the chemical pesticides hasdemanded greater attention towards the development of safer methods of crop andenvironment protection. One of the major targets of recent research studies is toenhance the defensive capacity of the crop plant by biological agents, includingphytoextracts and microbial antagonistsBiocontrol of phytopathogens was effected by phytoextracts(Gomathi, 2001),antagonistic microbes(Soureche et al., 2001), phytoproducts either individually orcombination of antagonistic microbes and Rhizobium and combination of

phytoextract and antagonistic microbes(Asha, 2006). Considerable attention hasbeen paid to plant growth-promoting rhizobacteria (PGPR), as the best alternative tochemicals, to facilitate eco-friendly biological control of soil and seed-bornepathogens.Farzana Banu ct al. (1990) reported that seed treatment with Mchodermaharzianum and T. viride reduced seed borne incidence of Colletotrichum dcmatium.Lewis and Papavizas (1991) confirmed the antibiotic efficacy of Trichoderma spp.and Gliocladium virens formulations against cotton damping off caused byRhizoctonia solani. Sariah (1994) established that the biocontrol of C. capsici and Cgloeosporioides with BaciNus subtilis was mainly due to antibiosis.Most of the bacteria which exhibit PGPR activity, belonging to thePseudomonads group (Gram-negative) and Bacillus spp (Gram positive) are mostwidely studied. Kannaiyan (2001) reported that fluorescent pseudomonads-mediated induced resistance acted as the main mechanism for the managementof multiple pathogens, insects and nematode pests and diseases in major cropplants. He further reported that the bro~d spectrum of pest management bypseudomonads could provide an effective, economical and practical way ofplant protection. Muthaiyan (2000) reported that the rice blast caused byI*ricularia oryzac was effectively managed with P, fluorcsccns formulation. Hereported that the application of Pseudomonas as seed treatment (10 gikg seed) andtwo foliat sprays at 45 and 60 DAT proved to be best for the effective control ofblast disease of rice.

Jetiyanon et a1.(2003) reported that mixture of Bacillus am.vlo/iquefaciens andB. pumilus significantly protected plants against all tested diseases viz, blight oftomato (Lycopersicon esculentum) caused by Sclerotium ralfsii, anthracnose of longcayenne pepper (Capsicum annuum var. acuminatum) caused by Colletotrichumgloeosporioides and mosaic disease of cucumber (Cucumis sativus) caused byCucumber mosaic virus (CMV).Enhancement of microbial biocontrol is possible when the pathogen pressureis reduced through microbial antibiosis (Rajathilagam and Kannabiran, 2001) orcompetition for nutrients on one hand and resistance induced in the plants, on theother hand. This has been achieved mainly through bacterization. Beneficial effectsof plant bacterization have been reported in several plants during the last fewdecades (Schroth and Huncock, 1982; Dileep Kumar er at., 2001).. In the recent past, research studies were conducted to bring out the potential ofemploying certain fungi and bacteria, including Pseudomonas spp and Bacillus sppas biocontrol agents of certain pathogens by virtue of their ability to producesiderophore under iron limited condition (Powell et a/., 1982; Bakker et at., 1986).Siderophores are defined as chelatiny compound, low molecular weight, secreted bymost of the microbes (bacteria and fungi) in the soil or in the culture media underiron starved condition which are virtually Fe(l1l) specific ligands. The termsiderophore is derived from two Greek words: 'Sidero-iron and phore-bearer' Theyact as scavenging agents in order to combat low iron stress.Siderophore microbial iron(II1) -transport agents produced by beneficialfluorescent Pseudomonads are partly responsible for enhanced plant and biocontrol.Beneficial fluorescent Pseudomonads and other microorganisms can promote plant

growth and induce disease suppresiveness by mechanism other than siderophoreproduction viz, through competition or through production of hormones, antibioticsor bacteriocins. Most arerobic and facultative anaerobic microorganisms respond tolow iron stress by producing extracellular, low molecular weight (500-1000 dalton)iron (111) - transport agents.Function of siderophore is to supply iron to the cell. Siderophores displayconsiderable diversity in chemical structure depending upon the producingorganism; fungi generally produced hydroxymate iron(l11)- binding groups whereasbacteria produce siderophore with catecolate iron(1II) binding legends.According to Berge.v's Manual of Svstemaric Bacteriology, two maincharacteristics of bacteria are: ( I) Presence of strict respiratory type metabolism withoxygen as the terminal electron acceptor in areobic bacteria and (2) Inability ofmost, if not all, species to grow under acid condition (pH 4.5). These two growthconditions justify the need for an efficient iron uptake system involving siderophorein order to meet the nutritional iron requirement of these bacteria. In Aerobicenvironment at neutral pH, it is not directly available to living cells (Palleroni,1984).Although siderophore was initially observed during Pasteur's time, thebiofunction of this compound was revealed only during the last four decades. Byeffectively chelating iron by siderophore, some of the microbes which may be thepathogens to the plants, could be deprived of this essential nutrient and thus theirgrowth may be inhibited. Suppression of the plant pathogens leads to an increme inthe plant growth ultimately resulting in a better yield( Schroth and Hancock, 1982).Interest in this biocontrol strategy system has increased considerably in recent times

due to possible roles of siderophore in several general phenomena such as plantdisease prevention, plant growth promotion. Siderophores may act as growth factorsand some are known for their potent antimicrobial activity (Neiland, 1981).Beneficial effects of siderophores secreted by microbes have been reportedin many plants during the last four decades. Some Fe-efficient plants have beenshown to acquire Fe from various microbial siderophores. However, the mechanismsinvolved and the relative importance of microorganisms and their siderophores forplant Fe acquisition arc not well understood (Crowley et al., 1988).The capacity of bacteria to utilize siderophores is important to the~r powthin the rhizosphere (Jurkevitch, et a/., 1992) and on plant surfaces (Loper and Buyer,1991). Specific siderophore-producing Pseudomonas strains rapidly colonize rootsof several crops and this colonization can result in significant yicld increases(Schroth and Hancock, 1982). Enhanced plant growth caused by these strains oftenis accompanied by reduction in the populations of fungi and other bacteria on theroots. These beneficial Psetddomonas strains suppress some soil-borne fungalpathogens (Leong, 1986) and there is convincing evidence to support a direct role ofsiderophore-mediated iron competition in the biocontrol ability exhibited by suchisolates (Leong, 1986; Loper and Buyer, 1991 ). Antagonism depends on the amountof iron available in the medium. Siderophore production by the biocontrol agent andsensitivity by target pathogens are expressed only under iron-limiting conditions(Kloepper et al.. 1980; Weger et al. 1988).Pyoverdine is the first siderophore recognized in Pseudomonas spp(Barelrnann, el al., 2002). Some other structurally different siderophores can beproduced together with pyoverdines by certain fluorescent Pseudomonas strains,

e.g., pyochelin, salicylic acid, quinolobactin (Audenaert, 2002a). Monocatecholatesiderophore, 2,3-dihydroxybenzol glycine (Ito and Neiland, 1958) are some of thesiderophores produced by Bacillus spp.Agrobacterium rhizogenes strain K84 (formerly called A. radiobacter) whichis used worldwide as a commercial agent for the biocontrol of crown gall diseasecaused by tumorigenic Agrobacterium strains (Farrand and Wang. 1992) is theproducer of the antibiotic agocin. However, under tield condition, this bacteriumcan protect plants against crown gall caused by pathogenic isolates resistant toagrocin 84 (Cooksey and Moore 1980, 1982; .Du Plessis er al., 1985; Lopez er a/,,1989, Lopez et a/., 1987; Penyalver and Ldpez, 1999; Vicedo et a/.. 1993) andagrocin 434 (Donner et a/., 1993). Recently, McClure ct al.(1998) suggested thatagrocin 434 plays a direct role in controlling agrocin 434-susceptible pathogens.Strain K84 also produces a third antibiotic-like substance named ALS84, whichinhibits many tumorigenic Agrobactcrium strains in vitro (Peiialver 1994). Thenonspecific inhibitory activity of ALS84 is observed in mamitol-glutamate mediumbut not in Stonier's medium (Peiialver, 1994). These two media differ in a number ofways including their relative iron concentrations, raising the possibility that ALS84produced in response to mannitol-glutamate in low iron conditions is identified assiderophore. Siderophores produced by certain isolates of Pseudomonas spp, play adiroct role in biocontrol of some plant pathogens (Leong 1986; Loper andIshimaru,l991). The siderophore produced by strain K84 may, in a similar fashion,contribute to the biocontrol of crown gall especially under conditions of ironlimitation.

Leong(1986) discussed the possible role of siderophores in the biocontrol ofplant pathogens. The involvement of siderophores in biological control processeshas been well documented by Powell et al. (1982) and Bakker et a1.(1986).Kloepper et al. (1980) reported higher yield in pea due to the activity ofPseudomonas bacterization and he discussed the role of siderophores in cropimprovement. Duijff et al.(1993) confirmed that the siderophore of Pscudomonas spacted as the main factor in the suppression of Fusarium Wilt, which in turn fetchedhigher yield in carnation plant. De Meyer and Hone (1997) found that reducedBotrytis infection by Pseudomonas treatment in tobacco was mainly due to theaction of siderophores. Higher yield in potato plant by bacterization was found to bedue to the activity of Pseudomonas siderophores( Dileep Kumar er al., 2001).Seed treatment with the rhizosphere bacterium Scrratia marcesrens strain 90-166 suppressed anthracnose of cucumber, caused by Colletotrichum orbiculare,through induced systemic resistance (ISR). When the iron concentration of aplanting mix was decreased, suppression of cucumbcr anthracnose by strain 90-166was significantly improved. Stram 90-166 produced 463.70 mg of catecholsiderophore per liter, as determined by the Rioux assay in deferrated King's mediumB. The hypothesis that a catechol sidcrophore produced by strain may be responsiblefor induction of systemic resistance by this strain was tested by evaluating diseasesuppression by a mini-Tn5-phoA mutant, deficient in siderophore production (Presset a!.,ZOO]), Similarly, Leeman et al. (1996) reported that ISR induced byPseudomonas fluorescens WCS374 against Fusarium wilt of radish is inverselyrelated to iron availability of the planting substrate.

It is evident from the above account that the studies conducted so far broughtout the beneficial role of siderophores produced by rhizosphere microbes(fungi andbacteria) in the biocontrol of pathogens.Review of literature reveals that there are only few reports on the effects ofthe foliar spray of synthetic chelators (EDTA) (Subrahmanyam et al.,lY92). Thereseems to be no report on the effects of the siderophore and siderophore-Fe complexextracted from culture filtrates of Pseudomonas spp and Bacillus spp applied asfoliar spray on the crop plants. Hence the present invest~gation was undertaken tofind out the efficacy of foliar spray of siderophores extracted from four soil bacteria,viz., Pseudomonas ,/luorescens, Bacillus am,yloliquqfacicns, Bacillus cereus andBacillus subrilis and their siderophore-Fe complex (chclates with iron) in controllingA. helianrhi causing foliar bl~ght in Helionrhus annuus .OBJECTIVES:To screen the crudc culture filtrates of four soil bacteria (Bacillus subtilis,Bacillus cereus. Bacillus amyloliquefacicns and Pseudomonas fluorescens) for theirinhibitory activities on Alternaria helianrhi in vitro.To screen the thrcc soil bacteria viz. Bacillus cerrus. Bacillusamj~loliqucfaciens and Pseudomonas,fluorescens with a view to find out 01C of thesiderophores of above three bacteria and their siderophore-Fe complex based onconidial gemination, radial mycelial growth and biomass of Alternaria helianrhi invitro.

To find out the effects of selected optimum concentration of selected bacterialsiderophores and their siderophore-Fe(lll) on cell wall permeability, protein and cellwall lytic enzymes of Alternaria helianthi in vifro .To investigate the phytotoxic effect of optimum concentrations ofsiderophore and siderophore-Fe complex of three species viz. P. fluorescenr. B.am.vloliquefaciens and B. cereus by studying the germination of seeds of host plant,Hclianthus annuus.To investigate the effects of the optimum concentrations of siderophore oftwo species viz. P. ,flltoresrens and B. am,yloliqucfacisns and thcir siderophore-Fecomplex and Fe(I1) on the host plant- pathogen interaction by studying thephysiological and biochemical parameters in the host plants under healthy, infectedand treated conditions .To investigate the effects of the optimum conccntrations of sidcrophore andsiderophore- Fe complcx of the above bacterial specics and Fe(ll) in reducing thedisease intensity in the host plant caused by A. helianthi.

2. MATERIALS ANDMETHeDS

The present investigation was taken up to find out the efficacy of foliarspray of siderophores extracted from four soil bacteria, viz., Bacillus subtilis,Bacillus cereus, Bacillus amyloliqueficicns and Pseudomonas,fluorescens and thesiderophore-Fe complex(chelates with iron)of latter two species in inhibitingAlternaria helianthi(Hansf.) Tubaki and Vishihara causing foliar blight inIielianthus annuus L.I. SOURCE OF THE PATHOGEN:Allernaria h'elianthi was isolated from the leaves of infected Helianthusannuus plants cultivated in the fields of Panmti, Villupuram district (Tamilnadu).They were surface sterilized with 0.2% mercuric chloride solution for one minute,washed thoroughly in sterile distilled water four times and the infected portions of theleaves were cut into small bits and placed on PDA (potato dextrose agar) medium with50 ppm of streptomycin and incubated in BOD incubator for seven days at 28 + 0.2OC.Culture characters and morphology of reproductive structures, conidia were tecorded.Identification of this fungus was done on the basis of reports of Allen el al.(l 983 a, b)and confirmation was done in the Center for Advanced Studies in Botany, Universityof Madras.MAINTENANCE OF THE PATHOGEN:The pathogen Alternaria helianthi was purified by single conidial isolationmethod. The purified culture was stored in the slants of Potato Dextrose Agar (PDA)and incubated in the BOD incubator at 28c0.2 'c. The pathogen was inoculated atleast once in three months in the Helianthus annuus leaf to wnfinn its virulence.

BIOCONTROL AGENTS:Pure culfures of four soil bacteria viz. Bacillus subtilis, Bacillus cereusAeudomonas jluorescens, Bacillus amyloliquefaciens were obtained from theInstitute of Microbiology Technique, (IMTECH), Chandigarh.2. CONFIRMATION TEST FOR SIDEROPHORE PRODUCTION:Bacillus subrilis, Bacillus cereus. Bacillus am,vloliquefaciens andPseudomonas fluorescens were grown in several masses of 200mL succinatemedium in SOOmL flasks at 2812 "C for 40-60 h. The production of pigment wasmeasured at 405 nrn of free cell medium.Screening of the siderophore at 10 % concentration:The culture filtrates (cell free medium) from the 7'h day old culture of theabove mentioned soil bacteria wcre prepared in 10% concentration using steriledistilled water, filtered through double-layered cheese cloth and the same was usedfor screening their effects on the conidial gemination, radial mycelial growth andbiomass production of A, heliunthi.2.1. Conidial GerminationThe culture filtrates of the four biocontrol agents were prepared in 10%concentrations using sterile distilled water. Conidial suspension of A, helianrhi wasadded to the 10% concentrations of the soil bacteria, so as to make the final countadjusted to 8000-12000 conidiafml with the help of haemocytometer. Conidial

germination studies were camed out in cavity slides. Triplicate slides weremaintained for each concentration. For control, conidial suspension was added to thesterile distilled water in the slide. The slides were incubated in moist chamber at30°C and conidial gemination was observed after 24 h. The percentage of inhibitionover control was calculated by the formula of Vincent (1927).Where, I = Inhibition over controlC = % of germination in controlT = % of germination in treated2.2. Mycelial growthThe mycelial gtowth of A. hrlianthi was measured by poisoned platetechnique. After the sterilization of petriplates (9 cm dia.), PDA medium, cork borerand other glass wares In an autoclave at 121.5 O C for 15 min. with 15 Iblsq inchpressure, the prepared crude culture filtrates of thc soil bacteria at 10% concentrationwere added to the warm PDA in three Petri plates. Then the plates were inoculatedby placing 9 mm (dia.) disc cut from the growing tip of 5 days old culture of A.hciianlhi. All this was camed out inside the laminar flow chamber under the sterilecondition. PDA without the culture filtrates served as control. The conidia ofA.helianthi of control and treated cultures were maintained in triplicates. Theinoculated plates were sealed with parafilm wax and incubated in the incubator at28+0.2 OC. On the 5 'h day of incubation, the measurement (cm) was taken along theradial line of the mycelial growth in the petriplates. The percentage of mycelialgrowth inhibition was calculated by using the formula of Vincent (1927).

C-TI = ------- * 100CWhere, I = Inhibition ofmycelial growthC = Diameter growth in controlT = Diameter growth in treatment2.3. Bio-mass ofpathogenThe crude culture filtrate was added to 50 mL of sterilized media in 25OmLErlenmeyer's conical flask, through a sietz filter of pore size 0.22nm and adjusted to10% concentration. Two discs of 9mm(dia.) each in size were cut with the help ofcork borer from the growing tip of 7 days old culture of A. helianthi and wereinoculated into each flask and incubated for 7 days in B.O.D. at 28°C. After sevendays, the fungal mat and liquid media were separated by filtering through doublelayered Whatman No.1 filter paper under pressure by vacuum pump. The freshweight of these mats was first determined. After this, the mycelial mats along withtheir pre- weighed filter papers were placed in a hot air oven at 70°C for three hours.After three hours, their dry weight was noted. Again they werc placed in hot air ovenfor one more hr. This procedure was repeated until two similar consecutive readingswere obtainedThe above tests were repeated using bacterial culture succinate mediumamended with 50pg of FeCll llitre to verify whether the inhibition was due to thepresence of siderophores.

3. ISOLATION OF SIDEROPHORESiderophore was isolated and purified by a modified method of Meyer and Abdallah(1978). The bacteria were gown in several lots of 200ml succinate medium in500ml flasks at 28i2 "C for 40-60 h. The production of pigment was measured at405 nm of free cell medium.4 mL of M FeCl 3 was added to one litre of the pooled broth and stirred for 20min, centrifuged at 10,000xg for 20 min and then the supernatant concentrated 50fold by flash evaporation at 55'C. The contaminating protein were precipitated bysaturation with NaCl and removed by centrifugation. The supernatant was thenextracted with an equal volume of chloroform and phenol [I:l(v/w)]. The organicphase was mixed with 3 volumes of diethyl ether and the pigment was thenpartitioned into deionized water (20ml) until color could no longer be extracted fromthe organic phase. The resulting aqueous extract was washed three times withdiethyl ether to remove the phenol. It was then reduced to dryness by lyophilization.The crude femc siderophorc extract (50-100mg) was further purified bypassing through a CM -sephadex C-25 cation exchange column (1.5X90cm)equilibrated with 50mM pyidine -acetate buffer ,pH 5.0. The column was elutedand elution were collected. Their absorbance was monitored at 405 nm and all the'peaks' were collected separately. The predominant reddish brown, non -fluorescent peak containing ferric siderophore was lyophilized, suspended inpyridine-acetate buffer(lm1) and once again passed through the column to increasethe homogeneity. The purity of the ferric complex was determined by thin layerchromatography using silica gel G uniplates developed with ethanol- water [70:30]70

solvent system. Homogeneity of fenic siderophore complex was confirmed by theappearance of only one sharp brown spot. No fluorescent spot were visible underUV light.Preparation of the irongree complexIron -free pigment was retrieved by the method of Meyer andAbdallah(1978). The purified fenic siderophore complex (SSOmg), obtained asabove was suspended in 50ml distilled water and pH was adjusted to 4.0 by using10%(v/v) aqueous acetic acid .A 5% solution (w1v)of 8-hydroxy quinoline inchloroform (150ml)was added to the ferric siderophore complex in a stoppered flaskand stirred vigorously. Thc pH of the aqueous phase was readjusted to 4.0 andwashed with chloroform to remove 8-hydroxy quinoline.11 was then concentrated to20ml and applied to a CM -sephadex C-25 cation exchange column (1.SX90cm)From which the elutes wcre collected(3mL each) and their absorption was detected at405 nm. The experiment was performed as quickly as possible to avoid anddecomposit~on of the pigment. The Iron -Free siderophore was reduced to dryness bylyophilization.Storage ofthe extract: The extracts were stored In dark chamber with sterilescrew cap bottles at -4 "c temperature.Testing: While evaluating the antimicrobial activity of the selected test extract,the following conditions were checked periodically.Firstly, the extract must be brought into contact with the microorganism whichhas been selected for the tests.

9 Secondly, the micro organism should be able to grow, even if not providedwith antibiotic.9 Thirdly, there must be some means of judging the rate of growth if any, madeby the test organism during the period of time chosen for the test.4. Determination of Optimum Inhibitory Concentration (OIC):To find out the OIC, required for the inhibition of the conidial germination, radialmycelial growth and biomass production of A.hclianthi, test siderophores wereprepared at different ppm concentrations (I, 5, 10. 50, 100, 500) and siderophoreiron complex was prepared at ratio of 10:l and for comparison, O1C of Fe(ll)(mg/g)was also found out.5.ELECTROLYTIC LEAKAGE:Mycelial mat of A. helianthi from 7 days old culture of control and treatmentwith ctude siderophore was collected and washed under tap water and then placed in6ml-deionised water. Electrical conductivity (EC,) measurement was taken in thebeginning to estimate the amount of electrolyte released from the cell In the initialstage. Then tubes were kept in dark at 25 "C and measured at different time intervals(ECf): 1.5, 3.5, 7.5 and 22.5 h. Following this, samples were autoclaved (EC,) andthen cooled at 25 O C and the total electrical conductivity was measured by using thefollowing formula:(ECr -ECd---------------*- XI00(EG-EC,)

6, CELL WALL LYTIC ENZYMES ASSA YS:Kwraction ProcedureThe pathogen was grown in Czapek's broth, supplemented with pectin ascarbon source replacing sucrose for pectinolytic enzyme production. Similarlymicrocrystalline cellulose and carboxylmethyl cellulose were used for cellulolyticenzymes. The siderophore and siderophore-Fa complex of BaciNus subtilis.Bacillus cereus Pseudomonas ,fluorescens, Bacillus amyloliquefacicns in theirOIC(0ptimum Inhibitory concentration)were amended to 50 ml sterilizedCzapek's liquid media in a 250 ml Erlenmeyer conical flask separately. Thetwo discs of 9 mm(dia.) were cut with the help of a cork borer from thegrowing tips of the 7 days old cultures ofA. helianthi. They were inoculated ineach flask and incubated in the BOD incubator at 28 i 0.2"C for 7 days. Thecontrol and treated flasks were maintained in triplicates. After incubation, thefungal mat and the liquid media were separated by double layered WhatmanNo.] filter paper placed in Buchner funnel under suction by vacuum pump. Thefiltrates were further centrifuged in a high speed, cooling centrifuge at 5000rpm for 10 min and the supernatant was used as the enzyme source for theest~mation of pectinolytic Enzymes1. Poly methyl Esterase (PME)End product estimation2. Polygalacturonase (PO)End product estimation

3. Polymethyl Galacturonase (Pectin Depolymerase) (PMG)Viscosity assayEnd product estimation4. Pectin Trans Eliminase (PTE)Viscosity assayEnd product estimation6.1. Polymethyl Esferase(PME) (EC 3.1.1.11)The enzyme hydrolyses pectin to methanol and pectic acid. Incrcase in freecarboxyl groups was monitored in a Control Dynamics pH meter. The PME wasassayed by the titration method of Muse e! al. (1972) with modification.Reagent1.5 g of pectin in I00 ml of 0.2 M NaCl8 0.02 N NaOHSubstrate Preparation: 1.5 g pectin dissolved in 100 ml 0.2 M NaCl was blendedwith the help of thc polytron homogenizer, then passed through two layers of cheesecloth and pH was adjusted to 7.Method10 ml of 1.5% pectin substrate was added to 3 ml enzyme and pH of thisreaction mixture was immediately adjusted to 7. After 24 h of incubation at 28 +O.Z°C, pH of the reaction mixture was measured in Control Dynamics pH meterand the solution was titrated back to pH 7 with 0.02 N NaOH. Control wasmaintained with boiled enzyme as enzyme source. The activity was expressed as

specific activity units (SAU). One unit = pml of 0.02 N NaOH required to maintainpH 7ih.62. Poly Galacturonase (PG) (EC. 3.2.1.6.7)6.2.1. Endo PG: The activity of the Endo-PG was mcasured by the reduction in theviscosity of the substrate caused by the enzyme. The activity of exo-PG was assayedby measuring the reducing sugar units produced and the activity was expressed asSAU. (Mahadevan and Sridhar, 1986).Reagent0: Acetic acid acetate buffer pH 5.2Substrate preparation: sodium polqpectate, 0.75% was dissolved in acetate buffer,pH 5.2, heated to 5060°C in a water bath and mixed wlth the help of a polytronhomogeniser (blender) and then passed through the two layered cheese-cloth. ThepH was adjusted to 5.2 using IN HCl or IN NaOH. Few drops of toluene was addedto the substrate and stored at 4OC.Viscosity AssayTo 4 ml of substrate, 1 ml of buffer and 2 ml of enzyme was pipetted outinto the Ostwald Viscometer-150. Suction was applied through the large arm of theviscometer to mix the contents and the suction was also applied to the small armand to determine the viscosity of the mixture (i.e. zero time). The efflux time of thereaction mixture at every 30 min intervals for 3 h was measured and the percentageloss in viscosity was calculated by the formula.

Where V = percent loss in viscosityTo = flow time of reaction mixture at 0 minuteTI = flow time of reaction mixture at a particular time intervalT, = flow time of distilled water6.2.2. Exo-PG:The activity of exo-PG was 'assayed by measuring the monomericgalacturonic acids released by the enzyme by catalyzing the sodium polpeptidedegradation. The results were expressed as specific activity units.Reagents.:. Dinitrosalicylate reagent: Ig of 3,5 Dinitrosalicylate, 30 g of sodiumpotassium tartarate and 1.6 g of sodium hydroxide were dissolved in 80 ml ofdistilled water and made up to 100 ml.8 Sodium acetate-acetic acid buffer, pH 5.2.OStandard maltose: Img/ml solution.MethodFrom the three hour incubated reaction mixture, 2.0 ml aliquots were pipettedout. To this, 2 ml of DNS reagent was added and heated in boiling water bath for 10minutes. Then it was cooled and diluted with 10 ml of distilled water. The orangered colour was read at 575 nm. Control was maintained with boiled enzyme reactionmixture. The enzyme activity was expressed as specific activity units. One unitrepresents pg of maltose releas&.

6.3. Polymethyl Galacturonase (PMG) (EC 3.2.1.6.7)6.3.1 Endo PMG:The activity of the Endo-PMG was assayed by measuring the reduction in theviscosity of the substrate caused by the enzyme. The activity of exo-PMG wasassayed by measuring the mono galacturonic units and the activity was expressed asSAU. (Mahadevan and Sridhar, 1986).ReagentAcetic acid acetate buffer pH 5.2Subsrraie preparation: Ig of pectin was dissolved in 100 ml of acetate buffer, pH5.2, heated to 5060°C in a water bath and mixed with the help of a polytronhomogeniser (blender) and then passed through the two layered cheese-cloth. ThepH was adjusted to 5.2 using IN HCI or 1N NaOH. Few drops of toluene was addedto the substrate and stored at 4OC.Viscosity AssayTo 4 ml of the substrate, 1 ml of buffer and 2 ml of thc enzyme was pipettedout into the Ostwald Viscometer-150. Suction was applied through the large arm ofthe viscometer to mix the contents and the suction was also applied to the small armand to determine the viscosity of the mixture (i.e. zero time). The eMux time of thereaction mixture at every 30 min intervals for 3 h was measured and the percentageloss in viscosity was calculated by the formula.

Where V =percent loss in viscosityTo = flow time of reaction mixture at 0 minuteTi = flow time of reaction mixture at a particular time intervalT, = flow time of distilled water6.3.2. Exo-PMGThe activity was exo-PMG was assayed by measuring the monomericgalacturonic acids released by the enzyme by catalysing the pectin degradation. Theresults were expressed as specific activity unlts.Reagents8 Dinitrosalicylate reagent: Ig of 3,5 Dinitrosalicylate, 30 g of sodiumpotassium tartarate and 1.6 g of sodium hydroxide were dissolved in 80 ml ofdistilled water and made up to I00 ml.*:* Sodium acetate-acetic acid buffer, pH 5.2.OStandard maltose: Img/ml solution.MethodFrom the three hour incubated reaction mixture, 2.0 ml nliquots were pipettedout. To this 2 ml of DNS reagent was added and heated in boiling water bath for 10minutes. Then it was cooled and diluted with 10 ml of distilled water. The orangered colour was read at 575 nm. Conhol was maintained with boiled enzyme reaction

mixture. The enzyme activity was expressed as specific activity units. One unitrepresents pg of maltose releasedh.64. Pectin Trans Eliminase (PTE) (EC. 4.2.2.10)The enzyme PTE cleaves pectin either randomly (Endo) or terminally (Exo),thereby reducing viscosity of substrate and produces TBA reacting substances. Endo-PTE activity was determined by measuring the loss in viscosity of reaction mixtureand Exo-PTE by determining the production of TBA reacting substances (Mahadevanand Sridhar, 1986).Reagent6 Boric acid - borax buffer, pH 8.7Substrate preparation: 1% of Pectin was prepared in boric acid-borax buffer. Themixture was kept at 50-5O0C in the water bath and then blended with the help of thepolytron homogeniser. It was then passed through two layered cheese cloth and pHwas adjusted to 8.7.64.1. Endo PTE:Viscosity loss was determined with the Ostwald Viscometer 150 at intervalsof 30 minutes starting from 0 to 180 min after preparing the reaction mixture.To 4ml of the substrate, 1 ml of the buffer and 2 ml of the enzyme was added andwere pipetted into the Ostwald Viscometer 150. The efflux time of the mixturewas measured at every 30 min interval for 3 h and the reduction in viscosity was

axpressed as percentage loss in viscosity and calculated by the formula as given$1 Endo-PMG.64.2. Exo PTE:Estimation nfTEA Reacting SubstancesReagent.:* 0.01 M TBAO0.5 N HCIMethod3 ml of the reactlon mixture incubated for 3 h was pipetted out into a 25 mltest tube, 10 ml of 0.01 M TBA and 5 ml of 0.5N HCI was added and placed in aboiling water bath for 60 min. It was cooled under running tap water and the volumeof the solution was adjusted to 18 ml w~th distilled water. The absorbance of thesupernatant was measured between 480 and 580 nm. The maximum absorbance ofthe solution was observed at 547 nm. Enzyme-substrate mixture drawn at zero timeincubation and boiled enzyme was used as blank. The activity was expressed inqpecific activity units. One unlt represents changes in the absorbance of 0.001h.6.5. Cellulolytic Enwmes:A. helianthi produces 1,4-P-Exo-glucanase(C~) and I ,4-$-Endo-glucanase(C,) whenWwn in Czapek's broth.65.1. I,#--0-Glucanase (CL)(EC. 3.2. L9.1)

The activity of CI produced by A, helianthi was assayed by measuring thereducing sugars released from microcrystalline cellulose and the activity wasexpressed in SAU. Exo-P-1.4-glucanase activity was measured by estimating thereducing sugars released by the breakdown of avicel with anthrone reagent(Mahadevan and Sridhar, 1986).Reagents** Sodium acetate-acetic acid buffer, pH 5.0OI% Avicel (Microcrystalline cellulose) suspended in buffer.*:* Orcinol reagent: Ig of orcinol dissolved in 50 rnl of distilled water.Gradually 20 ml of 67% H2S04 was added on ice. The volume was raised to 100 mlwith distilled water.Anthrone reagent: 200 mg of anthrone was dissolved in 100 ml of coldconcentrated HzSO~MethodTo I ml of enzyme source, 1 rnl of buffer and 0.5 ml of substrate wereadded in a test tube and incubated at room temperature for 2 h. The reactionmixture was mixed well with vortex mixer at regular interval of 30 minutes. Atthe end of the reaction. the volume of the reaction mixture was adjusted to 5ml with distilled water. The tubes were centrifuged for 15 min at 2000 g todeposit the residual avicel cellulose. Soluble sugar in the supernatant wasmeasured with the orcinol reagent. Two ml of the above supernatant, 3 ml oforcinol reagent was takcn in the test tubes and 10 ml of anthrone reagent wasadded on ice. The tubes were mixed well with the help of vortex mixture and

heated in a water bath at 80°C exactly for 20 minutes and immediately cooledunder running tap water. The colour developed was read at 485 nm inSystronics Spectrophotometer. A blank was prepared with 2% H2SOa insteadof orcinol. Control was maintained with boiled enzyme reaction mixture andwith zero time reaction mixture.6.5.2. I,4-PEndo-Glucanase (Cd(EC. 3.2.1.4)C, cleaves carboxyl methylcellulose randomly (endo-C,) and terminally(exo-C,). The activity of endo-C, was assayed by the viscosity loss caused byenzyme in the substrate CMC.Endo-P-l,4-glucanase (C,) activity was determined by measuring theviscosity loss in reaction mixture (Mahadevan and Sridhar, 1986) and byestimating the reducing sugars released by the enzyme sources in the same reactionmixture (Wang cr al.. 1997).ReagentSodium acetate-acetic acid buffer, pH 5.26.5.2.1 Endo C, :Ostwald viscometer 150 size was used to determine the viscosity loss of cellulosesubstrate. 4 ml of carboxyl methyl cellulose. 1 ml of the buffer and 2 ml of enzymewas pipetted out into the viscometer. The contents were mixed by drawing air gentlythrough the large arm of the viscometer. Suction was applied to the small arm andthe emu time of the mixture was determined at every 30 min interval for 3 h

incubation. The percentage loss in viscosity was calculated by employing theformula of the viscosity assay of Endo-PMG.6.5.2.2: Exo C,Exo activity of endo-P-1,4-glucanase(C, )activity was measured by theestimation of reducing sugar released by the breakdown of CMC with dinitrosalicyclic acid reagent as in the case of Exo-PMG.7. PROTEIN7.1. Extraction MethodProtein was extracted by the mcthod of Schneider (1945). 500 mg offresh mycelial mat of pathogen was collected from treatment and weighed. 5ml of 10% cold trichloro acetic acid was added to this on ice. This washomogenised with a polytron homogeniser and then allowed to stand for 30min and supernatant was discarded. To the pellet. 3 ml of 10% cold TCA wasadded and mixed thoroughly in a cyclomixer. It was centrifuged at 2500 rpmfor 10 min and supernatant was discarded. To the pellet 3 ml of isopropanolwas added and mixed thoroughly in a cyclomixer. This was centrifuged at2500 rpm for 10 min. The supernatant was discarded. lsopropanol washingwas repeated thrice. To the precipitate, 5 ml of 5% PCA was added mixed andheated in a boiling water bath for 15 min. The tubes were centrifuged at 3000rpm for 25 min. The residual pellet was dissolved in a 5 ml of 0.1 N NaOH

and centrifuged at 3000 rpm for 10 min. The supernatant was used for proteinestimation.7.2. EstimationProtein was estimated by the modified method of Lnwry (Furlong ct a!., 1973).ReagentsReagent A: 0.5 g copper sulphate (CuS04. 5H20) and 1 g of sodium citratedissolved in 100 ml of distilled water.*:* Reagent B: 20 g of sodium carbonate (NazCO3) and 4 g sodium hydroxidewere dissolved in 1 litre water.*:* Reagent C: To 50 ml reagent B, 1 ml reagent A was added.6 Reagent D: Folin-ciocalteau reagent was prepared by adding equalvolume of distilled water to the wmmercial reagent.Reagents C and D were prepared fresh at the time of use.03 Protein Standard: Standard curve of protein was prepared by using knownconcentrations of Bovine Serum Albumin (BSA).Method2.5 ml of reagent C was added to 0.5 ml of the protein extract and themixture was incubated for 5-10 min and then 0.25 ml of reagent D was added. Themixture was incubated for another 20-30 min. The wlour developed was read at awavelength of 610 tun. The pmtein standard was estimated by using BSA as a stocksolution. Pmtein content was expressed in rng of proteinlg of fresh leaf tissue.

8. IN VIVO STUDIES8.1. Seeds used for the pot experiments: The C02 variety of Helianrhus annus.(L).was used for the potted in vivo studies. The experiments were canied out in theSchool of Life Sciences, Pondicherry University. The pots were prepared acwrdingto the standard procedures. Triplicates were maintained for all treatments.8.2. Preparation of A. helianthi(pathogen): Seven-day-old mycelial mats of A.helianthi, grown on PDA were washed three times under running water, blotted inthe blotting paper and weighed. Weighed portion of the mycelium were fragmentedfor 3 min. at high speed polytron in 800 ml of distilled water. Triton-X was added inthe blended material. This prepared material was used for the inoculation of thepathogen, A. helianrhi in Helianrhw annuus plants.8.3. Pathogenicity test and study: The pathogenicity of A. hclianthi was tested onHelionthus annuus-C02. Seeds soaked in the distilled water were inoculated with A.helianfhi on 45 DAS (days after sowing) by spraying the pathogen culture (preparedas above) over the plant between 6.00-7.30 AM. Control plants were sprayed withdistilled water. All the plants were immediately covered with holed polythene bagssprinkled with sterile water on the i ~ eside r to maintain high humidity and keptundisturbed for 12 h.Seven days after the inoculation, the leaves were observed for the diseasedevelopment. The pathogen was reisolated from the infected area of the inoculatedleaves and wmpared with the original isolate.8.4. Assessment of the eflcacy of diflcnt concentrations of siderophore andsiderophore-Fe complex and Fern)

One kg of Helionthus annuus seeds (CO2) was soaked for 12 h in 400 ml. Excesswater was drained off and treated seeds were incubated in the dark for 24 h afterwhich they were sown. In control, sterile distilled water was used instead of theproduct. Pot culture experiment was laid out in three replications for each treatment.14 seeds were sown in each pot.The young leaves of 45-day-old plants germinated from the above seeds wereinoculated with A. helianrhi by spraying on the leaves. inoculation of A, helianfhiwas done in the early hours 6.00-7.30 PM. Control plants were sprayed with thesame volume of sterile distilled water. All the plants were immediately covered withpolythene bags sprinkled with sterile distilled water on the inner side so as tomaintain high humidity and kept undisturbed for 12 h. After 12 h, of siderophoreand siderophore-Fe complex of &ctmkand Fe(ll) was sprayed (46 DAS) on theleaves, The leaves were collected on 8Ih day for the estimation of the variousparameters.8.5. EXPERIMENTS (POTTED PUNTS)Experiments was designed as follows:8.5.1 HEALTHY PLANTS8.5.1.1 HEALTHY CONTROL: Helianrhus annltus plants raised from the seedssoaked with distilled water for 12 hrs overnight and left without any inoculationand any treatment.8.5.1.2 HEALTHY PLANTS UNDER TREATMENT8.5.1.2.1 TREATMENT WITH SIDEROPHORES: Healthy H.annuus plants (without inoculation of pathogen) sprayed with siderophores of P.

fluorescens (I0 ppm wnc.) and B. amyloliquefaciens (50 ppm conc.) individuallyon 46 DAS.8.5.1.2.2 TREATMENT WITH SIDEROPHORE-Fe(U1): Healthy H.annuus plants (without inoculation of pathogen) sprayed with siderophoreFe(ll1)(10: 1 ratio) of P. fluorescens and B. amyloliquefaciens individually on 46 DAS.8.5.1.2.3 TREATMENT WlTH Fe(l1): Healthy If, annuus plants(without inoculation of pathogen ) treated only with Fe (500mgil ) on 46 DAS .8.6. INFECTED PLANTS:8.6.1 INFECTED CONTROL: H annuus plants inoculated with A.helianrhi wnidia on 45 DAS and left without any treatment.8.6.2 INFECTED PLANTS UNDER TREATMENT8.6.2.1 TREATMENT WITH SIDEROPHORES: Infected H. annusplants(inoculated with A.helianthi conidia on 45 DAS) sprayed with siderophoresof P. fluorescens (10 ppm conc.) and B. amyloliquefaciens (50 ppm)on 46 DASindividually.8.6.2.2 TREATMENT WlTH SIDEROPHORE-Fe COMPLEX: Theinfected H annuus plants (inoculated with A.hclianthi conidia on 45 DAS)sprayedwith siderophore-Fe complex(l0:lratio) of P. Jluorescens and B.amyloliquefaciens on 46 DAS individually.8.6.3 TREATMENT WlTH Fe(l1): The infected H. annuus plantssprnyed only with Fe(1l) (500rngIl) on 46 DAS.Leaf samples were collected from healthy (wntrol and treated) and infected (controland treated) plants on 54 DAS and the following estimations wcre canied out

9. DISEASE INTENSITY STUDIES:Twenty five plants under each treatment were selected at random for recordingthe data on leaf blight incidence. The intensity of leaf blight disease infection wascategorized into five groups based on the number of disease spots and length of thespot on the leaf, stem and petiole and color of the stem (Balasubramanyam andKolte.1980). The disease index (%) was calculated by using the formula of(i) 0No symptom(ii) I=Light1-25 spots on the leaf of middle node ofstem. no defoliation: 0-1 spot/2.5cm lengthon stem and petiole; stem remains green.(iii) 2=Medium 25-50 spots on the leaf of middle(iv) 3=HighInode of stem, no defollatlon: 0-1 spol'2.5anlength on stem and petiole ; stem remainsgreen51 -75 spots on the leaf of middle node ofstem, no defoliation"-4 spoti2.5cm length(vi) 5=Very heavynode of stem. up to 50% defoliatiorr: 1-4spoV2.5cm length on stem and petiole;moderately discolored stemMore than 100 spots on the leaf of middlenode of stem.up to 50% defoliation: 3-6spots12.Scm length on stem and petiole;highly discolored and shntnken stem.

10. ELECTROLYTIC LEAKA GEFully expanded mature leaves were collected and washed under tap water andthen placed in 6ml-deionised water. Electrical conductivity (EC,) measurement wastaken in the beginning to estimate the amount of electrolyte released from the cell inthe initial stage. Then tubes were kept in dark at 25 "C and measured at differenttime intervals (ECf): 1.5, 3.5, 7.5 and 22.5 h. Following this, samples wereautoclaved (ECJand then cooled at 25 OC and the total electrical conductivity wasmeasured by using the following formula:(ECr -ECJ----------------- xl0(ECI-EC,)11. CELL WALL LYTICENZYMES11.1. Acetone Powder PreparationReagentsAcetone*> Diethyl etherPhosphate buffer, pH 6.6.MethodThe leaf tissues were weighed and cut into pieces of 1-2 cm each and thentransferred to a blender. Chilled acetone (-20QC) was added to wver the tissues andthen blended at high-speed 12,000 rpm for 3-5 minutes in wld condition with thehelp of polytron homogenizer. The resultant slurry was filtered through Buchnerfunnel using Whatman No.1 filter paper and the powder was washed with chilled

acetone in the Buchner funnel under auction. Then it was washed again with colddiethyl ether. The powder was dried over night under room temperature. The powderwas spread on Whatman No. l filter paper and air-dried for about 1 h. The powderwas stored in containers with tight caps in a freezer.11.2 Enqyme Preparation0.1 g of acetone powder was weighed and ground in 5 ml of phosphate buffer (0.1 MpH 7) at 4'C for 1C-15min in a mortar and pestle. The extract was centrifuged at2000 g for 20 min at 4'C and the supernatant was used as the enzyme source.11.3. Estimation of Pectinolytic En~mesEstimation of the pectinolytic enzyme was done by following similar procedureas mentioned in in-vitro studies.11.4. Estimation of Cellulolytic EnzymesEstimatw~ of cellulolytic enzymes was measured with similar methods asmentioned in in-1,itro studies.12. PHOTOSYNTHETIC PIGMENTS12.1. Total Chlorophyll ContentThe total chlorophyll content of the leaves was estimated according to themethod of Moran and Porath (1980) using the formulae suggested by lnskeep andBloom (1985). Fresh leaf discs of about 50 mg were cut and placed in a test tubecontaining 10 ml of DMF and stored for 24 h at 4OC. The wloured supematant wasused for chlorophyll and carotenoid estimation. By reading the absorbance at 647

nm and 666 nm In the spectrophotometer wrth DMF as blank, the total chlorophyllcontent was calculated uslng the followtng formulaTotal chlorophyll (rng g ' fw) = (I7Where+ A@7) (8 O8l000x w xax vA -Absorbance at spectfic wavelength (nm)w - Fresh we~ght of the sample (mg)V - Volume of the sample (ml)a - Length of the Irght path In the cell (1 cm)12.2. Carotenoid ContentThe carotenotd content was deterrnrned usrng the method of Ikan (1969)Absorbance values of the ledf extracts were detenn~ned at 480,647 and 666 nmCorrected 0 D = A480 f (0 1 14 X ,4666) - (0 638 X A647)1 1Total carotenotds (mg g ' fw) =Corrected 0 D x - x -100 w13. CARBOHYDRATES13.1. PREPARATION OF ALCOHOL EXTRACTFresh leaves were dr~ed In hot alr oven and powdered w~th the help ofmortar and pestle Drred leaf powder of about 50 mg was borled In a water bathwrth 10 ml of 80% ethyl alcohol The homogenate was first cooled and thencentrtfuged at 600 rpm for IS mtn The supernatant was saved and made upto20 ml wrth 80% ethyl alcohol Thrs extract was used for quantttatlve esttmat~on

of carbohydrates, phenols and nitrogen content. The residue was saved forstarch estimation.13.2. Reducing SugarsThe reducing sugar was estimated by the Nelson's modification of Somogyi'smethod (Nelson, 1944).ReagenrsCopper tartarate solution (A): 25 g of anhydrous sodium carbonate, 25 gof sodium potassium tartaratc, 20 g of sodium bicarbonate and 200 g of anhydroussodium sulphate were dissolved in 800 ml of distilled water, diluted to I litre, thenfiltered and stored in a brown bottle.*:' Copper sulphate solution (B): 15 g of copper sulphate was added to 100ml of distilled water. One or two drops of Conc.Hz SO1 were added.*:* Copper reagent: 25 ml of reagent A and 1 ml of reagent B weremixed.0: Arsenomolybdatc reagent: 25 g of ammonium molybdate was dissolved in450 ml of distilled water. To this, 21 ml of conc. HzS04 was added. Three g ofsodium arsenate dissolved in 25 ml of distilled water was added to the abovemixture, and incubated at 37OC for 48 h. The reagent was stored in a glass stopperedbrown bottle.MethodOne ml of fresh copper reagent prepared by mixing copper tartarate and coppersulphate solution (25:l vlv) was added to I ml of ethanolic extract. The mixture washeated for 20 min in a boiling water-bath and cooled. One ml of arsenomolybdate

eagent was added and the contents incubated for 15 min. The solution was thendiluted to 25 ml with distilled water and the colour intensity was read at 500 nm inSystronics Spectrophotometer. The reducing sugar was calculated using the standardgraph for glucose.13.3 Non-Reducing SugarThe amount of non-reducing sugar was determined by following the formulasuaested by Laomis and Shull (1937).Non-reducing sugar = Total sugar - free reducing sugar x 0.9513.4. Total SugarThe total sugar was estimated by the method proposed by Dubois el al.(1956).ReagentsAnthrone reagent: 100 ml of conc. sulphuric ac~d was added to 40 ml ofdistilled water. To 100 ml of the above mixture. 200 mg of anthrone was added andthoroughly mixed until a golden yellow colour appeared.MethodFour rnl of wld anthrone reagent was added to 1 ml of ethanolic extract. Thismixture was shaken vigorously and boiled for I0 min in a boiling water bath. Aftercooling in running tap water, the absorbance was read at 620 nm in SystronicsSpectrophotometer. A standard curve was prepared with known amount of glucose.

13.5. SucroseThe sucrose content was estimated by the method of Van Handel (1968).ReagentsAnthrone reagent30% KOHMethod0. l ml of 30% aqueous KOH was added to 1 ml of the 80% ethanol extract andkept in a boiling water bath for 10 min. The samples were cooled and 3.0 ml ofanthrone reagent was added and kept at 40°C for I0 min. The absorbance was readat 620 nm. Glucose of known concentration was used as standard.13.6. StarchThe starch content was estimatcd according to the method proposed byMcCrendy er a/. (1950).ReagentsHISOI.Anthrone reagent: Anthrone (200 mg) was d~ssolved in 100 ml of cold 95%Perchloric acid: 52 ml of commercial perchloric acid (70%) to 18 ml ofdistilled water was added to get 52% perchloric acid.

ExtractionThe residue left behind after alcoholic extraction of the leaf materials wasdissolved in 5 ml of 52% perchloric acid (PCA) for 1 h. The mixture was filteredthrough Whatman's filter paper (No. 42) and the filtrate was made upto 100 ml withdistilled water.Method4 ml of distilled water and 10 ml of freshly prepared cold anthrone reagent wereadded to 1 ml of the PCA extract carefully along the side of the tube. The contentsof the tubes were shaken vigorously and heated in a boiling water bath for 7.5 min.The tubes were then cooled immediately in running tap water and shaken wellbefore reading the colour intensity at 630 nm in Systronics Spectrophotometer. Thestarch content was calculated with reference to gluwsc standard and multiplied by0.9.14. NITROGEN METABOLISM14.1. ProteinProtein was extracted by the method of Schneider (1945). 500 rng offresh leaf materials from treatment was weighed. Estimation of the proteln wasdone with s~m~lar procedure as mentioned in in-rdtro stud~es.14.2. Amino Acid(1954).The amino acld content was estimated by the method of Moore and Stein

Reagents9 20.5 ml of 0.2 M solution of citric acid and 29.5 ml of 0.2 M solution ofsodium citrate were mixed and diluted to a total volume of 100 ml with distilledwater and the pH was checked in a pH meter.9 Citric acid solution (0.2 M): 21.09 g of citnc acid was dissolved in 500 mlof distilled water.9 Sodlum citrate solution (0.2 M): 29.41 g of sodium citrate was dissolvedin 500 ml of distilled water.9 Ninhydrin solution: 0.8 g of stannous chloride was added. Four gram ofninhydrin in 500 ml of methyl cellosolve was added to 500 ml of 0.2 M citratebuffer (pH 5.0) to the above mixture. The reagent was stored at 4°C in arefrigerator*:- Dlluents solution: Distilled water and n-propanol were equally added.MethodOne ml of rnnhydrin solution was added to 0.1 ml of alcoholic extractand shaken well. To this, 0.9 ml of distilled water was added and the above mixturewas heated in a boiling water bath for 20 min and cooled under running tap water.Flve ml of dlluents solution was added to the above mixture and kept for 15 min.The absorbance was read at 570 tun. The amino acid content of the sample wasdetermined with the help of a standard curve prepared for glycine.14.3. Nitrate Reductase ActiviwNitrate reductase activity was assayed by the method of Jaworski (1971)with suitable modifications (Muthuchelian er a[., 1990). Harvested fresh leaves

were washed and cut into 5 mm discs. Leaf bits corresponding to I00 mg freshweight were incubated in vials containing 5 ml of incubation medium. Theincubation medium was prepared by mixing 0.1 N KNOn (1 ml), 0.1 Mphosphate buffer of pH 7.5 (3.75 ml), 0.1% of Triton X-100 (0.01 ml) and 1%propanol (0.25 ml). Incubation was carried out in dark for one hour at roomtemperature (28* 2'C) giving occasional shakings. Aliquots of 0.2 ml from theincubation mixture were analysed for nitrite after 60 min. 1.8 ml of distilledwater, 1ml of 3% sulphanilamide in 3 N HCI and 1 ml of 0.02% N-(I-naphthyl) ethylene-diamine dihydrochloride were added to 0.2 ml of incubationmedium in quick succession. This was incubated for 15 min in darkness forcolour development and absorbance was read at 540 nm with suitable blank in aSystronic Spectrophotometer. The amount of nitrite formed was expressed asnmoles of nitrite produced per minute per mg fresh weight using a sodiumnitrite standard curve.14.4. Amino Nitrogen ContenrReagents** Citrate buffer: 21 g of citric acid was dissolved in 200 ml of I N NaOHand the same was made up to 500 ml with distilled water. The pH was adjusted to5.0 by adding I N NaOHIHCI.9 Ninhydrin reagent:oSolution A: 800 mg of stannous chloride was dissolved in 500 mlof Citratc buffer, pH (5.0).

oSolution B: 20 g of ninydrin was dissolved in SO ml of methylcellosolve.oSolution C: To 1 ml of Solution A, I ml of solution B was added.Standard: The known quantity of glutamic acid was used as standard.MethodThe pH of the alcoholic extract was adjusted to 7.0 by adding 0.1 NNaOHIHCI. To 1 ml of the above extract 1 ml of ninhydrin reagent was added.Then, it was heated for 20 min and cooled. 5 ml of distilled water was added and theabsorbance was measured at 475 nm in Systronics Spectrophotometer.IS. PHENOLSIS. I . Total PhenolThe total phenol content was estimated according to the method of Bray andThorpe (1 954).Reagents+ 20 Oh sodium carbonate: Twenty g of sodium carbonate was mixed withI00 ml of distilled water.*:* Folin-Ciocaltcau reagent: Commercial Folin-C~ocalteau was diluted withdistilled water in 1 :2 ratio.MethodOne ml of Fohn-Ciocalteau reagent and 2 ml of 20% sodium carbonate wasadded to 1 ml of alcoholic extract and shaken well. The mixture was heated in aboiling water bath for 1 min and cooled under running tap water. The blue solution

was diluted to 25 ml with distilled water and read at 650 nm in SystronicsSpectmphotometer. Phenols were quantified using catechol as standard.15.2. Ortho Dl-hydrary PhenolThe Ortho Di-hydroxy phenol content was estimated according to the methodproposed by Johnson and Shoal (1952).Reagent*:* Amow's reagent: Ten g of sodium nitrite and 10 g of sodium molybdatewere mixed in 100 ml of distilled water. The reagent was storcd in a brown bottle.Method0.5 N HCI and 1 ml of Arnow's reagent were added to I ml of alcoholicextract. To this, 2 ml of 1 N NaOH and 10 ml of distilled water were added. Apink colour appeared immediately on adding NaOH. The colour intensity wasreduced by diluting it to 25 ml with distilled water and the absorbance read at515 nm. The O.D. phenols were calculated using a standard curve withcatechol.16 PralineReagents-3 Aqueous Sulpho salicylic acid (3%): 3 gm of sulphosalicylic acid wasdissolved in 100 ml of distilled water.0: Acid Ninhydrin: 1.25 gm of Ninhydrin was dissolved in a warm mixtureof 30 ml of glacial acetic acid and 20 ml of 6 M Phosphoric acid with agitation.The reagent was stable for 24 hours when stored at 4'C.

4 Standard Proline: 5 mg of proline was dissolved in 10 ml of 0.1 NHydrochloric acid.ErtractlonThe extraction and estimation of proline was done according to themethod of Bates ef al. (1973). The midribs of a leaf were removed and 500 mgof the leaf tissue was weighed. It was homogenised with 10 ml of 3%sulphosalicylic acid in a mortar and pestle. The homogenate was filteredthrough a Whatmann No. 2 filter paper. The procedure was repeated wlth theresidue and the filtrates were pooledMethodTwo ml of acid ninhydnn and two ml of glactal acetic ac~d was added to 2.0 mlof the filttate. The tubes were ~ncubated for 1 h at 100°C on a water bath. Thc tubeswere transferred on Ice to terminate the reaction and four ml of toluene was addedand mixed v~gorously for 15-20 seconds. The chromophore containing toluene wasasplrated from the aqueous phase. It was allowed to reach room temperature and theabsorbance measured at 575 nm. A reagent blank was maintained. A standard curvewas obtained using a known concentration of authent~c prol~ne. The prol~ne contentwas expressed as mg of prollne per gram fresh weight.17. OXIDA TIVE ENZYMES17.1 Superoxide Dismutase (SOD, EC 1.13.1.1)SOD was determined by the method of Beauchamp and Fridovich (1371) asmodified by Dhindsa and Matowe (1981), which measures the ~nhibition in thephotochemical reduction of nitroblue tetrazolium. In the spechophotometric assay, 1

ml reaction mixture contained 50 mM phosphate buffer (pH 7.8), 0.1 mM EDTA, 13mM methionine, 75 pM nitroblue tetrazolium INTB), 2 pM riboflavin and I00 p1 ofthe enzyme supernatant. Riboflavin was added at last and the reaction was initiatedby placing the tubes under two 15-W fluorescent lamps.The reaction was terminated after 10 min by removal from the light source.Non-illuminated and illuminated reactions without supematant served as calibrationstandards. Reaction product was measured at 560 nm. The volume of the supematantcorrespond~ng to 50% inhibition of the reaction was assigned as one enzyme unit.17.2. Catalase (CA T, EC 1.11.1.6)Modified method of Luck (1974) was employed for the assay of CAT.To 50 p1 of the enzyme extract, 3 ml of hydrogen peroxide-phosphate buffer(pH 7.0) was added. The time required for decrease in absorbance from 0.45to 0.40 was noted. The enzymc solution, which contained hydrogen pcroxide-freephosphate buffer was used as control. The activity was expressed as units. Thechange in the absorbance of 0.001/minlml of enzyme was assigned as one unit.17.3. PeroxidasePeroxidase activity was measured by the method of Hampton (1962).Reagents03 Phosphate buffer (0.05 M) pH 6.5Ppgallol (0.001 M): 0.01261 g of py~ogallol was dissolved in 100 ml ofPhosphate buffer.

*'Hydrogen peroxide: 2% Hydrogen peroxide was prepared by adding 2 mlof 6% hydrogen peroxide in 4 ml of distilled water.Method1.8 ml of distilled water was added to 1 ml of 0.001 M pyrogallol in a cuvetteand the absorbance was adjusted to zero at 470 nm. Immediately 0.1 tnl of 2%(0.588 M) H202 and 0.1 ml of enzyme were added. The contents were mlxed welland placed In Systronlcs Spectrophotometer. The change In the absorbance atevery 30-second Interval for 3 mlnutes was measured. Suitable control with heat-k~lled enzyme was ma~ntalned.17.4. Polyphenol OxidasePolyphenol oxldase activlty was measured by the method of Matta and Dlmond(1963).Reagents*:* 0.2M Phosphate buffer pH 7.0.:* Catechol (0.1 M): 1.1011 mg Catechol dissolved In 100 ml of dlstllledwater.Method0.5 ml of the phosphate buffer (pH 7.0) and 1.5 ml of dlst~lled water wasadded to 0.5 ml of enzyme, and the absorbance was adjusted to zero at 495 nm inSystronics Spectropnotometer and lmmed~ately 0.5 ml of 0.1 M catechol wasadded into the cuvette and the changes In the absorbance at every 30 secondslntnvals upto 3 mln was recorded. Control was maintained with heat-killedenzyme.

18. MORPHoLOGfCAL CHARACTERS:18.1. Shoot length: Shoot length was measured with the help of scale and wettedtwine.18.2. Total leaf area: The leaf area was calculated following the formula of Kemp(1960). The total leaf area was obtalned by mult~plying the total number of leavesper plant.Total leaf area = L x B x K x NWhere, L = length of the leafB = breath of the leafK = Kemps Constant (0.66 for dicots)N = Number of leaves per plant18.3. Inflorescence and disc size: . Inflorescence and disc size was measured withthe help of scale and wetted twine.18.4. Dry Weight of Roots and Shoots (g): Roots and shoots of each plant werewrapped in the paper bags separately and placed in an oven at 70EC to a constantweight. After drying their dry weight was recorded.18.5. Yieldprameters: On the sampling day. seeds were removed from the control,Infected and treated plants and weight of hundred seeds and percentage of oil wererecorded.19. STA TlSTICAL ANALYSIS:The data obtained from the above experiments were analysed using statisticalpackage IRRISTAT, version 3/93, developed by Blometrics unit, International RiceResearch Institute (IRRI), Phillipines and software Microsoft Excel .

3. RESULTS

I. IN VITRO STUDIESI. I. Conidial germinationThe crude culture filtrates of the four soil bacteria viz. Bacillus suhrilis, B.cereus. Bacillus am,vloliquclfhciens and Pseudomonas ,flttore.vcens at 10%concentration were tested fbr the efficacy of their siderophores on conidialgermination of A. helianrhi (24 h). The results are prescnted in Table I. The controlshowed 96.22% conidial germination at the end of 24 h. Fe-amended culture filtratesof the all the four bacteria at 10% concentration showed very low percent inhibitionof conidial germination(P. ,flrtorescois 2.15%; R.ccrcrrs 1.75%: Bacillusam,vloliquefaciens 1.37%; Bac-illtts strhtilis 0.05%).All the culture filtrates of the four bacteria without Fc amendment exhibitedvarying deb~ees of inhibition of conidial germination. Maximum inhibition ofconidial germination was recorded in culture tiltrates of Bacillus am~~loliquefaciens(78.08%). It was followed by that of P. ,fluarescens (67.15%) and 5. cereus(66.22%). That of Bacillus sttbfilts showed minimum perccnt inhibition(l.4896).1.2. Radial mycelial growthThe effect of crude culture filtrates of 5, subtilis, B. cereus, B. am,vloliqueficicns.P.fluarcscem at 10°/o concentration on the radial mycelial growth of A. helianrhi was

Table 1. Effect of crude culture filtrate (10% conc.) of BaciNus subtilis, B.cereus, B. amyloliquefaciens and Pscudomonns ,fluorescens on conidialgermination of Alternaria helianthi(in vitro)TreatmentsFe-amendedculturemediumat 24hrs(%)Conidial germinationInhibitionovercontrol(%)(24hrs)Culturewithout Feat 24 hrs(%)Inhibitionovercontrol(%I/ B. subtilisI 1 II/ 96.18f 0.02 / 0.05 1 94.8i 0.12 11.48Table 2. Effect of crude culture filtrate (10% conc.) of Bacillus sribtilis. B. cereus,B, am,vloliquqfaciens and Aeudomonas ,flttorescens on radial mycelial gowth ofAlternoria hclianthi (in ~,irro).- -TreatmentsRadial mycelial growthControlB. subtilisB, cereusB.am.ylo1iquefbciensP. ,fluorescens' Fe-amendedculture filtrateon7"'day(dia.)(cm)8.6f 0.048.4 f 0.088.3 i 0.167.6 i 0.047.81 0.16lnhidzonovercontrol on7Ih day(%)(I2.333.4911.629.30Culture filtratewithout Feon 7Ih day(dia.)(cm)8.6f 0.048.21f0.124.210.033.7f 0.123.41 0.16Inhibitionovercontrol on7Ih day(%)04.5051.1656.9760.46

studied by poisoned plate technique. The PlRMG (Percentage of inhibition of radialmycelial growth) is presented in Table 2.The radial mycelial growth of A. helianthi was found to be 8.6 cm (dia). Fe-amended culture filtrates of the all the four bacteria at 10% concentration showedvery low percent inhibition of radial mycelial growth (Bacillus srrhtilis- 2.33%; B.cereus- 3.49%; A. amylolique~uciens- 1 1.62%; P. ,fluoresccns- 9.3%).The highest PlRMG was observed in culture filtrates without Fe amendment.P. ,fluoresccns treatment showed 60.46% inhibition followed by those of A.am.rloliquefaciens (56.97%) and A. cereus (5 1.16%) over control. That of 8. strbliliswithout Fe amendment showed lower rate of inhibition(4.50N) of mycelial b~owth.1.3. BiomassThe effect of crude culture tiltrates of B. sub~ilis. B, cereus, B, am.vloliquefacicn~and P, fluorescenr at 10% concentration on the biomass ofA helianfhi is presented inTable 3.The biomass of A. hclianrhi incubated in chapek's medium on 7Ih day wasfound to be 1.69 g. Minimum inhibition was rccorded in all the treatments of fourbacterial culture amended with Fe(B. subrilis-15.50%; H, cereus-13.20%; B.am,yloliqu~faciens-12.72% and P ,,fluorescens-10.30%).The highest rate of inhibition was observed in the culture filtrate P.,fluorescens without Fe amendment(91.30%), followed by those of B.amyloliquefacicns (89.85%) and B. cereus(84.05%) over control. The culturefiltrate of B. subrilis without Fe amendment showed lower rate of inhibition (23%).

Table 3. Effect of crude culture filtrate (10% conc.) of Bacillus subtilis, B,cereus, B. amyloliquefaciens and Pseudomonas ,fluorescens on biomass ofAlternaria helianthi(in vitro)TreatmentsFeamendedculturefiltrateon 7Ih dayInhibition overcontrol on 7Ihday(%)Control I 1.691 0.02 1.691 0.02H, subtilis 1.43M.10 15.50 1.301 0.02B. cerrlls 1.47iO.02 84.05 '12.72Dry weightlnhibiticulture filtrate on overwithout Fe 1 controlOn 7Ih day (g) on 7IhI daM%) ITable 4. Effect of s~derophore of Pseudomonas ,fluorrsccns on conidialgermination, radial mycelial growth and biomass of Alternaria hclianrhi(in vitro)1 Itt~ufrn~i('w!rd~ul y~~!minrr!~onl%) I Hiidiol rni

1.4. Opthum Inhibitory Concentration (OK)OIC (Optimum concentration at which highest inhibition of pathogen growthwas recorded) of the purified siderophore and siderophore-Fe complex of threebacteria was found out on the basis of the results obtained from wnidialgermination, mycelial growth and biomass studies. These concentrations were usedfor further study.1.4.1. Siderophore of P.fluorescensSiderophore extracted from the culture filtrate of P. .fluorescens at Ippm,5,ppm. IOppm, 50ppm, IOOppm and 500ppm concentrations were tested for theirefficacy on conidial germination (24hrs), radial mycelial growth (12 days) andbiomass(l2 days) of A. helianrhi. The results are presented in Table 4. The controlshowed 95.87% conidial germination. 8.2 cm mycelial growth. 0.63 grn biomass.All the concentrations exhibited varying degree of inhibition of conidialgermination. While total inhibition of mycelial growth and biomass was recorded in5 ppm concentration, total inhibition of conidial germination mycelial growth andbiomass was recorded at I0 ppm concentration. Hence 10 ppm concentration ofsiderophore of P. ,fluorescens was selected for in-1,irro and in-t8i10 studies.1.4.2. Siderophore of B. amyloliquefaciens and B. cereusSiderophore extracted from the culture filtrates of B. amylolrquefaciens andB, cereur at Ippm, Sppm, lOppm, 50ppm.IOOppm and 500ppm concentrationswere tested for their efficacy on conidial germination (24hrs). radial mycelialgrowth (12'~ day) and biomass(l2 days) of A. helianrhi. The results are presented

Table 5. Effect of siderophore of B, amylolique~ciens on conidial gemination,radial mycelial growth and biomass of Ahcmaria helianlhi(in v im)I 1 1 I I 1 I I10 ppm I! j 87.48 Nognrwth Nogroxlh IUiI ili~lrill~~ LIopro~lhIOUTable 6. Effect of siderophore of B, ccrcus on conidial germination, radialmycelial growth and biomass of Alternaria hclianthi (in ritro)

in Tables 5 and 6. The control showed 95.87% conidial germination, 8.2 cmmycelial growth and 0.63 g biomass. All the concentrations exhibited varyingdegree of inhibition of conidial germination. Total inhibition of radial mycelialgrowth and biomass was recorded at 10 ppm concentration, but total inhibition ofconidial germination was recorded at 50 ppm concentration. Hence 50 ppmconcentration of siderophore of B. amylo/iquefaciens and B, cereus was selectedfor in-~itro and in-vi~~o studies1.4.3. Siderophore-Fe complex of bacteriaSiderophore-Fc complex of the three bacteria viz. B. cereus. P. ,j7uorescens.B. am.v/oliquefaciens at l0:l ratio was tested for their efficacy on conidialgermination (24h), radial mycelial growth (71h day) and biomass(l2"'day). Theresults are presented in Tables 4, 5 and 6. The control showed 95.87% of conidialgermination, 8.2 cm mycelial growth, 0.63 g biomass at the end of 24 h.Siderophore-Fe complex of all the three bacteria at$lioexhibited total inhibition ofconidial germination, radial mycelial growth and biomass.1.5. Cell permeabilityThe change in the permeability of the mycelial mat under control and treatedconditions measured as electrolytic leakage is presented in Table 7. The controlshowed minimum percentage of electrolytic leakage (0.032 mslcm2 ), followedby mycelial mat treated with siderophore of B. cereus (0.038ms/cmz), B.amyloliquefaciens (0.052 mslcm2) and P. ,/luorescens(O.O6l mslcm') andsiderophore-Fe complex of B, ccreus (0.124 mslcm2). The maximum rate of

Table 7. Effect of siderophore and siderophore-Fe complex of PseudomonasJuorescens, B, amyloliquefaciens and B, cereus treatments on cell permeability ofAllernaria helianthi (in vitro )Cell permeabilityTreatments ms/cm2 ,IControl 1 0.032 i 0.001 / Siderophorc (IOppm) 0.061 10.02 1Table 8. Effect of siderophore and siderophore-Fe complex of P. ,fl~rorrscc.ns, B.am,vloliquc/bcicns and 5, cereus on Exo activity of polymethyl Esterase(PME),poly galacturonase(PG), polymethyl galacturonase (PMG) pectin transeliminase(PTE) of Afternaria helianthi (in vitro).TreatmenuEXO-PME EXO- PGEXO- PMG(SAU) (SAW) (SAU)

electrolytic leakage was observed in the mycelial mat treated with siderophoie-Fecomplex of P. fluorescens (0.298 ms/cm2) followed by that of 6.amyloliquefaciens (0.281 ms/cm2).1.6. Cell wall lytic enzymesThe activity of pectinolytic and cellulolytic enzymes produced by A, helianthiunder control and treatments was estimated and expressed in percentage of viscosityloss and in specific activity units(SAU).1.6.1. Polymethyl esterase (PME)The activity of PME estimatcd in the enzyme sourcc obtained from in ~pitroculture is presented in Table 8.The highest rate of activity was observed in the enzyme source of control (42.5SAU), while lower rate was recorded in the enzyme source treated with siderophore-Fe complex of B. cereus (12.92 SAU). B. am.vlolique/aciens (12.78 SAU) and P.fluoresccns (I 1. 86 SAU). Control recorded maximum inhibition of enzyme activity(96.62%). while the lowest rate of enzyme activity was found in the enzyme sourceobtained from siderophore of P. ,fluorcscens-treatment (01.44 SAU), while followedby siderophore of B. am.vloliquqfaciens (4.51 SAU) and B. cereus (6.25 SAU).Treatment with siderophore of bacteria showed higher rate of inhibition than thoseof siderophore -Fe complex.

Flg. 1. Effect of siderophore, siderophon-fe compkxof P. fluomnnr , Bacilius amyioliqurfrcims and 6.caws on rndo activlty of PMO ofA. hdiandhi (in vitro)

1.6.2. Pnlygalacturonase (PC)The activity of exo-PG was measured as change in the absorbance causedby TBA reacting substances, which was produced by the terminal cleavage ofpectin by exo-PG. The activity of exo-PG (expressed in SAU) in the controland treated cultures is presented in Table 8. Highest rate of activity of exo-PGwas observed in the enzyme source obtained from control (174.15 SAU). Theleast activity was obtained in the enzyme source from siderophorc of P.,fltrorescens (7.45 SAU), 5, am,r'loliquefaciens (8.13 SAU) and 5. cereus (12.57SAU) and siderophore-Fe complex of P. ,/luorescen.v(l4.13 SAU), B.am,vloliqirq/aciens (18.40 SAU) and X. cereus (20.47 SAU).1.6.3. Pnlymethyl galacruronase (PMG)The effect of siderophore and siderophore-Fe complex of 8, cereus. P.lluoresccns. 5. am.vlolique/aciens (in r9ilro) on PMG activity ofA, helianrhi in thepectin substrate is expressed as viscosity loss (%) and the amount of sugar releasedIn g.1.6.3.1 Endo-PMG:The endo-PMG of control reduced the viscosity of the substrate to 78.50% atthe end of 180 min(Fig. I). The least endo-PMG activity was observed in theenzyme source obtained from culture treated with siderophore of P. fluorescens(I 3.44% viscosity loss), followed by that of B. am,vloliquqfaciens (16.6%) and 5.cereus (23.5%). SiderophorcFe complex of above three bacteria showed higherendo-PMG activity (5, cereus -63.67%; B. am.vloliqucficiens-28.57%; P..J7uorescens-45.70%).

Fig. 2. Effect of sidorophom, 8Merophore-Fe comlpex ofP. ~UOWCM~, B.C/IIUS amyio/iqu.fici.ns md 6.c e m on endo- PTE activity ofA.hell8nthl (IN WTRO)0 BD w iiQTlm In(.ml (rnln.)+-CONTROL-m- P nmsens srderophom8.amyld~~n8 srderopkmB.cereusSMemphon,* P n~sswoptm-~,complex+- 8 amyhMquebdens SMempbm-Fe complex+ ~.-s giderophore :Fe_*y

1.6.3.2 Exo-PMG:The activity of exo-PMG was expressed in specific activity units (SAW) ispresented in Table 8. All the treatments inhibited the activity of exo-PMG at varyingdegree. Higher amount of mono galacturonic units was released in the enzymesource obtained from the control (403.51 SAU) while the lower amount was releasedin siderophore-Fe complex of R. cereus (26.09 SAU), B. amyloliqu~faciens (18.73SAU) and P. ,fluorcscens (17.66 SAU). Among the trcatments, the least amount ofsugar was liberated in the enzyme source obtained from siderophore of P.flitorcsccns treatment (1 1.76 SAU), followed by that of siderophore of B.amvloliquefacicns (12.32 SAU) and B. ccreu.9 (14.68 SAU).1.6.4. Pectin Transelimina.se (PTE)The enzyme PTE cleaves pectin randomly (endo) or terminally (exo), therebyrcducing the viscosity of the substrate and producing TBA reacting substancesrespectively. The data are present in Fig. 2 and Table 8.1.6.4.1 Endo-PTE:The activity of endo-PTE produced by A. hclianrhi grown in control andtreated Czapek's broth is presented in Fig.2. The enzyme source obtained fromcontrol culture showed maximum percent viscosity loss (67%). followed by thatobtained from that of siderophore-Fe complex of B. cereus (37.20?~0), B.am,vloliquefacicns (33.33%) and P. ,j7uorescms(30.5l0h) and siderophore of B.cereus (25.97%). The least amount of viscosity loss was observed in the enzyme

80Fig. 3. Effect of slderophore and slderophore-Fecomplex of P. tluomscens, Bacilius 8m~doIIqu.hc/.nsand 6. cerrus on endo acthrlty of Cx ofA. h.li.nbhl(ln vh)

source treated with siderophore of P. ,fluorescens (14.28%) followed by that ofsiderophore of B. amyloliquefacicns (17.83%).1.6.4.2 Ewo-PTE:The activity of exo-PTE was measured as change in the absorbance caused byTBA reacting substances, which was produced by the terminal cleavage of pectin byexo-PTE. The activity of exo-PTE (expressed in SAU) in the control and treatedcultures was presented in Table 8. Highest rate of activity of exo-PTE was observedin the enzyme source obtained from control(l 15.31 SAU). The least activity wasfound in the enzyme source treated with siderophore of P. fluorescens (4.56 SAU),followed by that of B, am,v/olique/aciens (4.83 SAU) and B cereus (5.74 SAU)and s~derophore-Fe complex of P. .fluorescens(8.36SAU). R. amyloliquefacicns(9.0hSAU) and B. cereus (9.79SAU).1.6.5. Cellulolytic enzymesThe act~vity of two ccllulolytic enzymes under control and treated conditionsIS presented in Table 9.1.6.5.1. p1.4 exo-glucanase (CJThe activity of P-1.4 exo-glucanasc (CI) assayed by measuring the reducingsugar released from CMC and expressed in SAU is presented in Table 9. Highestrate of activity was found in the enzyme source obtained from control (30.56 SALJ).Among the treatments, the least activity was observed in the enzyme source obtainedfrom siderophore of P. JIuorescens (2.22SAU). B. om.vlo~iquefaciens (3.50 SAU)and B, cereus(4.81SAU) and siderophore-Fe complex of P. ,fluorescens (6.74 SAU),B. amyloliquefaciens (8.55 SAU) and B. cereus(8.81SAU).

Table 9. Effect of siderophore and siderophore-Fe complex of P. fluorescens, B.amyloliquefaciens and B. cereus on the Exo activity of 1,4 p-exo-glucanase (C,) andp-I ,4- Endo glucanase (exo-Cx) of Alternaria helianthi(in vitro)TreatmentsControlSiderophoreSiderophore-FeEXO-CX(SAW1 I378.603r0.11EXO-CI(SAW30.56M.OlTable 10. Effect of siderophore and siderophore-Fe complex of Pseudomonasfluorescens. B am,vloliquefaciens and B cereus on protein content of Allernariahclrantht (in wtro )! : Protein LITreatmentsI/ mg1gf.wControl8.8 k 0.04I Siderophore2.3 k0.16P. fluorcscens i (I@pm)Siderophore-Fe0,71 0,23complex(l0: I )Siderophore(50pp2, 6 f 0.20B.m)am.vloliqurkciens Siderophore-Fe.5 + 0,complex(l0: I )Siderophore(S0~~3.7 * 0.11B.cereusm)SiderophOre-Fe3.2 i 0.07complex(l0: I )% ofinhibit~onoverinfectedcontrol73.869270.5184,3057.9563.63

1.6.5.2. P1,4 endo-glucanase (CdP-1,4 endo-glucanase cleaves CMC (carboxyl methyl cellulose) randomly(endo-C,) and terminally (exo-C,). The activity of endo- C, was assayed by theviscosity loss caused by the enzyme in the CMC substrate and the activity of exo- C,was obtained by measuring the amount of reducing sugar liberated by the breakdown of CMC substrate. The activity of C, is presented in Fig. 3 and Table 9.1.6.3.2.1. Enda activity.The enzyme source obtained from control showed(Fig.3 ) maximumactivity (67.53%), followed by enzyme sourcc obtained from culture treated withsiderophore-Fe complex of R. cereus (39.15%). 5. am,vloliqusfacicns (35.80%)and P. ,fluorcrcens(33.82%) and siderophore of R. ccrsus (27.27%). The leastamount of viscosity loss was observed in the enzyme source of siderophore of P./Iuorcfcms (l6.77%), followed by that of B. amyloliql~efarienr (22.50%).1.6 5.2.2. Exn activity.Highest rate of activity was obtaineq in the control (378.60 SAU)(Table9) Among the treatments. the least activity was observed In the enzyme sourceobtained from siderophore of P.,/lrroresccns (18.08 SAU), 5, amyloliq~ct:/acicns(21.34 SAU). B. cercus (24.92 SAU) and siderophore-Fe complex of 1'.,fluorcscens (35.12 SAU),B. amvl~liqucfacicns (35.29SAU) and B, cereus(36.45 SAU).I. 7. Protein contentThe change in the protein content of mycelial mat from control and treatedconditions is presented in ~abh, 10. Maximum protein was found in the control

Table 11. Effect of siderovhores and siderovhore-Fe comvlex of Pseudomonasfluorescens, B. amyloliquefaciens and B, cereus and Fe(l1) on the germination ofHelianthus annuus seeds infected with Alternaria hclianthi (in vitro).TREATMENTSHealthyInfectel(%) (%)I I ICONTROL 1100 3.44/ SiderophoreI 93 I 88P. /luorcsccnsSiderophore-FeSiderophoreS am,vlolrq ~qJacrcns Siderophore-Fecomplex92 8 194 89- -B ccrcus1 Siderophon 2 518

Table 12. Effect of siderophore and siderophore-Fe complex of P, j7uorescans,B, amyloliqu&ciens and Fe(l1) on disease intensity in Helianthus annuus causedby A, helianthi(in vivo)TreatmentDiseaseISidemphvrc No symptomSiderophoreP ,jluorc.~rcns1 1 9,6(]1&)Fe com lexHealth1siderophorc No symptom 1I/lderophore-Fe complex1 l.56(light)Fe(l1)Infected 1 62.70(heavy)

(8.8 mgig), followed by the culture treated with siderophore of B.ccrcus (3.7mglg), siderophore-Fe complex of B.ccreus (3.2 mglg), siderophore of B.amyloliquefaciens (2.6mglg) and P. ,fluorescens (2.3mg/g) and siderophore-Fecomplex of B, amyloliquefacicns (1.5mglg) and P. jluorescens(0.7 1 mglg).I. 8 .Phytotoxic studyPercentage of germination of seeds under infected and treated conditions ispresented in Table 1 1. Seeds infected with A. hcliunthi showed minimum (3.44%)percentage of germination. Infected seeds treated with siderophore of B, cereusshowed (1 8-2.5%) of germination of seeds (siderophore control-25% infected- 18%and siderophore-Fe complex control-22% and infected-17.5 %) P. ,/luorescenssiderophore(control-93%. infected-88%) and siderophore-Fe complex (control-94%and infected- 91% )and B. am,vloliqtrefuciens siderophore(control-92%, intected-81%) and siderophore-Fe complex (control-94%. infected-89%) showed higher rateof germination of seeds. Fe(1l)control-84% and Fe(ll) infected-72% of germination.2. IN- VI VO STUDIES2. I. Disease intensityFoliar blight disease intensity caused by A. helianrhi on the host plant,Helianrhus annuus under infected and treated conditions is presented in Table 12and Fig. 4. Highest disease intensity (87.77%) was recorded in the infectedplants(severe type). The lowest disease intensity was recorded in those treated withsiderophore-Fe complex of P. ,fluorescens (9.6%) and B. om,vloliquqfaciens (1 1.56%) (light infection), followed by plants sprayed with siderophore of P. .fluorcscens

Table 13. Effect of siderophore and siderophore-Fe complex of P.fluorescens and B. amyloliquefaciens and Fe(l1) on the cellof H, annuus leaves infected with Alternaria helianthi (invivo)ITreatments /CellIControlSiderophore-FeComplexlnfectedSiderophoreSldcrophore-FeSlderophoreSlderophorePiSiderophore-FecomplexHealthyInfected

(1 8%) and B, amyloliqucfaciens (20%)(medium infection) Severe disease intensity(62.70%) was recorded in the infected plants treated with Fe(ll).2.2. Cell PermeabilityThe change in the cell permeability of the host plant under healthy, treated andinfected conditions measured as electrolytic leakage is presented in Table 13. Leavesof control healthy plants showed lowest rate of electrolytic leackage (0.039mslcm2). While those of treated healthy plants showed slightly higher rate ofelectrolytic leakage (0.045-0.049mslcm2). It was found to be highest in the leaves ofcontrol infected plants (0.375mslcm2). Lowest value was recorded in those treatedwith siderophore-Fe complex of P. ,lluorcscens (0.063msicm2), followed by that ofR am.vloliqu~facicns (0.071mslcm2). siderophore of P. ,fluorc.scens (0.078mslcm2)and 8. am,vlolique~~ciens (0.081 mslcm2) in ascending order, Infected plants treatedwith Fe(ll) showed higher electrolytic leakage values (0.31 1mslcm2).2.3. LEAVES2:3:1 Cell wall lytic enzymes2:3:1 Pnlygalacturonase (PG)The activity of exo-PG in the healthy. infected and treated leaves ispresented in Table 14. All the treated healthy plants showed minimumactivity(04.01 SAU to 5.44 SAU).Highest activity of exo-PG (309.41 SAU) was observed in enzyme sourceobtained from the leaves of infected plants and those treated with Fe(11)(237.86SAU). Among the infected plants treated with siderophore and siderophore-Fe

Table 14. Effect of siderophore and siderophore-Fe complex of Pseudomonasfluorescens. B. amyloliquefaciens and Fe(ll) on the Exo activity of pectinolyticenzymes in leaves of H, annuus infected with A, helianthi (in vivo)CONTROL1L1 siderophOre-l Fc complex s,lni 0,018 1 06,86ioOOl ! S,l3k 0,065s~derophore-( 1 /Fe complex 15.lbM.021 13.44tO.040 17.31tU.014iderphor 4 . 00,093 03,92fl,OO? 4,0210,032iI

+ Control healthy+ Control infected. ..Healthy plank treated with siderophore of P fluorescens* lnfected plants treated with sidemphom-Fe complex of P.nuorescens+ Healthy plants treated with s~demphore-Fe complex of P.fluorescens+ lnfected plants treated with siderophore of P.nuorescens'+ Healthy plants treated with sidemphore of B.amy/dquefaciens-lnfected plants treated wRh sidemphore of B.amyloliquefeciens"- Healthy plants treated with sidewhore-Fe complex of B.amy/diquefecienslnfected plants treated with siderophore-fe complex of B.amyloliquefacmnsHealthy plants treated with Fe(l1)Infected plants treated wilh Fe(l1)

complex least enzyme activity was recorded in those sprayed with siderophoreof P. fluorescens (08.71 SAU) and B. amyloliquefaciens (12.58 SAU),siderophore-Fe complex of P. ,fluorescens (15.16 SAU) and B.am,vloliquefaciens (24.76 SAU).2.3.2. Polymethylgalacturonase (PMG)2.3.2.1. Endo-PMG activity:The activity of endo-PMG produced by A. helianthi in the leaves ofinfected and treated plants is prcsented in Fig.4. The highest activity of endo-PMG was observed in the enzyme source obtained from A. helianthi-infectedleaves at the end of 180 min (67.2% viscosity loss of the substrate). Controlhealthy leaves showed 3.01% viscosity loss of the substrate. All the treatments inthe healthy plants showed minimum reduction activity (3.35% to 7.78%). Amongthe treatments on the infected plants, the least loss of substrate viscosity wasrecorded in the enzyme source obtained from those treated with siderophore of P.lluorescens (6.39%). This was followed by that of B. amyloiiq~refaciens (7.1 I%),siderophore-Fe complex of P. ,fluoresccns (10.7%) and B, amyloliquefacicns(13.5%). Maximum viscosity loss in the substrate (45.84%) observed in theleaves of infected plant treated with Fe(l1).2.3.2.2. Exo-PMG activity:The amount of reducing sugars released by the activity of exo-PMGexpressed in SAU is presented in Tablel4. Healthy control showed minimumrelease of sugar (03.72 SAU). All the treatments in the healthy plants showed

Fig. 6. Effect of Sldemphom, SMarophore-Fe complex ofP. iluotw8cma and emvloIiau~ims and Fdllb , . on endo-PTE acttvlty of ~.h.llanth/(in-vhro)m+- Conhd Healthyt Control Infected. . - -- . . -- -Healthy plents treated with siderophon, of P. fluoresoens-*- Infected plants treeted with sidemphore of P, fluorescans+ Healthy plents bated with sidemphweFe complex of P. fluorems-c Infected plants hated with sMer0phom-h comp/ax of P. fluoI8scens-I- Healthy plents treatod with sidemphom d 8. amyloliqueMnsW- Infected p(snt8 heeted with sidemphom of 8. emylolquefeciens-Healthy plants trseted with s&mphorefe compkx of 8. emyldquefaciensInfected plents bated with sMer0phore-fe complex of P fluorescensHealthy plents treated with Fe(1I)Inhded plents bwted with fe(l1)

minimum release of sugar (03.92 SAU to 08.24 SAU). Highest rate of activity ofexo-PMG was observed in the enzyme source obtained from the leaves ofplants infected by A. helianthi (206.85 SAU). Among the leaves of treatedinfected plant least enzyme activity was recorded in those treated withsiderophore of P. ,fluoresccns(8.87 SAU), followed by that of B.amyloliquefaciens (I 1.16 SAU) and siderophore-Fe complex of P. ,ftuorescens(13.44 SAU) and B. am,vloliquefaciens (16.7 1 SAU) Fe(ll) showed higherectivity(82.47 SAU).2.3.3. Pectin Transelimincrse (PTE)2.3.3.1. Endn-PTE activityThe activity of cndo-PTE secreted by A. helianthi in the leaves of healthy,infected and treated plants is presented in Fig 5. All the treatments in the healthyplants showed minimum rate of activity (3.08% to 4.22 %) at the end of 180 min.Highest ratc of activity of endo-PTE was found in the enzyme source obtained fromthe infected leaves (43%), followed by that obtained from the infected leaves treatedwith Fe(ll) (31.8%). Among the treatments on the infected plant, the least activitywas recorded in the enzyme source obtained from siderophorc of P. ,fluorescens(5.84%), followed by that of B amyloliquejaciens (6.07%), siderophore-Fecomplex of P. Jluorescens (11.77%) and B. amyloliquefaciens (16.5%).2.3.3.2. Exo-PTE activityThe activity of exo-PTE in the leaves of healthy, infected and treated plantsis presented in Table 14. Control healthy leaves showed minimum activity of exo-PTE (4.18 SAU). All the treatments in the healthy plants showed minimum

fig. 6. Effect of slderophore, rlderophore-fe complex ofP. ~uonscons and B.8mylollqurfrcI~s and Fe(1l)onrndo acthrtty of Cx of A.hdianth/ (In vivo)Bar8 a52.0i/7'0 90-e Control healthy+- Contrd rnfected%nwi&yrn~Q'Heatthy plants treated with s ~etyhm of P.nuomscBnsx lnkted plants treated wp sidemphore of P Iu(~~scens+ Healthy plants treeted wth sidewhore-fe complex of P nuorescens-+ lnfeded plants treated with SiddmpWe complex of P. IUO~~SEB~S-+ Healthy plants treated with slderophom of B amyldiquefecienslnfeded plants treated with side- of B. amykdQuefeciens- Healthy plants treated with sirkqhomh complex of B amyld' uefaqnsInfected plank treated withe complex of B amyi$!i~efec~[)nstbahy ants treated with ~ v e~ d p l atreated n ~ with ~ehj -- .--lM

activity of exo-PTE(04.61 SAU to 10.54 SAU).The infected leaves recordedmaximum exo-PTE activity (96.4 SAU). Highest rate of activity of cxo-PTE wasobserved in the enzyme source obtained from the infected leaves treated withFe(11)(87.97 SAU). Among the treated infected plants least enzyme activity wasrecorded in siderophore of P. ,fluorcscens (11.63 SAU) and 5.am,vlo/iqucfacicns (15.88 SAU), siderophore-Fe complex of P. fluorcsccns(1 7.3 1 SAU)and 5. am.vloliquc/aciens (20.35 SAU).2.3.4. CelluloIytic enwrnes2.3.4.1. p1.4 endo-glucanase (Endo Cx):E&activity of Cx enzyme in the leaves of healthy, infected and treated plantsis presented in Tahle 15. Exo activity of C, is presented in Fig.6Control healthy plants showed 2.78% of endo glucanase activity. All thetreated healthy plants showed minimum reduction activity(3.34% to 5.67 %). Atend of 180 min. h~ghest rate of activity of endo Cx was found in the enzyme sourceobtained from the infected leaves (77.23%). followed by those treated with Fe(ll)(34.09%). Among those of treated infected plants, the least activity was found inthe enzyme source obtained from siderophnre of P. ,fluorescens (9.33%) and B.am,vloliquc/acbcrcns ( 1 1.72%). siderophore-Fe complex of P. ,fluorescens (1 6.18%)and B .amyloliquofacicns (22.35%).2.3.4.2 p1,4 endo-glucanase (Ero-Cx):The exo-Cx activity in the leaves of infected and treated plants is presented inTable 15. Leaves of control healthy plants showed minimum activity of exo-C,

Table 15, Effect of siderophore and siderophore-Fe complex of I'seudomonas,fluorescens and 5, amyloliquejacicns and Fe(1l) treatments on Exo activity of thecellulolytic enzymes in leaves of Helianthus annuus infected with A. helianthiCONTROLTREATMENTSIHealthyInfectedEXO-CX(SAU)EXO-CI(SAU)I s:2Slderophorc-Fe1 Fc(1,)$sk-----I $eB-2Ppd1IcomplexlnfectedSlderophoreS1derophore-FecomplexHealthvSlderophoreS1derophore-FecomplexlnfectedSiderophoreSiderophora-Fccomplex1 1 1Infected15.59M.2324.91M.0128.04M.0113.54M.2118.02M.0305.02kO.0105.95H.0112.73kO.0204.77k0.0205.34M.0327.85M.04 i12.39M.2130.59M.0214.25H.0120.14M.01 10.03H.01280.30M.03 18.31hO.13

(1 1.29 SAU) followed by those of treated healthy plants(] 1.72 SAU to 20.14SAU).Maximum activity of exo-C. was found in the infected leaves (397.38 SAU).Highest rate of activity of C, was observed in the enzyme source obtained fromthe infected leaves treated with Fe(11)(280.30 SAU). Among the infected plants,least enzyme activity was recorded in those treated with siderophore of P.,f7uoresccns (24.91 SAU). followed by that of B. amyloliqucfaciens (27.85SAU), siderophore-Fe complex of P. .fluorescens (28.04 SAU) and B.amvloliqucfacicns (30.59 SAU).2.3.5. PI,# exo-glucanase (CI):Activity of CI in the in the healthy, infected and treated leaves is presented inTable 15. Leaves of control healthy plants showed minimum CI activity (03.34SAU). All the treatments in the healthy plants showed minimum activity (04.56SAU to10.03 SAU).Maximum activity of C, (46.42 SAU) was observcd in theenzyme source obtained from the infected leaves followed by those treated withFe(11)(18.31SAU). Among the infected plants, least enzyme activity wasrecorded in the enzyme source obtained from the infected leaves treated withsiderophore of P. jluorescens (05.95 SAU), B, amyloliquefacicns (12.39 SAU).siderophore-Fe complex of P. ,/7uorcscens (12.73 SAU) and R.amyloliquefaciens (14.25 SAU).

Table 16. Effect of slderophore and slderophore-Fe complex of P fluorerrensB amylol~que/aclens and Fe(ll) on the Chlorophyll and Carotenold of H annuusleaves infected w~th Alternana helranlhi (rn v~vo)I Carotenoid 'chlorophyll content(mdg)I (mdn)i i hover !1 / HealthyControl1 I Infected 1 0 3801 0 0110 110i 0 021 0 630M 03 1 32 53 10 26M 0011 37 hX i~~dero~hore-PI1 Complex I 203t003 0 628t007 1 970500l I03 I4.-'I .1 4,Fe(Il)1II ( /I 1IS~derophore 0 66410 02 0 371t 0 04 I 12010 34 58 63 10 3610 01 1152 17S1dcrophore-FecomplexS~derophoreS1derophore-FecomplexHealthyInfected1 207.01 110+0 10 o 392to 05 1 $701 00104 0 592lO 01 I 91 I 1005 I00 05 0 66flOO2 95 650 72310 02 0 37810 02 I OROM 0420 739. 0 03 0 48410 05 1 2301 0611 392f 0 11 0 5Rli 0 05 1 906i 0090 620+ 0 06 0 458t 0 01 1 22 100382 5456 5464 5799 7963 870 5lM 0210 35M 0100 4610 03174 110 71M 029 102 890 46M 00550 7267 4066 34,hlorophylmg,gealthyontrol

2.3.5. Photosynthetic PigmentsThe photos~thetic piwents viz. chlorophyll a, chlorophyll b, totalchlorophyll and carotenoid extracted from the leaves of healthy, infected and treatedplants on the sampling day are presented in Table 16. Maximum amount ofchlorophyll a, chlorophyll b, total chlorophyll and carotenoid content was noticed inthe leaves of healthy control plants (1 322 mglg; 0.457 mug: I .910mglg: 0.69 mdg)and healthy plants sprayed with siderophore-Fe complex of 1'. ,fluorescenr(1.203mg/g; 0.628mglg; 1.97mug; 0.68 mgy). This was followed by siderophore-Fe complex of B.am.vlolique,faciens (1.207 mgg; 0.592 mdg; 1.91 1 mdg; 0.66mug) siderophore of P. .fluorescens (1.203 mug; 0.561 mug; 1.86mdg; 0.52mglg)and R. am,vloliqucfacrens (1.293 mgg; 0.437 mug; 1.83 mug; 0.65 mdg) andFe(ll) (1.392 mgg; 0.581 mdg; 1.906 mug; .71 mdg). Minimum amount of totalchlorophyll and carotenoid was recorded in the control plants . infected with.4. hclianthi (0.380 mdg: 0.110 mdg; 0.63; 0.26 mg/g). Among the infected plantstreatment with Siderophore-Fe complex of P. fluorescrns showed higher amount oftotal chlorophyll and carotenoid (1.1 10 mgig; 0.392 mdg: 1.571nug; 0.5Imdg).IThis was followed hy siderophore-Fe complex of B. am.vloliquefociens (0.739mug; 0.484 mdg; 1.23mUg; 0.46 mdg), Fe(11)(0.620 mug; 0.458 mgg.,1.22mglg;0.46mug), siderophore of P. ,flrrorcsccns (0.664 mug; 0.37 1mglg;1.12mgg; 0.36 mug) and B, am,vloliquefacicns (0.723 mglg; 0.378mg/g;1.08mglg;0.35 mug).

Table 17. Effect of siderophore and siderophore-Fe complex of P. ,fluorcscens,5. am,yloliquclfhciens and Fe(ll) on reducing sugar, non reducing sugar and totalsugar of Hclianthus annuus leaves infected with Altcrnaria helianrhi (in viva)1 1 InfectedP, flirorcscensREDUCING NON KEDUCINC;TO.I.AL SUGARSUGARSUGAKO'aTreatmentsh/oOverververmg/g healthymggHealthy 41.64i1.24 33.02i1.96 74.66il.05/20,48t093 49,I9 1 1 Fealthym0'4Lalthycontrolcontrol,control1l2,23d,92 17,051 32 19~1,36143.12 &&!&Sidcrophorc 40,1612.07' 96.44 32.68t2.02 98.97 72.8412.14 98.90,Siderophore-FeC.om lexwSiderophore41,5611.1423 51+1 22i -99.8056 46I32,9212.13 / 99.h920.7212.2769 7774.48M.9744.23i1.4499.7559.2427.7952 52 66.76 23.29k1.84 70 54 S1.08~.06 68.41~ ~ ~ ~ ~ h ~ ~&&yS~derophorc 39.81+2 52 95.60 / 30.0111.22 1 90.M 69.82i0.84 95.70Sidcrophore41.4Jt1.68(.omplexB, utn,v/oliqufoc~enslnfccredS~derophore 25 251l.hlE'e(1l)S~derophore.lexHealthylnfecled26.88i2.1240.20M 8620.30i1.3299.51hO.63M.5596,5448.7532.80i1.522 1 8911 .ISI23.04f1.9232.h211.X414.3hd.0499.3366.2962.7498 7843.4814.2413.5447.14k1.2549.92t1.6673.24a.8134.62tO.8799.43h3.1366.8698.0946.37

2.3.6. CarbohydratesThe amount of carbohydrates vlz reduclng sugar non-reduc~ng sugar, totalsugar, sucrose, starch and total carbohydrates In the leaves of healthy, treated andInfected plants IS presented In Tables 17 and 182 3 6 1 Reducrng sugarReduclng sugar In the leaves of healthy, infected and treated plants IS presentIn the table 17 H~ghest amount of reduc~ng sugar was observed In the leaves ofhealthy plants (41 64 mglg), followed by those of healthy plants sprayed w~thslderophorc-Fe complex ot P fluorrsccn~ (41 56mglg) and B amvlol~quefacrens(41 44mglg). slderophore of P fl~roresccns (40 16mgig) dnd H amvlolrqucfa~~ens(19 81 mglg) The least amount of reduclng sugar was notlced In the leaves ofInfected plants (20 4X mglg) Th~s was followed by thoqe of unfected plants sprayedw~th Fetll) (20 30 mdg) Among the treatments on the Infected plants, maxlmumamount of reduclng sugar was recorded In those sprayed with s~derophore-Fe complexof P flrrorc~ccns (27 79 mdg) and Ham~lolrqucfacrenr (26 88 mg/g) ands~derophore of R amvlolrqucfacren~ (25 25 mglg) and P fluorescens (23 51Wig)2.3.6.2. Non-reducing sugarNon reducing sugar In the leaves of healthy, Infected and treated plants 1spresent In the Table 17 Hlghest amount of non-reduclng sugar content was observedIn the leaves of healthy plants (33 02 mgig), followed by those of healthy plantssprayed w~th s~derophore-Fe complex of P flttorescens (32 92 mglg) and B

am.vloliqucfacicns (32.80 mgig) , Fe(ll) (32.62 mgg), siderophore of P.,fluoresccns (32.68 mg/g) and B. amyloliquefacicns (30.01 mdg). The least amountof reducing sugar was noticed in the leaves of infected plants (12.23 mdg), whichwas 62.95 % less than that of healthy plants. This was followed by those sprayedwith Fe(11)(14.36 mdg). Among the treatments on the infected plants, the maximumcontent of non-reducing sugar was recorded in those sprayed with siderophore-Fecomplex of P. ~Iuorcscens (23.29 mgg) and B. am.vlolique/acicns (23.04 mgig),followed by siderophore of B.am.vloliqucfaciens(2 1.89 mglg) and P. ,fluorescens(20.72 rngtg).2.3.6. 3. Total sugarTotal sugar in the leaves of healthy, infected and treated plants is present in theTable 17. Highest amount of total sugar was obscrved in the leaves of healthy plants(74.66 mdg), followed by those of healthy plants sprayed with siderophore-Fecomplcx of P. ,fluorc.c.cens(74.48 mgg) and 5. am,vloliquqfaciens (74.24 mglg).Fe(l1) (73.24 mglg), siderophorc of P. ,fluorcsccns (72.84 mgg) and B.am,vloliqurfucicns (69.82 mug). The least amount oftotal sugar was noticed inthe leaves of infected plants (32.19 mdg), which was 56.88 % less than that ofhealthy plants. This was followed by those sprayed with Fe(l1) (34.62 mgg). Amongthe treatments on the infected plants, the maximum amount uf total sugar was recordedin those sprayed with slderophore-Fe complex of P. .fluorescens (51.08 mug) and B.am.vloliqucfaciens (49.92 mglg), siderophore of B. am,vloliquefaciens(47.14mglg) and P. ,fluorescens(44.23 mgig).

Table 18. Effect of slderophore and s~derophore-Fe complex of P fluorcsccn~B amylolrquefacrcns and Fe(l1) on the carbohydrate contents of HeIranthus annuusleaves Infected wlth Alternoria hclianthr (rn vivo)-ControlTreatmentsSTARCHI "10I %Over Overmgig healthy mgig healthycontrol control1 Healthy 1 6 9 97 M9nn 93 I 92Infected 11 02i2 81 M 94 30 56t2 14HealthvSiderophore 16 R2*1 08 99 1745 63TOTALCARBOHYDRATI%1 overmd6 'health:contro5 3s 14 -43 34M 2346 832Fe complexFe(11)IHealthyInfected16 87k1 0712 68s 9099 46 66 85L? 2474 76 32 16*2 6399 8348 0289 21iO 1451 IOM 3396 4155 22

2.3.6.4. SucroseThe amount of sucrose content in the leaves of healthy, infected and treatedplants is presented in Table 18. Leaves of healthy plants showed highest sucrosecontent (16.96 mg/g) and among the treatments on the healthy plants, the maximumcontent of sucrose was recorded in those sprayed with siderophore-Fe co~nplex of P.fluorcsccns (16.93 mdg) and 6. am.vloliquqfaciens (16.88 mgig) and Fe(11)(16.87),followed by siderophore of P. ,fluoresccns (16.82 mglp) and 6.amvloliquqfacicns (16.72 mglg). Those of infected plants showed lowest sucrosecontent (1 1.02 mdg), which was 35.06 % less than that of the healthy ones. Thiswas followed by infected plants sprayed with Fe(11)(12.68 mg/g). Among thetreatments on the infected plants, the maximum sucrose content was recorded in thosesprayed with siderophore-Fe complex of P ,fllror~scens(13.93 mdg), followed bysiderophore-Fe complex of 6. amvloliqzrefaciens (1 3.18 mgig), siderophore of 6.omvloliqucfaciens (12.97 mglg) and P, fluoresccns (1 2.72 mglg).2.3.6.5. StarchThe starch content in the leaves of healthy, infected and treated plants ispresented in Table 18. Healthy plants showed 66.96 mdg of starch. Among thetreatments on the healthy plants. the maximum starch was recordod in those sprayedwith sidemphore-Fe complex uf P.,fluo,~scmm (67.01 mg'g) and R, amvloliquqfaciens(66.85 mglg). Fe(ll)(66.85). siderophore of P. ,fluorcscens (66.82 mgig) and 6.omyloliquefacicns (66.79 mg/g).The leaves of the infected plants showed loweststarch content (30.56 rngg), which was 54.35 % less than that of the healthy ones.This was followed by those of infected plants sprayed with Fe(11)(32.16 mglg).

Among the treated infected plants, the maximum starch was recorded in those s~ayedwith siderophore-Fe complex of I? ,fluorcscens (47.77 mg/g), followed by that of B.am.vloliqucfacicns(43.68 mglg), siderophore of 6'. amyloliqucfhciens(38.19mglg) and P. ,fluorcsccns (35.56 mglg).2.3.6.6. Total carbnhydrareThe Total carbohydrate in the leavcs of healthy, infected and treated plants ispresented in Table 18. Highest amount of carbohydrate was observed in the Icaves ofhealthy plants (41.64 mg/g), followed by those of healthy plants sprayed withsiderophore-Fe complex of H. amyloliqtcq/acirn.s (92.86 mug) and P. ,fluurescens(92.03 mglg). siderophore of B. am,vloliqncfaciens (92.49 mdg) and P.,fluorescens (91.42 mgg). The least amount of rcducing sugar was noticed in thelcaves of infected plants (43.34 mdg). This was followed by those of infected plantssprayed with Fe(l1) (51.10 mp'g). Among thc treatments on the infected plants,maximum carbohydrate was recorded in those sprayed with siderophorc-Fe complex off'.,flitoresrcn.s (78.04 mdg) and 8, am~~loliquefacirns (69.70 mg/g) andsiderophore of H. amvloliqu~focicn.s (63.38 n~gig) and P. ,fluorcsccns (54.95wig).2.3.7. Nitrogen metabolism2.3.7.1 ProteinThe protein content in the leaves of healthy, infected and treated planls ispresented in Table 19. Leaves of healthy plants showed 14.01 mg/g protein. Among thetreatments on the healthy plants, the maximum protein content was recorded in those

Table 19, Effect of siderophore and siderophore-Fe complex of P. ,fluorc,wensand B. amyloliquefaciens and Fe(I1) on protein content and amino acid content ofH. annuus leaves infected with Altcrnaria hclianthi (in vivo).IControl'tg,TreatmentsHealthyInfectedHealthvSiderophoreSiderophore-FeComplexInfectedSiderophoreSiderophore-Fecomplexm ,SiderophoreProtein%Overmglg healthycontrol14.01 +_ 2.14 1003.88a.072 27.7613.23 1t 0,08315.42tO.0507.12i0.0211 1 .I 910.0812.98t0.0741Aminoacid%Overmd6 healthycontrol34.741t1.05 10011.69*1.73 33.6794.43 33,8212,81 97.35I110.0650.8279.8792.6534.191t1.7727.16* .6729.89+1.3833.57t1.2498.4178.1886.0396.63e.B4,Siderophore-FeComplexInfectedSiderophorc13.96i0.0296.04i0.046I99.6343.1134.02H.9525.30+0.8497.9272.829$siSiderophore-Fecomplex10.73M.08276.5827.31*1.09i78.61Fe(ll)HealthyInfected13.39*0.0265.56M.08195.5739.6632.88tI .2616.40i0.9194.6547.20

sprayed with siderophore-Fe complex of P. .fluorescans (15.42 mglg) and B.amylo1iquefacicns (13.96 mglg), Fe(l1) (13.39 mglg) and siderophore of P.fluorescens (13.23 rnglg) and B. am.vloliqucfaciens (12.98 mgig). Maximumreduction was observed in those of infected plants (3.88 mg/g). Among the treatedinfected plants, the higher amount of protein content was recorded in those sprayed withsidemphore-Fe complex of P. ,fluoresccn~(1 1.19 mg/g) and B. umyloliquefaciens(10.73 mglg), followed by siderophore of P. /l~torescens(7.12 mgig) and B.amvloliquc/aciens (6.04 mglg) and Fe(l1) (5.56 mgig).2.3.7.2. Amino acidsThe amino acid content of the leaves of healthy, infected and treated plants ispresented in Table 19. Higher quantity of amino acid content was recorded in theleaves of healthy plants (34.74 mglg). Among the treatments on the healthy plants, themaximum amino acid content was recorded in those sprayed with sidcrophore-Fecomplex of P. ,fTuoresccns (34.19 mdg) and B. am.vloliquefaciens (34.02 mglg).This was followed by siderophore of P, fltroresccns (33.82 mgig) and B.urn~loliqurfacio~s (33.57 ~nglg) and Fe(ll) (32.88 mgig). The lowest amino acidcontent was recorded in the leaves of infected plants (1 1.7 mug), which was 66.33%lesser than that of the healthy plants. This was followed by those of infected plantstreated with Fe(ll) (16.40 mg/g). Among the Weated on the infected plants, themaximum protein content was recorded in those sprayed with sidemphore-Fc complexof P. fluorcscens (29.89 mdg), followed by that of B. am.vloliquefacians (3.3 1mgig), siderophore of P. ,fluorescans (27.16 mglg) and B, amyloliqucfaciens(25.30 mug)

Table 20. Effect of siderophore and slderophore-Fe com~lex of Pfluorescscens B arnvl~l~~ucfaa@ns and Fe(ll) on the aminonltrogei content ofHel~anrhus annuus leaves infected wlth Alternar~a hellanthi (In vlvo)Control2$LS%4TreatmentsHealthyInfectedLlealthvSlderophoreSlderophore-Fe complexInfectedS~derophore-Slderophore-Fe complexAminonltrogen27 0151 6810 2612 8527 04+1 3419 3451 5024 76+1 95(mdd%Overhealthycontrol100 003R 00100 l l71 6091 67EC12'ShBEs&&SlderophoreSlderophore-Fe complcx!&adS1derophoreSldcrophore-Fe complex26 96M 9829 31f2 0918 94i2 3023 l9il 6099 8 l10R 5170 1285 851Fe(ll)HealthyInfected27 07k1 8623 0312 21100 2285 26

Fig, 7. Eflert of ~Merophore, rMerophoreFe complex of P.flvomcens and B. umyloUquefackns md Fe(ll) on Nitratereductrue activity in leave# of H. annuus Infected withI CoMd healthyIContrdhWM y @ants bbrted wifh sidemphore of P, nuorescensinfected plants mted with sidemptnnw of P. nuorescensIhrthy plants t m M Mh siUewWe complex of P. nuoremsInfected planfa trrPeted with siderophona of P. rluonascensI Heathy plants hated with siderophore of 6. amyldiquefaciensOlnfected plants treated Mth sMerophore of 8. amylo!iq~~LuensI Hedlhy plants treated with 8Me-ecomplex d 8, a myldique~sI lnlbcted plant8 tmated wilh ddemphore+e complex dB. amyldlqueleciensOHedlhy @ant$ hated &h Fe(H)I Inlbdsd plant$ hated with Fe(ll)

2.3.7.3. Amino nitrogenThe amino nitrogen content in the leaves of healthy, infected and treated plantsis presented in Table 20. The highest amino nitrogen content was recorded in theleaves of healthy plants (27.01 mg/g).Among the treatments on the healthy plants,sidcrophore-Fe complex of P..fluorescenr exhibited 29.50 mgg followed by that of B.am,vloliquqfaciens (29.31mgig), Fe(11)(27.07 mglg). siderophore of P.,fIuorcsccns (27.04 mdg) and 8. am.vlolique/aciens (26.96 mglg).The lowest amount of amino nitrogen was observed in the leaves of theinfected plants (10.26 mgg), which was 62% less than that of the healthy plants.Among the treatments on the infected plants, least inhibition in amino nitrogen wasrecorded in those sprayed with siderophore-Fe complex of P. ,fluorescen~(24.76mug). followed hy that of B. amy1oliqrrcfacien.v (23.19mgig). Fe(11)(23.03mgig),siderophore of P. ,fluorcsccns (19.34 mglg) and B. am.vloliquefacicns (18.94mg/g).2.3.7.4. Nitrate reductase (NR)The NRA in the leaves of healthy. infected and treated plants is presented in Fig.7. The activity of nitrate reductase (NR) enzyme was found to be hi&er in the healthyplants (1.48 nmollg). Among the treatments on the healthy plants, sidemphore-Fecomplex of P. ,fluoresccnv showed 1.65 nmollg, followed by that of B.amyloliqu~facicns (1.64 nmolig), Fe(11)(1.54 nmolig), siderophore of P.,/luorcscens (I .47 nmollg) and B. um,vloliquqfacicns (1.47 nmolig).

Table 21. Effect of siderophore and siderophore-Fe complex of P. ,fluorcscens,8. amyloliquefaciens and Fe(1l) on the total phenol, 0 D Phenol and prolinecontent of Helianthus annuus leaves infected with Allernaria helianthi(in viva)Control2f~iE392'5PTreatmentsHealthyInfected!jg&ySiderophoreSiderophore-FecomplexLnfectedSiderophoreSiderophore-Fecomplex&&SiderophoreSiderophore-FecomplexlnfectedSiderophorcTOTALPHENOLmg/g13.78i0.6155.07i9.25I4.03f0.3113.86M.2629 0 2 416.38k0.1 114.26k0.5613.92M.1433.26i1.12ODPHENOLmdg, 8.08i0.1928.55M.149.12fl.08 18.61M.11I8.02*0,0713.12M.129.23i0.31X.72f0.16PROLINEmgis1.01iO.003.64i0.02I .OlM.Ol1.03M.01I 210.06 11.04Ri0.011.04fl.011.032M.01-1 ,18.1 1f0.10 1.059i0.02Siderophore-FeHealthylnfected

The least activity was recorded in the leaves of infected plants (0.873 nm~l/~),showing a reduction of 59% compared to that of healthy plants. Among the treatedinfected plants, maximum activity was recorded in the leaves of plants sprayed withsiderophore-Fe complex of P. ,fltroresccns (1.29 nmollg), followed by that of B.amyloliquefacicns(l.24 nmollg), Fe(11)( 1.18 nmollg), siderophore of P.,fluorescens ( 1.16 nmoWg) and B. am,vloliquefaciens( I. 15 nmolig).2.3.8. Phenol contentsThe total phenol and O.D. phenol contents in the leaves of healthy, infectedand treated plants are presented in Table 21. The leaves of healthy plants exhibitedminimum total phenol (13.78 mgg) and O.D. phenol (8.08 mglg). Among thetreatments on the healthy plants, the lowest content was recorded in those sprayedwith siderophore-Fe complex of P. ./l~roresccns (13.86mgg; 8.61mgg) and R.om.vlolique/aciens (13.92 mgig: 8.72 mglg), siderophore of P. ,fltrorcscens(1 4.03 mglg; 9.12 mglg) and R, amyloliqtrc/aciens (1 4.26 mg/g;.9.23mg/g) andFe(ll)( 14.29mglg;9.1 5mgIg).The leaves of infected plants exhibited higher amount of total phenol (55.07mug) and 0.D. phenol (28.55mdg). which was 4 times that of the healthy plants(13.78 mdg; 8.08 mgg). All the treatments both on the healthy and infected plantsshowed generally higher total phenol and O.D. phenol content than the controlhealthy plants. Highest total phenol and O.D. phenol were noticed in the leaves ofinfected plants sprayed with Fe(ll) (47.83 mdg; 23.84 mdg), followed bysiderophore of B. amyloliqucfacicns (33.26 mglg; 18.1 I mglg) and P. ,fluorescens

aFig. 8. E W of rld~rophom and rid.rophore-Fs complex ofP. nuonacms and 8, rmylollquehclms and FHII) on SODactlvlty In haof H. annuus Infected wlth A.hrlirndhiI Control healthyI C4nW InfectedHeeiYhy plants treeted with sidewhore of P, fluoresoensI n W plants mated wilh sidemphore P. nuomsoensI HeeIthy plant bwted with sidemphors-fe complex of P. numsc8nsInfected planb hated wrth sdmpkmFe comp4ex of P. numsc8nsI Healthy plants treated with siderophore of 0. emylolquefaciensE Infscted planb halsd with sidemphm of 6. amyldquefscisnsHealthy plants hated with siderqphmfe complex of 6. amylolquefeciensI Infected plants bated with Sidemphore-Fe comlpex of 6, amyld~uefeciensHeelthy plants treated with Fe(l1)Infected plants treeted with Fe(Il) -. ------ -- -- -

(29.17mglg; 18.02mgig), siderophore-Fe complex of B. amyloliquefaciens (2 1.20mgig; 13.6 1 mgig) and P.fluorescens (I 6.38 mgig;l3.12mglg).2.33 PralineThe proline content in the leaves of healthy, infected and treated plants ispresented in Table 21. The lcavcs of control healthy plants exhibited l.Olrng/g ofproline content. Leaves of healthy plants sprayed with siderophore-Fe complex ofP. ,/luorssccns, siderophore-Fe complcx of B. amvloliquefaciens, siderophore off'. .fluorescens, siderophore of H. am.vloliqitefaciens and Fe(l1) showed 1.03 mdg,1.032mdg, 1.01 mg/g ,I .04 mdg and I .08 mg/g respectively.The proline content showed a sharp increase of about 3 times (3.64 mgtg) inthe lcavcs of infected plants compared to that of healthy plants. Among the treatedinfected plants, those sprayed with siderophore-Fe complex of P. .fluorescensshowed minimum proline content (1.048 mdg) which was only 3.77% higher thanthe healthy one. This was followed by those sprayed with siderophore of B.am~loliqir~~aciens (1.059 mug) (4.89 % higher than the control healthy plants)and P ,fluorescens (I. 12 mug). siderophorc-Fc complex of B, am.vloliqrr~furicns(1 .I4 mg/g). Fe(ll) treatment showed 3.05 mdg of proline which was 83.73% thatof control infected one.2.3.10. Oxidative enwmes2.3.10. 1. Superoxide dismutase(S0D)The activity of SOD in the leaves of healthy, infected and treated plants ispresented in Fig.8. The leaves of healthy plants exhibited 14.1 1 U of superoxide

3aFig. S. Elhct of slderophore and siderophon-fe complex ofP. tluomsnns and B. rmy/ollqmhcIws and Fqll) oncablase actlvlty In leaves of H. annuus infected wlthA. hellanthlContEfhealthyConhdunwedO Heelthy plants trseted WIUI auierophorr, ot P nuQR)sa,nsInteded plants hated with srderqdron, of P iluorescensHealthy plants trseted mM Ydemphm-Fe complex of P, iluOTBsa,nS8mfected dents bated wiM sMeropbcfe&9 Ewnplex of P iluQR)sa,ns"'8 Heellhy Ants haled with siderqphore of B amyldiquefauens8 Infected plants bwfed with auierophore of 8. amyldquefauens8 Heelthy plants hated with sidsrophoncFe mpx d B amyldquefa~nsInfected plants hated with auiewhwafe complex of 8. amyldquefeciensHealthy plants b ww with FYI)8 InbcW plants treated _with Fe(l1) --- -

dismutase. Among the treatments on the healthy plants, the leaves of those sprayedwith Fe(ll) showed higher activity (20.00 U), followed by those sprayed withsiderophore-Fe complex of P .fluorescens(15.89) and B, amyloliquefuciens (15.23),siderophore of P. .fluuresccns(14.64 U) and B. am.vlolique/aciens(14.43 U).The highest SOD activity was recorded in the leaves of infected plants(40.02 U), which was 2.83 times higher than that of healthy plants. This wasfollowed by Fe(11)(34.71 U). Among the treated infectcd plants, the leaves ofthose sprayed with siderophore-Fe complex of P. ,fIuorescens showed higheractivity of SOD (19.62 U). followed by siderophore-Fc complex of R.amvloliqire/acicns (19.05), siderophore of P. fluorcscrns (17.89 U) and B.um,vloliquqfuciens (I 6.49 U).2.3.10. 2. Caralase(CA 7)The catalase activity(CAT) in the leaves of healthy, infected and treated plants1s presented in Fie.9. The leaves of healthy plants exhibited 8.3 U of catalaseactivity. Among the treatments on healthy plants, the leavcs of those sprayed withFe(ll)showed higher activity (13.92 U), followed by those sprayed withsiderophore-Fe complex of 1'. j1uorescr.n~ (8.41) and B. umyloliqtiefacicns (8.35),siderophore of 1'. Jluoresccns (8.22 U) and siderophore of B, am.vlolique/acicns(8.15 U).Highest catalase activity was observed in the leaves of infectcd plants (25 U),which was 3 times higher than that of healthy plants This was followed by theinfected plants treated with Fe(ll)(21.2 U). Among the treated infected plants. theleaves of those sprayed with siderophore-Fe complex of P. .fluoresccns showed

Fb. 10. Effect of sld.rophom, sidorophore-fe complex ofP. ilmmcons and 6. amylollqmhei.ians and FNII) onporoxldne rcthrlty of luvea of H. annuus Infected withConbd healthyConWinkWHealthy plant hated with asiderophore of P, fluoremnsInfected plants treated with &smphom of P. nmscens8 Healthy plants trssted with sidemphom-Fe complex of P. fluoresoens8 InbcM plants hated with side-Fe compbx of P fluomsoensHeathy plants trssted wlih suierophore of 8, amyWiquefach9nsII Infected plants biMW with sidemphom of 8. amyloliquefacmns8 Heathy plants bated with sidemphon+Fe complex of 6. emyldiqoefeciensInfected plents hated with siderophotu-Fe complex of 8. amyloliquefaciensHeellhy plents hated with Fe(ll)Infected plants bated with Fe(ll)-- - -- --

$40Flgure 11. E M of rlderophore and slderophore-fecomplex of P. fluomcens and 6. amylollquefaciensand Fe(ll) on activlly of Polyphenoloxidase of leavesof H. annuw Infected wlth A. hdlandhlConbd heallilyI cbntrd infectedHealthy plants treated with siderophom of P, fluoroscsnsInfecfed plants hated with siderophom of P. fluomsc~nsW Healthy plants mHlted with sldemphomFe wm@x of P. fluomscansI Infected plants trwted with siderophoefe complex of P. nuorescsnsHealthy plants hated WM srdemphore of 6. amylcficluefaciensq Infecfed plants heated with siderophars of 6. amylo/iquefaciensI Healthy plants heated mth slderoph016Fe complex of 6, amyldiquefeciensW Infected plants trwted with 6. amylcfquefaciensM y plants trwted wilfrfe(ll)I lnfeded plants hated with Fe(ll)

higher activity of CAT (15.96 U), followed by that of B. amyloliquclfaciens(15.38 U), siderophore of P. ,f7uorescens (14.29 U) and B. amyloliqucfaciens(14.02 U).2.3.10.3. PeroxiduseThe peroxidase activity in the leaves of healthy, infected and treated plants ispresented in Fig 10. The leaves of healthy plants showed lowest peroxidase activity(27.56 SAU).Among the healthy plants, those sprayed with siderophore-Fe complcxof I? .flttorcsccn~ showed least value (27.71 SAU), followed by that of 8.amvloliqurfacicns (28.08 SAU), siderophore of P. ,/lirorcsccns (28.72 SAU) and8, amvloliqu~faciens (28.79 SAU) and Fe(11)(29.15).The leaves of infccted plants showed highest peroxidase activity (96.49 SAU)which is 3.5 times higher than that of the healthy plants. This was followed byinfccted plants sprayed with Fe(11)(32.16 mdg). Among the treated infected plants,the maximun activity was recorded in the plants spraycd with siderophore-Fecomplex of P. ,fluorestmr(3 I .76), followed by that of B. am.~~loliq~rc~acicns (3 I .28SAU), siderophore of P. ,fluor-sscens (3 1.22 SAU) and B, umyloliquefac~cns(30.73 SAU).2.3.10.4. Polyphenol oxidase (PPO)The activity of plyphenol oxidase(PP0) in the leaves of healthy, infected andtreated plants is presented in Fig I I. All the treatments both on the healthy andinfected plants showed generally higher activity of PPO than the healthy controlplants. The leaves of healthy plants showed lowest plyphenol oxidase activity

'Table 22. Effect of siderophore and siderophore-Fe complex of P. ,fuoresccns,B. amyloliquefaciens and Fe(l1) on carbohydrate and protein content of seeds ofHelianrhus annuus infected with Alternaria helianthi(in vivo)-controlTreatmentsInfectedHealthySiderophoreI1Carbohydrate I Proteinmgrg146.00i7.3071.6515.8140.67i5.32I % I 1 %Overhealthycontrol-49.0796.6 3mgg170.21+1.0194.78r1.04167.94t1.01Overhealthycontrol-55.6898.49edCSiderophore-FecomplexInfectedSiderophareE~~~~orehore.Fc&&!y140.18+3.05~124.00t4.99 84.93137,57r3,l7 , 94.2296.01 1 l75.82M.091 Siderophore ! 138.04i2.68 1 94.54 1 l67.76t1.09I139.34i0.12IS2,08tlO,5103.2981.8689.3498.56 Ig,@Fe(ll)complex--Siderophore 1 1 7.83i2.31 80 70 138.94t0.151 81.62Siderophore-FecomplexHealthyInfected,33,78k3,01145,9311.94154.77f3.3391.6399.95106.00149.19+2.00172.68il.50194.22i1.22 114.10I87.65101.45

(19.44 SAU). Among the treated healthy plants, the lowest activity was recorded inthe plants sprayed with sidemphore-Fe complex P ,fluorescem (19.61), followed bythat of 5. amyloliquejuciens (19.90 SAU), siderophore of P, jluorescens (20.30SAU) and B. amyloliguclfaciens (20.73 SAU) and Fe(11)(22.21 SAU).The infected leaves showed maximum activity (I I7 SAU) compared to that ofthe healthy plants. Among the treatments, highest activity was noticed in theinfected plants sprayed with Fe (11) (98.3 SAU). Lowest activity was noticed in theinfected plants sprayed with siderophore of 5. am.vloliqucfacicns (31.37 SAU)and P. ,fIitoresccns (30.18 SAU), sidcrophore-Fe complex of B, am.vlolique/bciens(26.67 SAU) and P. fluorcscens (25.39)2.4. SEEDSCarbohydrate, protein, and saturated fatty acid and unsaturated fatty acid of theseeds collected from healthy, infected and treated plants are presented in the tables22 and 23.2.4. I Carbohydrates and ProteinThe highest amount of carbohydrate (146mgfg) and protein (170.21 mdg) wasrecorded in the healthy plants (Table 22). The treatments on the healthy plantsshowed higher content in siderophore-Fe complex of P. ,fluoresccns (cnrbohydrate-140.18 mgg; protein- 175.82mgig). followed by Fe(ll)(carbohydrate- 145.93mglg;protein-1 72.68 mglg); siderophore-Fe complex of B, amyloliqrrefaciens(carbohydrate-143.36mg/g; protein- 169.29 mglg). siderophore of P. ,fluorescens(carbohydrate- 140.67mglg; protein- 167.94 mglg) and B. dmyloliqtrqfaciens(carbohydrate-1 38.04mglg; protein- 1 67.76 mglg).

Table 23. Effect of siderophore and siderophore-Fe complex of P. ,fluorrsccns,B. amyloliquefhciens and Fe(l1) on the saturated fatty acid, unsaturated fatty acidof seeds of Hclianthus annuus infected with Allernaria hclianthi (in \'ivo)SATURATED UNSATURATEDFATTY ACID FATTY ACIDTreatmentsbh%Overealthyms/s ye:yihy mdK fontrolcontrolllealthy 58.93k4.15 100 95.55i4.23 100ControlInfected 34.74k2.23158.9552,19?2.45 54.62-w 'Siderophore !58.0414,20i 98.48IsiderOphOx- 18,5014.03 9P27 9719il69Fe comdex -82Siderophnrc 40,07+3,M 67,99 59.34i4.7l.v'.gsiderOphore- 50.6it4.28 85.95Fe complex!j&jySiderophore 57.0613.371 98.35Siderophorc- 58.99t3.66, 100.10Fe complex!&%IdSiderophore 39.48t5.24 66.99I92.9713.79197.29101.82--I92.26+2.3495.31k2.5858.94k1.9296.5599.7561.68Siderophore- 49.19i2.08Fe complexpealthy 58,43i4ilInfected 52,8h24983.4789~6867.23i2.8670.36100,32fl.15 104,99 (I

The lowest amount of carbohydrate (71.65mg/g) and protein (94.78mgIg) wasobserved in the seeds of infected plants, which was 50.92% and 44.3 I% less thanthat of the healthy plants respectively. Among the treated infected plants, maximumcarbohydrate and protein content of seeds was recorded in those sprayed with Fe(ll)(carbohydrate-154.77mg/g; protein- 194.22mgg). This was followed hy siderophore-Fe complex of P. .fluorcscens (carbohydrate-] 37.57mgg; protein- 152.08mgg) andB, amyloliquefaciens (carbohydrate-1 3 3.78mgig; protein- 149.1 9mgIg).siderophore of P. fluorescens (carbohydrate-l24mglg; protein- 139.34 mglg) andR, amvloliquefacicns (carbohydrate-! 17.83mglg; protein- 138.94 mglg).2.4.2 Fat@ acidsThe mono unsaturated fatty acid and saturated fatty acid contents of seeds ofhealthy, infected and treated plants is presented in Table 23. The highest monounsaturated fatty acid and saturated fatty acid were recorded in the seeds of controlhealthy plants (95.55 mug, 58.93rnug). Among the treatments on the healthyplants highest fatty acid was recorded in siderophore-Fe complex 1'. ,j7uorescem(97.29mg/& 58.50 mdg), This was followed by Fe(11)(96.87mglg.58.43mgig),siderophore-Fe complex of B, amvloliqlrqfaciens (95.3 1 mglg. 58.99mg/g),siderophore of P. ,jluorcscens (92.97mdg. 58.04 mglg) and R.amyloliquqfacicns (92.26mglg.57.96 mgig).The lowest amount of unsaturated fatty acid and saturated fattyacid was observed in the seeds of infected plants(52.19 mgg; 34.74mgg). Amongthe treated infected plants, maximum mono unsaturated fatty acid and saturated fattyacid were recorded in the plants sprayed with Fe(11)(100.32mgg; 52.85mgg). This

Table 24. Effect of siderophore and siderophore-Fe complex of P. ,fluorescens,B. amyloliquefaciens and Fe(l1) on morphological characters of Helianthusannuus infected with Alternaria helianthi (in vivo)TreatmentsPlantheight(cm)Total leafarea(cm2)InflorescericesizecHealthy 112t0.91 498.lt0.10 9.4i0.004Infected 94S.46 438,2820.05 8.7H.002&&ySiderophore 112+0.39 490.4710.12 9.5*0.001.D~sk size(cm)4.9t0.0054.4i0.0164.8M.018Dty weight(g) ,32.39?r0.05315.77*0.09235.00i0.015 il3r0.l I 4955O;OlY ( 9.4iO.Ol4" Infected! 2 Siderophore 98i0.03 448.56k0.28 9.1iO.009Q*Fe(ll)Siderl'phore1 101027 460,29@.06 9.2H.051 4,7d,062 / 27,5633.064-Fe complexHealthy I 12fl.22495.50i0.039.4H.071 s.om.013 132.44M.075lnfected 99kO.31 456,1933.35 9.2i0.003 4.693.024 i 25.81+_0.010

was followed by siderophore-Fe complex of P. ,fluorescem (79.21 mug; 50.6Smdg)and B. amyloliquefaciens (67.23mglg; 49.19mg/g), siderophore of P. ,fluorescens(59.34mglg; 40.07 mglg) and B. amyloliquefaciens (58.94; 39.48 mglg).2.5. MORPHOLOGICAL CHARACTERSMorphological characters which include plant height, total leaf area, head(inflorescence) size, disk size, plant dry weight are presented in Table 24. Thehighest values were recorded in the control healthy plants (plant height-l 12 cm; totalleaf area-498.1 cm'; head (inflorescence) size-9.4 cm; disk sizc- 4.9cm; plant dryweight-32.39 g). Treated healthy plants showed more or less same values as that ofcontrol healthy ones. Plants sprayed with siderophore-Fe complex P. ,fluoresc.errs(plant height-l 17cm; total leaf area- 497.23 cm2: head (inflorescence) size-9.4: disksize-5; plant dry weight-35g) and B. am,vloliqucfacicns ( plant height- 113 cm; totalleaf arca- 495.50 cm'; head size- 9.4 cm; disk size- 5.0 cm; plant dry weight- 32.0g )showed similar values. This was followed by Fe(ll) treatment (plant height-l 12 cm;total leaf area- 495.50 cm'; head size- 9.4cm; d~sk size- 5cm; plant dry weight-32.44g). siderophore of P. fluorescc~ns (plant height- 112 cm; total leaf area-490.47 cm'; head size-9.5cm; disk size-4.8cm; plant dry weight- 35.00g) and B.o~vloliquqfacicns (plant height-110; total leaf area- 488.19 cmZ; head size- 9.4;disk size- 4.8 cm; plant dry weight- 32g).The lowest values were recorded in the infected control plants (plant height-94cm; total leaf area- 438.28 cm2; head size- 8.7 cm; disk size- 4.4 cm; plant dryweight- 15,776). This was followed by those treated with siderophore of B.am,y/o/iqucfaciens (plant height- 98cm; total leaf area- 448.56 cm2; head size- 9.1

Table 25. Effect of siderophore and siderophore-Fe complex of P. ,fluoresccns,B. ornyloliqueJaciens and Fe(l1) on seeds weight and oil content of Helianthusannuus infected with Altcrnaria hclianthi (in vivo)Control-TreatmentsHealthyinfecledHcalthvSiderophore 1SEED WEIGHT / Off. CONTENT% IOverhealthy giK6WeighV 100seeds4.43r0.011.75i0,l l4.194tO.04control-395094.67245.1 1r7.l 16l47t4.83237.44r6.29%Overhealthycontrol10025.08 I96.87 )lnfectedSiderophore-i w IHcalrhv 'loRrOo7 1 92'73Sidcmphore234.7112.9791.76jc_Siderophore-272fl,01 96.43 240.06t4.33 97.93Siderophorc 2.61 7iO.20 1 59.07 183.56*6,54 X8.i$/ Siderophore-1 Fe cotnplexI Healthy1 FC(Ib I4.342r0.12 / 98.01 240.00Y2.65 97.95Infected 3,207iO.03 72.41 1 88.90i3.20/ 36.27 I

cm; disk size- 4.6crn; plant dry weight- 23.6283 and P. ,/luorcscens (plant height-99crn; total leaf area- 452.89 cm2; head size- 9.1; disk size- 4.7cm; plant dry weight-23.99g), Fdll) treated plants (99cm-plant height; total leaf area- 456.19 cm2; headsize- 9.2 cm; disk size- 4.6 cm; plant dry weight- 25.81g), siderophore-Fe complexof B. amyloliquefaciens (plant height- 1 IOcm; total leaf area-460.29 cm2; head size-9.2 cm; disk sizc- 4.8 crn; plant dry weight- 27.56 g) and P, ,fluorescem- sprayedplants ( plant height- 110; total leaf area- 467.36 cm2; head size- 9.3 cm; disk size-4.7 crn; Plant dry weight- 29 g).2.6. YIELDThe yield of the healthy. infected and treated plants which includes weight per100 seeds and % of oil content are presented in Table 25. The highest yicld wasrccordcdin the healthy plants (seed weight -4.43&'100 seeds; oil contcnt245.1 Igkg). Among the treated healthy plants, the siderophore-Fe complex of P.,fluuresct~~ showed almost similar weight of the seeds (4.533@,100 seeds) and oilcontent was 240.35 gkg (98.06%). This was followed by siderophore-Fe complex ofR, amvloliq~~cfocic~ns (seed weight 4.272&'100 seeds: percentage of oil content240.06g!kg (97.93%). Fe(ll) (seed wcight 4.342g/lOO sccds; percentage of oilcontent was 240.00 glkg (97.91%). siderophore of P. ,fluorc~sccns. (seed wei&t -4.194g/100 seeds: percentage of oil contcnt 237.44 g!kg (96.87 %), siderophoreof B. am,vloliquefacicns.(seed weight 4.108g/100 seeds: oil content 234.71 glkg(95.76%).The lowest yield was recorded in the infected plants showing an yield loss oiabout 74.92% (seed weight -1,75g/100 seeds (39.50%): percentage of oil content

61.47gkg (25.08%). This was followed by the infected plants treated with Fe(ll).(seed weight -3.207glIOOseeds (72.41%); oil content 88.90glkg (36.27%). Amongthe treated infected plants. the highest yield was noticed in the siderophore-Fecomplex of P. ,fluorescenr- sprayed plants(seed weight -2.975g1100 seeds, oil content213.73 gkg), followed by siderophore of B. amyloliquqfaciens.(seed weight -2.61 7gl100. oil content- 183.56 glkg), siderophore-Fe complex of B.am,vloliquqfacirns.(sced weight- 2.540gil00 seeds: oil content-179.27 glkg) andsideraphore of P. ,fluoresc.ens(seed weight -2.149d100 seeds; oil content- 169.63$'kg).

The present study brings out the effects of siderophores of four soil bacteria viz.B. subrilis, B. cereus, P. ,fluorescens and B. am.vloliquefaciens and the siderophore-Fe complex of the latter two on Alternaria helianlhi, the infectant of foliar blight ofHelianthus annuus under healthy, infected and treated conditions.IN VITRO STUDIES.Screening of culture fitrates of Bacterias+a r dDrlDScreening of crude culture fitrates of soil bacterianfor their effects on conidialgermination, mycelial gowth and biomass ofA11emaria hclianthi.. There was no significant record of inhibition on the wnidial germination,radial mycelial growth and biomass of Allernaria hclianthi when Fe-amended crudebacterial culture filtrates were subjected to screening tests. This supports the findingof Bakker er a1.(1986) that microorganisms did not produce siderophore when therewas Fe in the medium.Crude extracts of the three bacteria viz. B. cereus, P. Juoresccns and B.am,vloliquclfacienr grown in the media under iron-starved condition exhibited higherrate of inhibition on the wnidial germination, radial mycelial growth and biomass ofAlternaria hclianrhi. This might be due to the fact that siderophore is produced bythe bacteria as a special device for absorbing Fe from the environment. (Powell eta/., 1982) Further, this finding proved that siderophore has strong antifungal orantifungistatic activity.

Crude extract of B subtrlrs w~th or wlth out Fe showed mmlmum percent rnhlb~t~onon wnld~al gcrmlnatlon, rad~al mycellal growth and b~omass, hence ~t was not usedfor further stud~esDetermination of OIC of siderophore and siderophoreFe complexIn the present m vrrro study, the results obtalned on con~dlal geimlnatlon,mycel~al growth and b~omass of A helranrh~ showed that out of SIX(I,S. 10,S0.100.S00 ppm) concentrations of tested s~derophore, 10 ppm concentratlonof P fluorercens, SO ppm concentratlon of s~derophore of B cereus and Bomvlohqucfocrens and slderophore-Fe complex (10 1ratlo) of above bactenaproduced cent percent lnh~bltlon The lnhlblt~on of con~dlal germination, mycel~algrowth and hlomass of A hclranthr by s~derophores of the above bactena mlght beattnbuted to thelr funglc~dal or fung~stat~c actlon The suppresston of pathogengrowth by antagonlstlc m~crober was malnly attnbuted to the activlty of secondarymetabolites present In the culture filtrate or hy the hyperparastt~sm The synergstlcactlon ot both b~ocontrol factor and numtron competltlon may also lead to effectrve~nh~b~t~on of the growth of the pathogen Lemanceau er a1 (1992) found that thegrowth ot Fusarrum sp, causrng wilt of camatton was lnh~blted by the actlon of theslderophore, pseudobactln present In the Pseudomonas culture filtrateThe s~derophore produced by the so11 bactena might lnhlb~t the conld~algeimlnatlon, leadlng to the d~slntegratron of the vlable fungal spores D~leep Kumarer al(2001) proved the ant~fungal actlvlty of s~derophore extracted from thePseudomonas straln agalnst Rhrzopus orvzae, Fusarrum oxvsporrum andAphanomvces eurerches In rn wrro cond~t~on Slderophore-Fe complex of the three

species of bacteria viz. B. cereus, P.,Juorescens and B. amyloliquefaciens was foundto show lesser inhibiting effects than those of siderophores alone. Similarlypseudobactin, the pyoverdin produced by Pseudomonas strain, inhibited the growthof the phytopathogens Fusarium osysporum and Gaeumannomyces graminis var.lrilici (Kloepper el al. 1980) while ferric pseudobactin did not. Misaghi el a1.(1982)demonstrated that pyoverdin produced by Pseudomonas Juorescens wasantagonistic against Gaeumannom.yces candidum and he reported that the generalfungistasis associated with the pyoverdines was regulated by iron. Similarly. apyoverdin produced by P. Iolaasii decreased mycelial growth of Pvthium ulrimum,while ferric-pyoverdine did not (Meyer er a1..1978).These studies suggest thatsiderophore is antagonistic through chelation of iron from the environment (mediumor soil) of the target pathogen. Free ligand siderophore competitor for the iron waspresent in the medium, while siderophore-Fe complex did not compete for iron andhence showed lesser inhibitory effects.Phytotoxic studies of siderophore, siderophore-Fe complex and Fe(l1)Seeds of infected plants showed minimum rate (3.44%) of germination. Out ofs~derophore of three soil bacteria, that of B, cereus showed minimum rate (18-25%)of germination of seeds of both treated healthy and treated infected plants. Thismight be due to phytotoxic effect of B.cercus siderophore on the seeds of H, annuus.Similar report on the phytotoxic effects of siderophore-agrobactin fromAgrohclerium rum~facirns was made earlier (Leong and Neilands, 1981).Pvricularia oryzae, produced a metal-binding compound, both free ligand and metalcomplex which was found to be toxic towards plants, animals, and bacteria (Lebrun

et a1.,1985). On the contrary, siderophore and siderophore-Fe complex of P.fluorescens and B. am,yloliquefaciens showed highest rate of seed germination(76%-94%) in both control and treated healthy and treated infected plants, thus revealingthat siderophore of the two species are not phytotoxic to H. annuw.IN VI VO STUDIESThe non phytotoxic siderophore of P. ,fluorescens and B. amyloliquefbciensand their siderophore-Fe complex were sprayed on the leaves of H. annuus in thepotted plants. The effects of the above spray on the leaves of host plants underhealthy, infected and treated conditions were found out by studying differentphysiological and biochemical parameters from the leaf samples collected on 54IhDAS.DISEASE INTENSITYIn the present study, the highest disease intensitfivery severe type ofinfection) was recorded in the plants sprayed with only A. helianthi (87.77%),Thetoxin produced by A. helianlhi such as alternin or the action of enzymesproduced during the pathogenesis, led to the formation of the symptoms anddisease development in the host plant. The present finding is in agreement withthe finding of Chattopadhyay and Appaji(1999) who concluded that the toxinproduced in A, helianthi was the major factor for the pathogenesis in Hetianthusannuus. In the present study. P. ,flrrorescens siderophore-Fe complex-sprayed plantsrecorded lowest percentage (9.6%) of symptom development (light infection). Plantsprayed with siderophore-Fe complex of B. am,vloliqueficicns, siderophore of P.

,fluorescems and B. amyloliquefaciens showed medium rate of infection compared tothe infected plants. FHII) caused very severe type of infection.Significant suppression of disease intensity in the present study was attributedeither to the effect of the siderophore on the pathogen or by ISR (induced systemicresistance) in the host plant. The rapid defense reaction exerted by the foliar spray atthe site of fungal entry delayed the infection process and allowed sufficient time forthe host to build up other defense reactions to restrict pathogen growth. This findinggains support from the observation of Manoranjitham ct a/. (2001) that thePseudomonas inhibited the symptom formation of damping off caused by firhiurnsp, in tomato. De Meyer and Hofie (1997) found that reduced Bottytis infection byPscudomonas treatment in tobacco was mainly due to the action of threesiderophores viz. pyoverdin. pyochelin and salicylic acid. Leeman et a/. (1995)reported that Pscudomonas treatment in radish induced the host defense reactionsuch as structural modification or accumulation of lignin content. The activateddefense reaction resists the pathogen entry and thus the establishment and blightsymptom formation by the pathogen was greatly reduced. Seed treatment with therhizosphere bacterium Scrratia marccscems strain 90-166 suppressed anthracnose ofcucumber, caused by C'ollcrotrichum orbicularr, through induced systemicresistance (ISR). When the iron concentration of a planting mix was decreased bythe addltion of a synthetic iron chelator, suppression of cucumber anthracnose bystrain 90-166 was significantly improved. Strain 90-166 produced 463.70 mg ofcatechol siderophore per litre, as determined by the Rioux assay in deferrated King'smedium B. The hypothesis that a catechol siderophore produced by the strain mightbe responsible for induction of systemic resistance was tested by evaluating disease

suppression by a mutant, deficient in siderophore production which failed tosuppress the anthracnose of cucumber (Press, 2001). The mutant no longer inducedresistance in cucumber to C, orbiculare thus supporting the hypothesis that thecatechol siderophore produced by the wild-type strain 90-166 is necessary for ISR.Role of siderophores in ISR was reported by Maurhofer et a1.(1994), who observedthat a pyoverdin deficient strain of P. jluorescens CHAO (CHA400) no longerinduced resistance against Tobacco necrosis virus in tobacco.CELL PERMEABILITYThe present study rcveals that A. hetianthi- infected leaves recordedmaximum electrolytic leakage (0.375 ms/cm2), which was 9.6 times higher than thatof healthy and treated plants. The change in the host membrane permeability afterexposure to a pathogen appears to be the earlier stage of pathogen recognition. Theaction of fungal toxin on the host cells resulted in the disintegration of semipermeable membrane, which in turn led to higher electrolytic leakage. Similarobservation was made by Thoithoi Singh et at. (2001) who found that fungal toxinor its enzymes contributed to the higher electrolytic leakage from the infected riceseeds. This higher leakage led to gradual loss of semi- permeable nature of the seedmembrane. Padma Singh (2000) showed that the higher electrolytic leakage in theAllernaria sp.-infected onion leaves was mainly due to the toxin produced by thepathogen. The damaged cells lost their ability to accumulate the metabolic solutes,which were required fcr the growth and development of the cells. The leastelectrolyric leakage was recorded in those treated with siderophore-Fe complex ofP.,fluorescens and B. amyloliquefacienr. Treatments on the healthy plants did not differ

much from the healthy control. This proves that siderophore and siderophore-Fecomplex did not create any damage to the cells of the Helianthus annuus plant.The leaves of infected plants sprayed with siderophore-Fe complex ofbacteria exhibited least electrolytic leakage. This might be either due to the action ofthis complex, which inhibited the establishment and activity of the pathogen or theyinduced the host cell to develop systemic resistance, which resisted the pathogeninvasion. Thus the host cell is protected from the damage caused by the pathogen.This finding is supported by the finding of Abad el a/. (1996) that minimumelectrolytic leakage in the treated tobacco leaves was mainly due to the action of PRprotein, osmotin. Similar report was made by Liu er a/. (1994) in potato plant.CELL WALL L YTIC ENZYMESThe fungal plant pathogens produce different t9es of pectic and celluloselytic enzymes, which act as the major factors for the plant disease development.Maximum pectinolytic activity of A. helianthi was observed in the leaves ofinfected plants. The higher activity of pectinolytic and cellulolytic enzymes of A.hrlianrhi is in conformity with the findings of Cano-Canchola et al. (2000) whoreported that extracellular lytic enzymes of Ustilago sp. were capable of degradingZeo ma,vs cell wall components. These enzymes transform the polysaccharides of thehost into simple carbon sources. which are used by the pathogen for their gmwth anddevelopment. Kaur and Deshpande (1980) reported the production of pectinolyticand hydrolytic enzymes in the culture and in infected leaves. The activity of theseenzymes was responsible for the symptom formation in the host cell. Chakrabartiand Basuchaudhary (1979) observed that the pectinolytic and cellulolytic enzymes

produced by Furanurn spp, dissolved m~ddle lamella and d~ffused Into the xylemvessels resultlng In xylem parenchyma maceration, m~ddle lamella d~stortron, vesselblockage and disease symptoms. The pectlnase enzyme IS produced pnmanly todlssolve the cell wall of the host and to reach the Intenor tlssue Dunng colonlzat~on~t secretes cellulase to attack the pnmary and secondary cell wall resultlng Inthlnnlng of cell wall followed by dlstntegratlon According to Ruesink (1971) andRueslnk and Thlmann (1965) the pectlnolyt~c enzymes w~th d~fferent pH optlma,~soelectnc polnts and them mechanisms of substrate cleavage were the cause of celldeath Aktar cr a1 (1960) brought out the fact that the cellulolyt~c enzymes secretedby Fusarrum sp attacked the pnmary and secondary cell wall of thc host (tomato)and d~slntegated them The degraded products might get Into the transp~rat~onalstream, whlch block the vessels lead~ng to wilt symptoms format~onMaxlmum lnh~bttlon In the actlvlty of PMG, PTE, PG and CI C, ( In ~~rrroand in ~~rvo) was recorded In the P fluorerrens s~derophore treatment S~gn~ficantlnh~blt~on In the pectlnolytlc and cellulolyt~c actlvlty of A helionrhr was ev~dent Inthose treated w~th the s~derophore of B amvlolrguefacrens and Fe(ll).The treatments mght lnh~b~t the actlvlty of lyhc enzymes of the pathogen bylnduclng ISR In the host cell such as th~ckenlng of cell wall or ~t Induces the host cellto produce toxlc compounds which m~ght lnactlvate or tnh~b~t the lytlc en7ymes ofpathogen The accumulat~on of phenol~c compounds In the treated leaves may betoxlc to the enzyme actlvlty of the pathogenSystemrc defences lnvolve the accumulat~on of ant^-m~crob~al compoundssuch as protelnase lnh~b~tors, phytolaxln and cell wall components In parts of theplant d~stant from the site of tnfect~on The leaves of healthy plant and Infected plant

treated with siderophore-Fe wmplex and siderophore of both bacterial species mightproduce some amount of oligogalacturonides (OGU) which are cell wallcomponents, can induce the plant to produce the inhibitors of proteinase of thepathogen and also induce the plants to produce phytoalexin and stimulate ligninsynthesis (Cote and Hahn, 1994). Lignin precursors are themselves directly toxic topathogens (Hammerschmidt and Kuc, 1982) and their polymerisation makes cellwalls more difficult to penetrate and degrade (Ride, 1980). In cucumber, the HRoccurs in SAR tissues and the cell wall becomes heavily lignified under the germtube of the pathogen, preventing its ingress (Richmond.. 1979). Duijff et a/. (1997)observed that the Pseudomonas treatment in the tomato plant induced higherlignification in the cell wall of conical cells. These thickenings added mechanicalstrength and resisted the lytic enzyme activ~ty of the pathogen. Similar ISR byPseudomonas treatment was observed in cucumber plant against R. solani(Brog1ie eta/.. 1991). Ph.vrophfhora sp. and Allernaria sp. (Yoshikawa cr al. 1993 a; Van Loomet a1.,1998) and fithiurn sp. in the same host plant by Serratia sp. treatment(Benhamou el a/., 2000) In addition to lignification. some PR proteins produceddue to SAR are also components of cell walls (e.g. glyclne-rich glycoproteins)which accumulate at the site of infecting cells during the pathogensis and theiroxidative cross-linking by peroxidase further increases the strength of the cell wall(Sticher el a/., 1997).PHOTOSYNTHETIC PIGMENTSThe photosynthetic pigments are responsiblethe process of photosynthesis in the host plants. Any101 I'. .- .

would be reflected tmmed~ately on the photosynthettc efficacy of the plant andulttmately on ~ts growth and yeld Hence the analysts of p~gment contents tn thehealthy, Infected and treated plants becomes essenttalThe present study shows that maxlmum reductton In the total chlorophyll(77.5%) and caroteno~d (62 5%) In A heltanthl-tnfected plantsThe toxlc effect of the fungal metabol~te caused the synptom formatton Inthe host ttssue Slmtlar observat~on was made by find~ngs of Pero and Ma~n (1970)In tobacco rnfected w~th Allernarta sp Pant ct a1 (2001) reported that malze leafInfected w~th bllght dlsease resulted In the least C02 fixatton The reduct~on In thephotospthettc acttvlty (suppressed ATP format~on) was malnly due to the toxtccffect of the pathogen Tu and Ford (1968) observed a heavy reductlon tn thephotospthet~c actlvlty In malze due to v~rus lnfectton Thls might be due to theactlvlty of the pdthogen. which ~n~t~ated chloros~s and necror~s In the loaves andreduced the number and size of the chloroplasts D~ener (1963) explained that thereductlon of photospthet~c plgnent contents and thew actlvlty In the vl~~-lnfectedleaves was malnly due to the lnductlon of chlorophyllase activlty by the pathogenThis mtght he due to the acttv~ty of toxln produced by the pathogenLeast reductlon in total chlorophyll (17 81%) and carotenotd (27 54%) wasnotlced In the P fluorcsccns and B amvlol~quefactens treatments Under tnfectedmndttton, s~derophore-Fe complex treatment showed htghest values In totalchlorophyll content and caroteno~d compared to the heavy reduct~on In the Infectedleaves The treatments might suppress the chlorophyllase actlvlty of the pathogendue to the presence of Iron tn the complex Though Iron IS not a component of thechlorophyll molecule. ~t 1s requtred as a cofactor In the reactlon leadlng to

chlorophyll synthcsts (Hull, 2000) Thts was supported by the hlgher valuesobta~ned from the leaves of healthy plants treated wtth s~derophore-Fe complex of Pfluorcscens and B amylolrque/aciens and Fe(ll)CARBOHYDRA TESThe total carbohydrate content was found to be drastically reduced In theleaves of Infected plants (total sugar by 65%, reduc~ng sugar by 69%. non-reducing sugar by 55%, sucrose content by 77%, starch content by 69%)The pathogen In the Infected tlssue nomlally uses the host carbohydratemetabolttes for thew growth and survlval (Chakrabarty et a /, 2002) Debnath er a1(1 998) reported that the decreased starch content In the Brass~ca leaves Infected wlthAlhugo sp was matnly due to the fact that these substances were utiltzed by thepathogen for tts growth and development Padma Singh (2000) found that the loss ofcarbohydrates from the Alternarra sp -infected onton leaves was due to heavy loss ofthese contents from the ~mpatred membrane Then these contents were used by thepathogen for ~ts growth Dropkln (1972) concluded that the reduced carbohydratewas malnly due to the acttvlty of the en7ymes of pathogen, whlch cleaved thc hostcarhohydrate and used 11 for thew growth and development Schlffer and Mtrocha(1968) found that the depletton of starch content In the rust-lnfwted bean leaves wasmatnly due to the acttvatton of amylase (starch hydmlylng enzyme) by the fungalmetabolttesAmong the treatments on the Infected plants, s~derophore-Fe cornplex-sprayed leaves exhlblted least reduction In the carbohydrate wntent (13% In total

sugar, 9% in reducing sugar and 3% in non-reducing sugar ) as compared to theirhighest reduction in the infected leaves.The least reduction in the siderophore-Fe complex-sprayed leaves might bedue to induction of the treated plants to utilize the carbohydrates for the biosynthesisof phenolic compounds, which in turn were used for the defense reaction against thepathogen infection. This finding confirms the earlier work of Rajavel (2000).NITROGEN METABOLISMProtein and Amino aridChakraharti and Basu Chaudhary (1979) found that maximum reduction inthe protein and amino acid content in safflower wilt caused by Fusarium sp. wasmainly due to their breakdown by proteolytic enzymes such as protease secretedhy the pathogen. These enzymes enabled the pathogen to use the host protein assource of nitrogen and amino acid for their growth and development. This wassupported by the works of Howell and Krusberg (1966) and Chakrabarty et 01.(2002) in alfalfa and pea plant infected with Dit,vlenclus sp. and in cotton plantinfected with grey mildew disease respectively.Least reduction in prote~n (20.13%) and amino acid content (14.02%) wasnoticed in the leaves sprayed with siderophore-Fe complex of P. .flrrorescens.Moderate reduction (50%) was recorded in other treatments.The reason might be that these treatments induced the host plant to utilizethe amino acids of protein and sugars of the nucleic acids for the synthesis ofphenolic compounds which acted as the defense agents against the fungalinfection. The present finding substantiates the work of Veeramohan et a/. (1994)

that the decreased proteln content of chlll~ Infected w~th Alternarza solant m~ghtbe due to the~r partlclpatlon In the synthes~s of phenol~c compounds Dasht~ et a1(1997) reported Increased proteln content in the Pseudomonas-treated leaves ofsoybeanRichmond and Lang (1957) reported that the klnetln product~onpreserved the proteln content by the decrease of the proteolyt~c enzyme actlrlty Indetached Xanthtum leaves Kloepper (1994) reported that the PGPR treatmentcaused higher protein content and total ammo ac~ds In rice and wheat plants andhlgher sugar content In sweet potato and In sugar beet Th~s was malnly due to thesuppression of the disease and Increase In the activation of enzymes that wereinvolved In nltrogen metabol~smNitrate reductase and Amino nitrogenThe leaves are the mdjor sites of utll~zatlon of nltrates The nltrates that areabsorbed by plants are reduced to nltnter and then ~mmed~ately to ammonla Finallythey are converted to amino ac~ds and proteins The nltrate reductase 1s the keyuruyme In nltrogen metdbolism, wh~ch converts the nitrate to nltnteMaxlmum reduct~on (62%) in ammo nitrogen and (59%) In the actlvlty ofNR en7yme was observed tn the A hel~an1h1-lnfected leavesHeavy reduct~onIn nttrogen content in the host plant under fungal pathogenes~s m~ght be due tothe surv~val of the pathogen at the expense of host nltrogen pool leading to theenhancement of disease development,as reported In banana(Prasad.1981)Padma Slngh (2000) attnbuted decreased nltrogen content In Alternarra sp -infected onlon leaves to the d~srupt~on of cell structure coupled with enhancedproteolyt~c enzyme actlvlty

Leaves treated with P. ,fluorescens siderophore-Fe complex showed higheramino nitrogen and NR activity over other treatments. This might be due to theenhancement of inducing activity of the enzymes involved in the nitrogenmetabolism by the treatment. The least reduction might be due to participation ofamino nitrogen and NR activity in the defense reaction against the pathogeninfection (Krikham, 1954; Murthy and Bagyaraj, 1978). The present findingsubstantiates the work of Asanuma et a/. (1980) where the increased nitrogenfixation was found in the rice plant treated with Pseudomonas,/luorescens. Lifshitzst a/. (1986) and Chanway ct a/. (1989) reported the enhanced nitrogen fixation inthe Pseudomonas-treated L. esculcn~a and P. sati~rm in the field and laboratoryconditions. Similar enhanced nitrogen metabolisni due to the application ofI'seudomonas was wnfirmed in chickpea (Parmar and Dadarwa1.1999).PHENOL AND PROLINEPhenol~c compounds are fungitoxic in nature. Accumulation of phenoliccompounds increases the physical and mechanical strength of host cell wall resultingin the inhibition of fungal invasion. The phenol and proline compounds act asadaptive mechanism in the host plant against the fungal infection.The present study recorded three-fold increase in the total phenol, O.D.phenol and proline in the infected plants over healthy and treated plants. This mightbe due to the hindrance of the glywlysis by the activity of the pathogen. which inturn activated the pentose pathway leading to the formation of 4-carbon compoundsfor the synthesis of phenols. Farkas and Kiraly (1962) and Jaypal and Mahadevan(1968) found that sharply increased phenol content in the infected plants might be

due to the fact that the accumulation of phenols in the infected tissue might comefrom the surrounding healthy leaves in order to resist the advancement of thepathogen towards the other healthy cells. The leaves sprayed with siderophores andsiderophore-Fe complex of both the bacteria exhibited an increase of about 1.5 timesin the phenol and proline content over healthy plants.This might be attributed to the induction of systemic resistance in the hostplant due to treatments. The over-production of phenolic compounds resists theadvancement of the pathogen towards other healthy cells. Similar findings werereported by Ramamoorthy and Samiyappan (2001) that the higher phenol contentin the Pseudomonas-treated chilli plants infected with Colletotrichum capsici wasmainly attributed to the fact that the phenols are fungitoxic in nature and theiraccumulation increased the physical and mechanical strength to the host cell wallresulting in the inhibition of pathogen invasion.Leaves of the infected plants treated with siderophore and siderophore-Fecomplex showed a higher quantity of phenolic compounds than the healthy controland healthy treated plants. It can be assumed that these compounds caused inhibitionof the pathogen. In another study, the commercial product Milsana (a plant extractfrom Revnuufria sachalincnsir) stimulated synthesis of phenolic compounds (Daafyct a/., 1995). Application of salicylic acid and its analoyes(benzo(l,2.3)thiadiazole-7-carbothioic acid S-methyl ester (BTH) and dichloroisoniwtinic acid (Dcina) led tothe increase in phenols in the cell wall (Siegist et al., 1997) which reduced thebacterial growth. Thus accumulation of phenolic compounds at the infection site hasbeen correlated with the restriction of pathogen development (Heath, 1980). Toxiceffect of phenols can kill bacteria and other microorganisms in plant tissue. As

components of physical barriers in the form of lignin, they prevent pathogens frompenetrating into the host tissue. Phenols act as chemical barriers in cell walls in theform of tannin substances (Treutter, 1996).Several studies established an increase in phenolic compounds anddevelopment of resistance (Goodman et al., 1986; Ojalvo er a/., 1987:Daafy et al.,1995). The resistance may be further enhanced by phenol esterification in the cellwall (Nicholson. 1992). It was also proposed that changes of the cytoplasmic pH(Ojalvo el al., 1987) in plant tissue, due to increased phenolic acid content, resultedin inhibition of pathogen development. Gallic acid, due to its monomer structure,can easily be decomposed by phenolic oxidation (Feucht and Treutter, 1989).Similar process may occur in plant tissue after inoculation. Bonhoff cr al. (1987)reported the oxidation of monomer phenolic compounds and their accumulation nearthe infection site. Generally, many resistance reactions of the host plant againstpathogens are characterized by very rapid synthesis of phenolics and theirpolperisation at the cell wall (Matem and Kneusel, 1988).These compounds led to considerable cytological changes in the pathogensuch as cytoplasmic disorganization and loss of protoplasmic content. Similarreport on higher phenol content due to Pseudomonas treatment was made intomato plant infected with I'.syringe (Alstrom, 1995) . Anderson and Guerra(1985) found that Pseudomonas treatment induced lignification of cell wall ofbean leaves which in turn resisted the invasion of the pathogen. Beckman (2000)reported that the accumulation of phenol resisted or reduced the wilt symptoms byinactivating the pathogenic enzyme activity. Several host defenses have beenreported to be involved with ISR elicited by PGPR. These defenses include

strengthening of epidermal and cortical cell walls and deposition of newly formedbamers beyond infection sites including callose, lignin and phenolics (Benhamou era/., 1996, 2000; Duijff el al., 1997; Jetiyanon et a/., 1997; M'Piga et a/., 1997).Increase in phenolic compounds in cell wall appositions in pea due to SAR isreported by Benhamou(1996) and Van Wees et a/.(1997)who suggested that a singlebacterial species may induce systemic resistance through more than any onemechanism and increase the yield: (1) The synthesis of phenolic compounds andtheir subsequent oxidation to quinones by polyphenol oxidase and peroxidase and(2)Systemic defenses involving the accumulation of anti-microbial compounds inparts of the plant distant from the site of infection.ANTIOXIDANT ENZYMESThe active oxygen species (AOS) such as superoxide anion, hydrogenperoxide and hydroxyl radical are generally produced in the plants as a result of themetabolic processes that take place in chloroplast. mitochondria and plasmamembrane-linked electron transport system. During the infection process, pathogentnterfercd wlth the electron transpon system in the host cell thereby resulting in theleakage of electrons. These electrons altered the structure of the molecular oxygenresulting in the production and accumulation of AOS within the cell. Theaccumulation of AOS damaged the cell and caused lipid peroxidation, proteindenaturation, DNA mutation, molecular malfunction and ultimately led to cell death.Generally the plants produce many antioxidant and low molecular weight enzymessuch as SOD(superoxide dismutase), CAT(catalase), PO@eroxidase), ascorbateperoxidase and glutathione reductase. The SOD converts the superoxide radicals to

hydrogen peroxide. Then the CAT and PO reduced hydrogen peroxide to water andoxygen molecules.Significant increase in the antioxidant enzymes activity wasfound in A. helianthi-infected H. onnuus.The enhanced antioxidant enzymes activities in the infected host tissue mightbe due to induction of systemic resistance in response to the pathogen infection.These enzymes participate in rapid detoxification of reactive oxygen species(supemxide anion (0; ); hydrogen peroxide (H202); singlet oxygen (OH) into water.The rapid conversion inhibited the toxic effect caused by AOS to the host plant. Inaddition, the host cell under pathogenesis might accelerate the terminal respiratorypathway, leading to the increase in the CAT activity. The higher activity of PO andPPO in the infected tomato leaves might be due to their participation in the oxidationof phenolic residues into cell wall polymers in the pathogen-infectedcells(Soureche, 2002). Asha and Kannabiran (2001) reported that the higher PO and PPOactivity in the chilli leaves infected with Colletotrichrtm capsici was mainly due tothe enhanced respiratory rate induced by the pathogen activity. Similar finding wasmade in Cap.sicum annurtnl (Gomathi. 2001). Nonaka (1959) found that increasedPO and PPO in the host plant infected with fungal pathogen might be due to thetriggering of the enzymes to meet the catabolic reaction exerted by the pathogeninfection. Naffaa ct 01. (1999) found that higher activity of PO and PPO in perennialryegrass leaf spot disease infected with endophytic bacteria was due to the lyticenzyme activity of the pathogen, which involved in the activation of the latent PPOactivity of the host plant. Similar finding was reported in anthracnose disease ofcucumber (Zhang et 01.. 1996)and in pearl millet infected with Sclcrospora sp.(Smdhara et a1..1995). Kwon and Anderson (2001) reported that the enhanced SOD

and CAT isozynes activity in the wheat leaves infected with Fusarium like fungusisolate.Baker and Orlandi (1995) explained that the accumulation of active oxygenspecies in the plant cell during interactions with potential pathogens affect manycellular processes (proteins, lipid, polysaccharides and nucleic acids) that areinvolved in the plantlpathogen interaction. Yim ct at. (1990) reported that theelevated concentration of SOD in the infected plant cell generally induceddisfunction of all metabolic activities and led to cell death. The fungi generatehydrogen peroxide as part of its weaponry to enhance the penetration process intothe host cell.Levine er a/. (1994) and Jabs er al. (1996) concluded that hydrogen peroxideproduced as a result of pathogen activity in the plant-pathogen interaction, led todirect cause for cellular death of the infected tissue. Bowler er at. (1992)explained that increased SOD activity in a susceptible host infected with avirulent pathogen was mainly responsible for the detoxification of theoxyradicals. Rusterucci er at. (1996) brought out that the elicitin (capsicin andcryptogein)-treated Nicotiana cell exhibited increased activity of active oxygenspecies. This was mainly due to their participation in the detoxification of thereactive oxygen species. Wojtaszek (1995) discussed the relationship hetween theoxidative bunt and plant defense responses. which included oxygen consumption.production of phytoaelaxins, SAR. change in membrane permeability and ion fluxand hypersensitive cell death.

Keen (1999) explained that the scavengers of active oxygen species (SOD,PO, CAT) act like antibiotics against the invading pathogen. The enhanced activityof SOD, PO, CAT recorded in the treated leaves generally restricted the pathogenactivity. Schinkel er a/. (2001) brought out the fact that the main function of SODwas to scavenge the superoxide anion radicals, generated in various physiologicalprocesses and prevent the oxidation of biological molecules. Significant increase inSOD. CAT, P0,PPO activity (about 1.5 times) was evident in the leaves of treatedinfected plants over healthy plants. The treatments generally induced systemicresistance in the host cell, which in turn enhanced activation of these enzymes in theconversion of reactive oxygen species or radicals to water in order to reduce theinfection. Thus the rapid conversion reduced the severity of the infection caused bythe pathogen. The systemic induced resistance by Pseudomonas treatment (VanLoom er a/., 1998) causes cell wall structure modification in response to pathogenattack (Chen er a/., 2000; Benhamou er a/.. 2000), accumulation of phenoliccompounds (Ramamoorthy and Samiyappan. 2001). induces biochemical andphysiolog~cal changes in the treated plants such as enhanced chitinase activityagainst red rot of sugarcane (Viswanathan and Saniyappan, 1999) and synthesis ofphytoalexin and other secondary metabolites (Maurhofer er a/., 1994).The ROSproduced in the oxidative burst could serve not only as protectants against invadingpathogen. but could also be the signals activating further plant defence reactions,including HR of infected cells (Tenhaken et a/.. 1995).

YIELD AND ITS REU TED PARAMETERSThe efficiency of any crop rs judged only by the yeld The knowledgeabout the lnter-relat~onsh~p between the yeld and yield-related parameters 1srmportant to understand the physrolog~cal basls of the yeld Lowest yeld of seedswas rewrded In the control Infected plants showlng loss of 74 92%. followed by theInfected plants treated wlth Fe(ll) (63 73% ) The lowest yeld was recorded In theA hal~anrht-rnfected plants Decrease In the yeld of the rnfected plants IS a commonphenomenon as the entrre metabolism related to the plant growth and development rsaffected by fungal rnfectlon Srmllar conclusron was drawn earher(Balasubrahmanyam and Kolte, 1980)Treated healthy plants (95 76%-98 06%) showed more or less slmllar values as thatof wntrol healthy plants (100%) Slgnlficant Increase rn the or1 content was rewrdedrn the Infected plants treated wrth P fluorercens srderophore-fe complex (87 20%),followed hy srderophore of B am) lolrquefacrens (74 89%)The h~gher yeld (87 2%) was recorded m the infected plants treated wrths~derophore-Fe complex of P /7uorescens This might be attnbuted to the control ofthe dlsease or due to the productron of ISR, wh~ch allows the plant to wrthstand evenrn pathogenesrs Increased yeld mrght be due to the ~nductron of plant pwthregulator actrvrty rn the treated host plant Srmrlar yeld enhancement In the rnfectedplant under treatment was observed earlier (We1 er a/, 1996) They observed that thehrgher yeld rn the Pseudomonas-treated cucumber plants was mainly due to thesuppressron of angular leaf spot, anthracnose and bacterial wilt drsease by theantagonrstrc treatmentMcCullagh er ol (1996) found that the applrcatlon ofPseudomonas reduced the root rot rncrdence rn cucumber plant and thus rncreased

the yield. Vidhyasekaran et al. (1997) established that the seed bacterization andfoliar spray of Pseudomonas in rice reduced the incidence of R. solani, which in turnled to higher grain production. Similar reports on higher yield due to suppression ofcollar rot disease caused by Fusarium oxysporum by Pseudomonas treatment weremade earlier in peanut and chick pea (Dileep st al. 1998, 1999; Dileep Kumar., 1999;Dileep kumar and Dube, 1992). Yoshikawa et a/. (1993 a) observed thatPseudomonas-treated soybean plants are able to synthesize organic acids likesuccinic and lactic acid that increased the plant growth even in pathogenic condition.This was made earlier by Lynch (1976). Duijff et al. (1993) confirmed that thesidorophore of Pseudomonas acted as the maln factor in the suppression ofFusarium wilt, which in turn fetched higher yield in carnation plant. Similar reporton higher yield in pea and potato due to the activity of Pseudomonas siderophorewas evident from the works of Kloepper et al. (1980) and Dileep kumar el al.(2001).All plants possess active defense mechanisms against pathogen attack. Ifdefense mechanisms are triggered by a stimulus prior to infection of a virulent plantpathogen, disease can be reduced (van Loom el a/.. 1998). Induced diseaseresistance can be defined as the process of active resistance dependent on thephysical or chemical barriers of the host plant, activated by biotic or abiotic agents(Kloepper el 01.. 1992). The resulting elevated state of resistance in plant pansdistant from the site of primary triggering is referred to as systemic acquiredresistance (SAR) or induced systemic resistance (ISR). SAR is characterized by anearly increase in endogenously synthesized salicylic acid (SA), which appears to bean essential signalling molecule in the SAR pathway (Metraux eta/., 1990). Selected

non-pathogenic plant growth-promoting rhizobacteria (PGPR) are also known toinduce systemic resistance (Bakker et al., 2003; Kloepper eral., 1992; van Loometal., 1998; Van Peer etal., 1991; Wei ef al., 1991). The term rhizobacteriamediatedISR is used to differentiate this type of induced resistance from pathogeninducedSAR.Bacterial determinants of ISR have been identified as lipopolysaccharides(LPS) and iron-regulated compounds. Lipopolysaccharides(LPS) of Pseudomonas,fluoresrens strain WCS417 have been implicated in triggering ISR againstPseudomonas svringac pv. tomato in Arabidopsis (Van Wees eral., 1997) andFusarium wilt in carnation (Van Peer and Schippers, 1992), radish (Leeman el a/.,1995) and tomato (Duijff er a/.. 1997). In radish, the 0-antigenic side chains of theLPS of both P.,/lrrorcsrens WCS417 and P.,fl~roresccns WCS374 were majordeterm~nants of ISR against Fusarium wilt under iron-replete conditions, but notunder iron-limiting conditions. Unknown iron-regulated factors in WCS374 andWCS417 caused ISR under low-iron conditions. Likewise, in Pseudomonas puridaBTPI. an unknown iron-regulated metabolite Cx appeared to be responsible for ISRagainst Aotptis rinerea in bean (Ongena et 01.. 2002).De Meyer and Hofte (1997) found that the siderophore- salicylic acid @A)-producing PGPR Pseudomonas aeruginosa 7NSK2 triggered ISR against B. cinereain bean when the bacterium was grown on iron-poor media but not on iron-richmedia and that SA-negative mutants of this swain had lost their ability to inducesystemic resistance. SA production by P. aeruginosa 7NSKZ appeared to beessential for ISR also to tobacco mosaic virus (TMV) in tobacco (De Meyer er al.,

1999) and to Colletotrichum lindemuthianum in bean (Bigirimana and Hofte, 2002).Accumulation of SA appears to be critical for the induction of the SAR-signallingpathway because transgenic plants unable to accumulate SA are incapable ofdeveloping SAR (Gamey et a/., 1993).Likewise, expression of siderophore-SA-biosynthetic genes inPscudomonas ,fluorescens P3 improved induction of resistance to tobacco necrosisvirus (TNV) in tobacco (Maurhofer eta/., 1998). However, the siderophorepyoverdine,rather than SA in Pseudomonas fluorescens CHAO, appeared to beimplicated in induced resistance against TNV (Maurhofer eta/., 1994). Similarly, inthe SA-producing PGPR Scrratia marccscens 90-166, an unidentified catechol-typesiderophore rather than SA is involved in ISR (Press ct a/., 1997. 2001).Audenaen er al. (2002 a. b) showed that the phenazine antibiotic pyocyaninIn combination with SA or the SA-containing siderophore pyochelin produced byP, aeruginosa 7NSK2 act as determinants for induced resistance in tomato againstR cincrea. P.~eudomonas purida WCS358 is a PGPR originally isolated from therhizosphere of potato (Geels and Schippers, 1983a. b). It can suppress soil-homeplant diseases by siderophore-mediated competition for iron (Bakker el al.. 1986,1993). WCS358 strain can also induce systemic resistance in Arabidupsis rhaliunaagainst Pseudomonas s,vringae prr, tomato and Fusarium ovsponrm ,fsp. ruphani(Van Wees era/.. 1997), but the strain is unable to induce resistance againstFurarium wilt in carnation (Duijff er a/., 1993) or radish (Leeman er a/.. 1995).

Pseudobaotin of Pseudomonas putida strain WCS358 was able to inducesystemic resistance in Arabidopsis against P. syringae pv. tomato, in bean againstColletotrichum lindemuthianum and B. cinerea and in tomato against Botptiscinerea. In bean and tomato, pseudobactin- and LPS-containing cell walls inducedresistance when applied to the plant roots.Both flagellin and siderophore induce resistance against B. cincrea in tomatobut purified flagellin failed to induce resistance in tomato (Felix ct at., 1999; Meindlera/., 2000). It is possible that the resistance pathway induced upon flagellinreceptorrecognition in tomato is not effective against B. cinerea and siderophore-SA-negative mutants of Pseudomonas aeruginoso 7NSK2 that failed to inducesystemic resistance in bean, tobacco and tomato (Audenaen era/.. 2OO2b; De Meyerand Hiifte, 1997: De Meyer cr a/.. 1999)Like LPS, purified WCS358 pseudobactin induced resistance in Arabidopsis,bean and tomato. Leeman eta/. (1996). however, showed that the pseudobactin ofWCS358 did not induce resistance to Fusarium wilt of radish. In bean plants. thepseudobactin mutant PB- and purified pseudobactin of WCS358 were as effectiveas, or even more effective than the wild-type.Likewise. the purified pseudobactin siderophorc of WCS374 inducedresistance in the Fusariun wilt in radish, while a pseudobactin mutant of WCS374still induced resistance under iron-replete and iron-limiting conditions (Leemanet al., 1996). However, in tomato plants, the pseudobactin mutant PB lost its ability

to Induce reslstance whereas ~t s~gn~ficantly protected leaves against B crnerea Inwmblnat~on w~th mutant OA- lacktng the 0-ant~gen of LPS This means that e~therboth LPS and pseudobact~n are needed for ISR In tomato, or that then effect ISadd~tlve It 1s poss~ble that the mutants produce too llttle pseudobact~n or LPS to beeffectwe In induclng res~stance In the absence of the other compound At present ~trema~ns unclear how pseudobact~n tnggers ISR Several host defenses have beenreported to be lnvolved w~th ISR elrc~ted by PGPR These defenses tncludeI~pificat~on, perox~dase, and superox~de dlsmutase (SOD) product~on In cucumber(Jetlyanon el a/, 1997). product~on ot perox~dase (Albert and Anderson, 1987) andphenolic compounds In cell wall apposltlons In pea (Benharnou et a/ 1996).phytoalexrn product~on in camatlon plant(Van Peer, 1991) and l~gn~ficatlon In bean(Anderbon and Guerra, 1985)More recently ~t has been shown that the b~o-control agent Pseudomonasfluorcscens strain CHAO (Maurhofer er a/, 1994) Induces SAR-assoc~ated proteins,confers systemlc reslstance to a vlral pathogen and Induces accumulat~on ofsallcyllc ac~d. whlch plays a role In s~gnal tranbductton In SAR (Gaff'ney er a1 1993.Ryals ct a/. 1996) Mutants of CHAO that do not produce the s~derophorepyoverdln do not Induce SAR, suggesting a novel role of s~derophore In d~seasesuppression by the synthes~s of bactenal metabolrtes (Maurhofer eta/. 1994)The ~nvolvetnent of the fluorescents~derophore (pyoverdln) In thesuppress~on of Pvthlum- Induced damp~ng-off of tomato by Pseudomonasoerugrnosa TNS K2, has been demonstrated uslng pyoverd~ne-defic~ent mutants

(Buysens ct a[, 1996). The hyperactive mutants (Flu++ Sid++) (RBL 1015 and1011) with higher siderophore production suppressed the wilt disease to a greaterextent than the wild type. Similar increase in the biocontrol potential of thefluorescent siderophore over producing mutant MPS 16 M-1 of Pseudomonas sp.against Rhizocronia solani in chickpea has been reported (Goel et al., 2000). Thus,the study has proved fluorescent siderophore production as a mechanism ofbiocontrol of the bacterial wilt disease in the fluorescent pseudomonad isolates, RBL101 and RSI 125.The efticacy of siderophore-Fe complex of P. ,fluorescens in the control of A.helianrhi infection suggests that there was a cumulative inhibitory effect bysiderophore and iron as foliar spray against A. helianrhi.Ferritins are the iron-storage proteins that accumulate in the plastids of leavesand seeds (Clarkson and Hanson, 1980; Caris er at.. 1995). Apparently theintercellular capture of Fe by the ferritins protect plants from oxidative damageinduced by various types of stress. Nevertheless, the results of Becker et a/. (1998).hased on mutants of pea (Pirum sativum) with defects in the regulation of theadsorption of iron, show that plants accumulate Fe in femtins and precipitate iron indeposits formed in the cytoplasm, mitochondria and endoplasmic reticulum. Thiswas a defense mechanism against accumulation of excessive soluble Fe. whichcaused oxidative stress. Similar conclusion was drawn by Deak er at. (1999) whoworked with transgenic tobacco plants that synthesized alfalfa femtins. Thetransgenic tobacco retained nonnal photosynthetic function in the presence oftoxicity caused by free radicals generated by excess of Fe or treatment with

paraquat.Additionally, the offspring of transgenic plants that accumulate foliarfenitins exhibit tolerance to necrotic damage caused by viral pathogens (tobacconecrosis virus) and fungi (Alternaria alternata and Botrytis cinerea).Plants are also able to activate defense mechanisms aiming at decreasing ironavailability at infection sites. Deak etal. (1999) reported that transgenic tobaccoplants ectopically expressing alfalfa fenitin, exhibited tolerance to necrotic damagecaused by viral or fungal infection. In addition, Mata eral. (2001) found that theinfection of potato leaves by the firngal pathogen, Phytophthora infistans resulted inthe accumulation of ferritin mRNA. Otherwise, siderophores produced by P.,fluorcscens strains participate in the induction of systemic resistance against fungalpathogens (Audenaert ct a/. 2002a; Leeman eral., 1996). However, the molecularmechanisms by which plants can control their iron metabolism in response topathogen attack have not been elucidated so far.A possible role of ferritin in plant resistance was reported by Deak cr al.(1999)who showed that transgenic tobacco plants over expressing alfalfa femtinwere more tolerant to infection with fungi and viral pathogen. Transgenic tobaccoplants that synthesize alfalfa ferritin in vegetative tissues--either in its processedform in chloroplasts or in the cytoplasmic non processedform--retainedphotosynthetic function upon free radical toxicity generated by iron excess orparaquat treatment. Progeny of transgenic plants accumulating ferritin in their leavesexhibited tolerance to necrotic damage caused by viral (tobacco necrosis vims) andfungal (Alrernaria alrernara. Bolptis cinerea) infections. These transfomantsexhibited normal photosynthetic function and chlorophyll content under green house

Table 26. Effects of siderophore and siderophore-Fe complex of P. ,fluorcscensand B. amyloliquefaciens and Fe(I1) on Helianthus annuus infected withAlrernaria helianthi.Infected plants treated withElectrolyticTotalch'orophyllL1content (mdg fw) -1,910 ( 0,63 I 1.57 1.23 1 I12 1 1.08/ /~[Oil content (YO) 1 100 1 25.08 1 87.20 73.13 69.20 74.89

condit~ons Sequestering intracellular Iron lnvolved in the generation of the veryreactlve hydroxyl radicals through Fenton reactlon Femtln protects plant cells fromox~dative damage Induced by a w~de range of stressesS~demphore-Fe complex of P fluorescens and B amylobquefaciens mlghtInduce Helranrhus annuus to produce fenttln, wh~ch In turn could stmulate hostplant to develop the above Fenton mechanism, thereby producing hlgher degree ofantlfungal effects This mlght lead to greater rate of lnh~b~t~on of pathogen growthand ultimately Increase the crop yeldIn the past. 5everal workers have reported that s~derophore of mlcrobes (fung~and bactena) lnh~b~ted $011 phyropathogens because of their capaclty to absorb Ironfrom ~ron-starved environment, depnving pathogens of iron For the first tlme, thepresent study bnngs out the potentla1 of the punfied s~derophore e~ther lndivlduallyor combined w~th ~ron(Fe(lll)) not only to control the pathogen but also to enhancethe growth of the host plantA companson of vanous parameters In H annuus due to the follar spray otslderophore-Fe complex and siderophore of P fllrorescens and B amvloliquefac~cns t Ft(11)1s made In Table 26 lnh~blt~on of Aheltanrh~ lnfectlng H annuus by the foliar$pray of s~derophore and s~derophore-Fe complex of above two bactena IS attributedto the following reasonsMax~mum lnhibtt~on of the actlvlty of all the tested pectlnolytlc and cellulolyTicewymes produced by A hehanthl In the host plant H annuusMlnlrnurn electrolytic leakage in H annuusReduced disease ~ntenslty In H annuus

Increase In carbohydrate and proteln metabollsrn In H annuusThe enhanced actlvrtles of antloxldant enzymes and phenol~ compounds In theleaves of the host plant, H annuuslnduct~on of SAR In H annuusOf the s~derophore and s~derophore-Fe complex of two bactena, slderophore-Fewmplex of P fluoresccns was found to be most efficient In wntrolllng A helranth~and also In malntalnlng the 011 ycld under pathogenes~s to the level of controlhealthy plants Th~s was followed by stderophore-Fe wmplex of Bamvlolrquefacrcns, s~derophore of Pf7uorcscens and R amvlol~qrrefacrcns Indescending orderThe following facts emerge tiom the present study1 Fe(l1) mtxed wlth EDTA sprayed on the leaves of ~nfected plantsIncreased the photosynthet~c actlvlty hut d~d not lnh~b~t the growth ot pathogenlcadlng to severe d~sease lntenslty Hence I! IS concluded that Fe w~thouts~derophore cannot be used to control A hclranrhr? The culture filtrates of Bacrllrcs srrb~rlr~ wlth or wlthout Feamcndment showed mlnlmum lnh~blt~on of contd~al germlndtlon, myceltal growthand blomass of A hclranrhr rn 11tro S~derophore and s~derophore-Fe complex ofBacrllus ccreris were found to be phytotox~c to the host plants exh~b~tlng loweslpercentage of gennlnatlon of seeds Infected w~th Ahelranthr Hence Bacrll~issubtrl~s and B cereus could not be employed for the b~ocontrol of A helranthr3 S~derophore of Psertdomonas fluorescens and BacrNusamvlolrqucfac~ens and thew s~derophore-Fe complex sprayed on the leaves of

healthy plants did not produce any toxic effect. But they inhibited the growth ofpathogen and increased the physiological processes such as photosynthesis,synthesis of carbohydrates and nitrogenous compounds, nitrate reductase activityand induced SAR in the host plants, resulting in the higher yield in the host plantsultimately.4. Of the above two spp. P. ,fluorcsccns exhibited the bestbiowntrol effects. This might be due to its genetic constitution and synthesis ofthe most efficient siderophore which could cause maximum inhibition of A.helianthi.

5. SUMMARY

The present study brings out the effects of siderophores of four soil bacteria viz.B. subtilis. 6, cereus. P. ,fluorescens and 6. amyloliquefaciens and the siderophore-Fe complex of the latter two on Allernaria helianthi, the infectant of foliar blight ofHelianthus annuus, L.(sunflower) under healthy, infected and treated conditions.The optimum inhibitory concentration (OIC) of siderophore and siderophore-Fecomplex of the soil bacteria was found out based on their efficacy in the control ofAlternaria helianfhi sporulation and growth under in ~dtro condition. The OIC ofsiderophore of P. ,fluoresccns was found to be 10 ppm and that of B. cereus and B.am,vloliquqfaciens was found to be 50ppm and siderophore-Fe complex of all thethree soil bacteria was 10:l ratio. These concentrations were used for both in vitroand in r,ivo studies. Fe(l1) at 50 mdlitre of water was used for comparison.The pots were prepared according to the standard procedure and triplicateswere maintained for all the treatments. Leaves of 45 days old plants were sprayedwith A, helianlhi culture containing 3000 to 7000 conidia iml. AAer an interval of12 hours (46 DAS) thc siderophore of P. ,fluorescens and B. am,vloliquefaciens andtheir siderophore-Fe complex (1O:lratio) and Fe(l1) were sprayed on the leaves ofhealthy and infected plants. The leaves were collected on 54 DAS for biochemicalestimation. Acetone powder was prepared from the leaf samples of healthy (controland treated) and infected (control and treated) plants and stored at O°C. The crudeenzyme sources were prepared from the acetone powder as and when needed. Theeffects of siderophore and Siderophore-Fe complex were investigated underhealthy, infected and treated conditions.

The following results were obtained in the in vivo study:bSevere disease intensity was observed in the control infected plants(87.77%). In all the treated infected plants the disease intensity was reduced. Plantstreated with siderophore-Fe complex of Pseudomonas ,fluoresrens exhibited lowestdisease intensity (9.6%), followed by that of B. amyloliquefaciens (1 1.56%) (lightinfection) and siderophores ofP. ,fluroscens (18%) and B. amyloliquqfaciens2O%(medium infection).kLeaves of control healthy plants showed lowest rate of electrolytic leackage(0.039 mslcma), while those of treated healthy plants showed slightly higher rate ofelectrolytic leakage i0.045-0.049ms!cm2). The electrolytic leakage was found to behighest in the leaves of control infected plants (0.375ms/cm~). Infected plantstreated with Fe(ll) showed higher electrolytic leakage values(0.3l lms/cm2), whileminimum value (0.063msIcm~) was recorded in those treated with siderophore-Fecomplex of P. ,Ruorcsc.cns.>. Leaves of control infected plants showed minimum quantity of chlorophyll a.chlorophyll b and total chlorophyll and carotenold. Maximum values in the totalchlorophyll pigments (82.54%) and carotenoid(74.11%) were recorded in theinfected plants sprayed with siderophore-Fe complex of P. .fluorrsccns. followed bythat of 6. am.v/oliquc/acirns(64.57%. 67.40%) and Fe(ll) (63.88%. 66.34).kControl healthy and treated healthy plants showed no cell wall lytic activity.Maximum activity of cell wall degrading enzymes (pectinolytic and cellulolytic)was observed in the leaves of control infected plants. followed by the infected plantstreated with Fe (11). Among the treated plants, infected ones sprayed with P.

fluorescens siderophore showed higher rate of inhibition of cell wall degradingenzymes.Higher carbohydrate content was observed in the leaves of plants sprayedwithP.,fluorescens-siderophore-Fe complex. The total carbohydrate content wasfound to be drastically reduced in the leaves of infected plants.Minimum protein content (27.76%) and amino acid (33.67%) was observedin the control infected plants compared to those of control healthy pIants(lOO%).Healthy plants treated with siderophore-Fe complex of P. ,fluorescens showedslightly higher protein content than that of control healthy plants. Higher amount ofprotein (79.88%) and amino acid content (86.03%) was found in the leaves ofinfected plants treated with siderophore-Fe complex of P .fluorescens.bMaximum reduction in amino nitrogen (62%) and nitrate reductase(NR)enzyme (59 Oh) was found in the leaves of control infected plants compared to thoseof controlhealthy plants(IOO%). Leaves of infected plants sprayed withsiderophore-Fe complex of P. ,fluorresccns showed maximum amino nitrogen(91.67%) and NR activity (87.66%).hThe proline content showed a sharp increase of about 3-times in the leaves ofwntrol infected plants compared to that of healthy(contro1 and treated) plants.Leaves of infected plants treated with Fe(ll) showed maximum(83.73%) prolinecontent. Those of infected plants treated with P. ,fluorescens siderophore-Fe complexshowed minimum value of proline (3.77%). followed by siderophore of B.am,yloliquefaciens (4.89%). This might be due to the induction of systemic acquiredresistance in the treated host plants against the infection caused by the pathogen.

kHigher amount of total phenol and 0.D phenol and in the control infectedplants was followed by the treated infected plants. This clearly indicates that the hostplant actively resists the pathogen by the production of phenols.kThe antioxidant enzymes viz. peroxidase, polyphenol oxidase, superoxidedismutase and catalase showed an enhanced activity in the leaves of control infectedplants and those treated with Fell). Other treated infected plants showed lowervalues than those of control infected plants but higher than that of the controlhealthy plants, Increase in the activity of antioxidant enzymes was might be due tothe siderophore, which stimulates the host plant defense system by induced SAR toresist the pathogen.). Control infected plants and infected plants treated with Fell) showed highestyield loss of about 74.92% and 63.73% respectively. Control healthy plants andtreated healthy plants (95.76%-98.06%) showed maximum yeld. Significantincrease in the 011 content was recorded in the infected plants treated withsiderophore-Fe complex of P. ,fluorescens (87.20%). followed by siderophore of B.am~loliqrrefacicns (74.89%).The enhancement of all the metabolic activities in the treated H, annuusultimately resulted in an increased yieldSeveral reports are available on the potential of siderophore of soil microbesto act as antifungal agents of phytopathogens because of their capacity to absorb ironfrom iron- starved environment. The present study brings out for the first time thetUi Foliw SPllLypotential of siderophore of P. ,/llrorcscens and 0. am.vloliquefaciensAto controlAirernaria helianthi by ~nhibiting its metabolic activities and by enhancing the SARin the host plant concomitantly. It is also interesting to observe that siderophore and

siderophore-Fe complex of P. .fluorescens and B. amyloliquefaciens sprayed on theleaves of healthy plants did not cause any adverse effect on them. On the contrary,treated healthy plants exhibited increase in the metabolic activities. Though Fe(ll)treatment on the infected plants showed higher chlorophyll content in the leaves andhigher amount of protein and carbohydrate in seeds, it increased thediseaseintensity (82.06%) and decreased the oil yield. This might be due to the fact thatFefll) treatment without siderophore neither showed antifungal activity nor inducedthe host plant defense system.Most effective inhibition of A. helianthi infecting H. annuus is brought out bythe foliar spray of siderophore and siderophore-Fe complex of P, fluorescens andB, am,vloliqueJbciens because of the following effects:Maximum inhibition of the activity of all the tested pectinolytic andcellulolytic enzymes produced by A. helianthi in vitro.Minimum electrolytic leakage in the host plant. H, annuusReduced disease intensity in the host plant, If, annuusIncrease in carbohydrate and protein metabolism in the host plant.The enhanced activities of antioxidant enzymes and phenolic compounds in theleaves of the host plant, H. annuusInduction of SAR in the host plant.Of siderophore and siderophore-Fe complex of two bacteria, siderophore-Fecomplex of P. ,fluorescens was found to be most efficient in controlling A.helianthi and also in maintaining the oil yield under pathogenesis to the level ofcontrol healthy plants. This was followed by siderophore-Fe wmplex of 8.

amyloliquefaciens, siderophore of P. ,fluorescens and B, amyl~liqu~faciens indescending order.The following facts emerge from the present study:I. Fe(1I) mixed with EDTA sprayed on the leaves of infected plants increasedthe photosynthetic activities but did not inhibit the gowth of pathogen leading tosevere disease intensity. Hence it is concluded that Fe(ll) without siderophore cannotbe used to control A, helianthi.2. The culture filtrates of Racillus strhtilis with or without Fe amendmentshowed minimum inhibition of conidial germination, mycelial growth and biomassof A, hclianthi in 1,itro. Sidcrophore and siderophore-Fe complex of Hacillus cereuswas found to be phyotoxic to the host plants as it exhibited lowest percentage ofgermination of seeds infected with A, hclianthi. Hence Bacillus subrilis and B.c.cvrrus could not be employed for the hiocontrol of A, hc~iiantlri.3. Siderophore of IJ.se~tdomonas fluorcsccns and Bacillus um~~loliquefaciensand their siderophorc-Fc complex sprayed on the leaves of healthy plants did notproduce any toxic effect.But they showed inhibition of gowth of pathogen andIncrease in the physiological processes such as photosynthesis, synthesis ofcarhohydrates and nitrogenous compounds, nitratc reductase act~vity and induceSAR in the host plants. Thc above resulted in the higher yield in the host plantsultimately.4. Of the above two spp P. ,fluore.sc~ns exihibited the best biocontrol effects.This might be duc to ~ts genetic constitution and synthesis of most efficientsiderophore which cnuld cause maximum inhibition of A. helianrhi.

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