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Scientific Papers, Series B, Horticulture, Vol. LVI, 2012ISSN Online 2286-1580, ISSN-L 2285-5653EFFICACY OF SOME BACTERIAL BIOPRODUCTS AGAINSTSCLEROTIUM CEPIVORUMSorina DINU 1 , Oana-Alina SICUIA 1 , Florica CONSTANTINESCU 1 , Ctlin LZUREANU 1 ,Florin OANCEA 1,21 Research and Development Institute for Plant Protection, 8 Ion Ionescu de la Brad Blvd., 013813Bucharest, Romania, tel. 004-021-2693231, 33, 34, 36, fax. 004-021-2693239, e-mails:sori.dinu@yahoo.com; sicuia_oana@yahoo.com; cflori@yahoo; catalinlazureanu@gmail.com2 The National Institute for Research and Development in Chemistry and Petrochemistry,202 Spl. Independentei 060021, Bucharest, Romania, tel. 021-315.32.99, fax: 021-312.34.93,e-mail: florino@ping.roCorresponding author email: cflori@yahoo.comAbstractThe aim of this paper was to determine the efficacy of some bioproducts based on useful bacteria from the genusBacillus and Pseudomonas against the phytopathogenic Sclerotium cepivorum fungus wich causes white rot in onioncrop.The selected bacterial strains for this study were: Bacillus subtilis Us.a2, isolated from rhizosphere of greengarlic, with deposited DSMZ number 23654; Pseudomonas chlororaphis Sal.c2, isolated from rhizosphere of lettuce;Bacillus pumilus OS.15 and Bacillus amyloliquefaciens OS.17 strains isolated from rhizosphere of onion and Bacillussubtilis 98a isolated from wheat straw. The five strains, besides other important biological features such as lithicenzyme production, mobility, action to stimulate plant growth, showed in vitro antagonistic ability against Sclerotiumcepivorum. In order to be tested in vivo, the strains were formulated as microemulsion (which can be applied as seedtreatment) and alginate beads (which can be distributed in rows along with the seeds). The tests were performed ingrowth chamber conditions by using artificially infected soil.Results showed a moderate or similar efficacy ofbioproducts compared with the chemical control Topsin 500 SC.Key words: bacterial bioproducts, biocontrol efficacy, Sclerotium cepivorumINTRODUCTIONBiological means of useful crop plantmicroorganisms is one of the directions forreducing the dependence of chemicalsproducts. Biopesticides are widely used infarming because of their advantages: (i) equalor higer efficacy than conventional productswhen used in integrated control systems; (ii)high applicability hence the use ofunqualified staff; (iii) are biodegradable,important feature for the groundwater andsurface; (iv) can be successfully used alone orin combination with some pesticides having asynergistic effect; (v) microbial biopesticidesare obtained by fermentation and often usedas conditioning substrate materials/agricultural bioproducts.In our country, the first bioproduct wasconducted by N. Zinca in 1970 based onavirulent Agrobacterium vitis bacteriadesigned to control the crown-gall of vines.Subsequently were obtained a series ofbioproducts for important soilborne pathogens(Pythium, Rhizoctonia, Fusarium) such as:Vamfix (based on spores of mycorrhizal Glomusfungi), Bacillin (based on Bacillus subtilisstrains), Trichosemin (Trichoderma viride), orbased on plant growth promoting rhizobacteriasuch as: Azostim (based on Azospirillumbacteria), Nitrostim (Bradyrhizobium), Nitrofix(Rhizobium).On the plant protection market in USA there areseveral bioproducts including bacteria and fungiof genera Agrobacterium, Bacillus,Pseudomonas, Streptomyces and Trichoderma,Candida and Coniothyrium. In the last years,there was a slight increase (2.5%) compared tothe chemicals products, the highest use (55%)being in the horticulture sector.The European Union is focused on developingorganic farming systems in order to reducenegative side effects that may occur from the useof chemical fertilizers. Some of these bioproducts81

are: Kodiak, Companion (Bacillus subtilis),Actinovate (Streptomyces lydicus),Mycostop (Streptomyces griseoviridis),PlantShield (Trichoderma harzianum).One of the most important soil phytopathogenis Sclerotium cepivorum Berkfungus, causing white rot of Allium species,being a major limitation factor of onion cropproduction and the incidence disease infavourable conditions being over 65% [2].The fungus penetrates the root epidermis andthen invades the cortical parenchyma [5] sothat, infected plants suffer from water stressand often die before harvesting or of rotting inwarehouses. The pathogen persists in soil inthe absence of host plants as sclerotia and cansurvive in this form more than 20 years [1].Sclerotia remain dormant in soil for 1-3months and can only germinate in thepresence of host plants. The stimulus forgermination is the exudates of alkyl-cysteinesulphoxideswhich are metabolized by the soilmicroflora to produce volatile thiols andsulphoxides that trigger dormant sclerotiagermination [1, 6]. Biological control of thedisease can also be done directly, by addingantagonists or indirectly by changingenvironmental conditions in favour ofselective antagonists [3]. Among antifungalmicroorganisms active against this pathogenare Trichoderma viride and Coniothyriumminitans that have proven an over 60%efficacy in reducing the onion bulbs attack.Also, the bacteria from the genus Bacillus andPseudomonas were found to have a stronglyantagonism against this pathogen [4].The aim of this work was to test the efficacyof some bioproducts based on Bacillus andPseudomonas useful bacteria formulated asmicroemulsion and as sodium alginate beadson the growth of Sclerotium cepivorum fungalphytopathogen, under controlled conditions.MATERIAL AND METHODMicroorganismsBacterial strains used in this study were:Bacillus subtilis Us.a2 isolated fromrhizosphere of green garlic, with depositnumber DSM 23654, Pseudomonaschlororaphis Sal.c2 isolated from rhizosphereof lettuce, Bacillus pumilus OS.15 and Bacillusamyloliquefaciens OS.17 strains isolated fromonion rhizosphere and Bacillus subtilis 98aisolated from wheat straw, identified bybiochemical and molecular tests (Biolog GENIII, ARDRA technique or sequencing analysis of16S rRNA gene). Bacteria were grown in LuriaBertani broth medium (LB) (bactotryptone – 10g;yeast extract – 5g; distilled water up to 1000 ml;pH adjusted to 7.4 with NaOH 5M or HCl 1Msolutions) with a starting inoculum of a 10%aqueous suspension of 1x10 8 cfu/ml, inErlenmeyer flasks on a rotary shaker (150rpm) at28°C for 48 h. Biomass was harvested bycentrifugation at 3000 rpm for 20 minutes.Fungal inoculum was the strain DSM 63024 ofSclerotium cepivorum which was previouslycultivated on PDA medium (decoction from 200gpotato, 20 g dextrose, 20 g agar-agar and 1000 mldistilled water, pH 7.0) which was inoculated inRoux plates on barley seeds substrate andincubated 20 days at 20°C. The fungal inoculumwas mixed with the soil (previously sterilized bygamma radiation) at a rate of 2% of soil weight(18 g / tray).In vitro selection of bacterial strains was carriedout by testing the antagonistic activity towardsdifferent phytopathogenic fungi: (Botrytiscinerea, Fusarium oxysporum f.sp. radicislycopersici ZUM 2407, Fusarium graminearumDSM 4527, Sclerotium bataticola, Sclerotiumcepivorum DSM 63024. Bacterial strains (24 hfresh culture) were inoculated on PDA mediumby streaking a straight line at 3cm distancetowards the calibrate mycelium disk (5mmdiameter). Inoculated Petri dishes were incubatedat 28°C and analyzed after 48-72 h regarding theinhibition zone (mm). The experiment wasrepeated three times.The bacterial strains conditioning procedurewas developed in two forms:1. microemulsion consisted in: biomass ofsporulated or nonsporulated bacteria as activeingredient – 10 g; sucrose as suspending agent –5 g; soprofor FL as anionic surface surfactant anddispersant agent – 9.6 g; emulsifier CL3 as nonionicsurface surfactant agent – 2.4 g ; PEG 400as antifreeze agent – 3.0 g; 5% carboxy-methyl-82

celullose solution as thickening agent – 5.0 g;sodium benzoate as stabilizer – 0.2 g; organicsolvent – methyl esters of fatty acids in canolaor sunflower oil – 30.0 g; distilled water – upto 100 ml.Preparation of organic phaseIn a conditioning recipient with stirring wereintroduced 30 g solvent, 9.6 g soprofor FLand 2.4 g emulsifier CL3. The mixture wasmaintained under stirring until obtaining ahomogenous solution. Resulted 42 g ofemulsifier solution.Preparation of the aqueous phaseIn a 250 ml conditioning gradually recipientwith stirring and dropping funnel 10 gbiomass, 5 g sucrose, 0.2 g sodium benzoate,3 g PEG, 5 g carboxy-methyl-celullose of 5%solution and about 80% of the amount ofwater needed (~ 27 ml) were placed. Themixture was homogenized under stirring for10 minutes.Preparation of microemulsionOver the aqueous phase the organic phase wasadded under stirring. After adding the organicphase, stirring continued for another 15minutes. 100 ml of bioproduct resulted ashomogenous microemulsion.2. Alginate beadsBacteria were cultured in LB liquid mediumin a rotary shaker (150 rpm) incubated at28°C for 48 h up to a final concentration of10 8 cfu/ml. Entrapment of bacteria within thealginate beads was carried out under sterileconditions in a laminar flow hood; 20 ml ofbacterial culture with 80 ml of 2% (w/v)sodium alginate was aseptically mixed. Themixture was homogenised by stirring gentlyfor 1 h at room temperature and then it wasadded drop wise with the aid of a 10-mlsterile syringe in CaCl 2 2% (w/v) solution.The resulting alginate beads (with 3mmdiameter) were maintained in the solution atroom temperature for 1 h in order to obtainregular solid beads. Afterwards the beadswere washed twice with 0.4% sterile NaCland then stored at 4±1°C up to use.Test of the antagonistic capacitypreservation was achieved in vitro on PDAmedium by inoculating phytopathogenicfungus (disk of 5 mm diameter, from a fiveolddays culture on PDA incubated at 22°C) andbioproducts in the same Petri dish (9 mmdiameter). After 5 days of incubation at 28°C,linear growth of tested pathogen was recorded.The percent of growth inhibition was calculatedusing the following formula:GC GT% IG 100GCwhere: IG = inhibition of growth; GC = growthin control and GT = growth in treatmentThe preservation of the biological qualities ofbioproducts was tested in vivo under controlledconditions on the efficacy against Sclerotiumcepivorum phytopathogen which attacks mostlythe seedlings, causing severe damages. Theexperiment was carried out in growth chamber onseeds of onion (Allium cepa) cultivarRIJNSBURGER, disinfected in a first stage with70% ethanol and in the second stage with 4%sodium hypochlorite solution. 30 seeds/variantwere planted in soil previously sterilized bygamma ionizing radiation, and artificiallyinfected with Sclerotium cepivorumphytopathogenic fungus at a rate of 18 ginoculum/tray. The alginate beads bioproductswere applied in rows along with the seeds (6beads / row, 18 beads / tray) and thoseconditioned as microemulsion were applied asseed treatment by immersion for 20 minutes.Chemical treatment was performed using soilTOPSIN ® 500 SC at a dose of 0.25% and rowsowing application (5 ml / row, 15 ml / tray).Experimental variants were: V1 – Pseudomonaschlororaphis Sal.c2 vs. Sclerotium cepivorum;V2 – Bacillus subtilis Us.a2 vs. S. cepivorum; V3– B. subtilis 98a vs. S. cepivorum; V4 – Bacilluspumilus OS.15 vs. S. cepivorum; V5 – Bacillusamyloliquefaciens OS.17 vs. S. cepivorum; V6 –Chemical control TOPSIN 500 SC vs. Sclerotiumcepivorum; V7 – Negative control withSclerotium cepivorum; V8 – Positive control –sterilized soil.After four weeks, the plants were analyzed interms of specific symptoms of disease likeyellowing and wilting of the leaves, particularlybrowning and growth of white fluffy mycelium atthe base of the stem. Roots that showed nocharacteristic symptom of the disease wereconsidered healthy (photo 4).83

Efficacy data were calculated according toAbbot’s formula and represent the mean ofthree replicates.RESULTS AND DISCUSSIONSSelection of the bacterial strainsTable 1 shows the activity of bacterial strainsin vitro their antagonistic activity on thegrowth of phytopathogenic mycelium fungistudied (observations at three days of incubationat 28°C for Botrytis cinerea, Fusariumgraminearum, Fusarium oxysporum f. sp. radicislycopersici and Sclerotium bataticola fungi, fivedays of incubation at 22°C for Sclerotiumcepivorum fungus respectively, according toManka & Manka method [7] (Photo 1).Table 1. In vitro antagonistic activity of tested bacterial strains against to different phytopathogenic fungi (3-5 days ofincubation at 22-28°C)FusariumBacterial strainsBotrytis Fusarium oxysporum f. Sclerotium Sclerotiumcinerea graminearum sp. radicis bataticola cepivorumlycopersiciBacillus pumilus OS.15 +++ ++++ +++ +++ +++Bacillus amyloliquefaciensOS.17++++ +++ ++++ ++++ +++Bacillus subtilis Us.a2 +++ +++ +++ +++ ++++Pseudomonas chlororaphisSal.c2+++ +++ +++ +++ ++++Bacillus subtilis 98a +++ +++ +++ +++ ++++Legend: ++++ = very strong inhibition of mycelium growth and formation of propagation organs; + + + = stronginhibition of mycelium growth; ++ = moderate inhibition of mycelium growthOS.15 OS.17 Us.a298aS.cSal.c2Photo 1. In vitro antagonism of bacterial strains vs. Sclerotium cepivorum (5 days of incubation at 22°C)The bacterial strains, tested in vitro regardingtheir antagonistic activity produced a series ofantifungal metabolites which inhibited thegrowth of the phytopathogens studied,causing large aria of inhibition (>5mmequivalent to ++++; >3mm equivalent to+++).The conditioning of the selected bacterial strainswas performed in order to obtain bioproducts(Photos 2), which were afterwards tested, both invitro and in vivo for establishing the biologicalcontrol agent qualities.84

a.Photo 2. Bacterial bioproducts: a. Microemulsionformulation, b. Alginate beads formulationVerification of preserving bio-antagonisticcapacityThe results presented in table 2, indicated thatboth types of bioproducts based on the bacterialstrains previously selected showed a stronginhibitor effect against Sclerotium cepivorumpathogen growth in vitro. Thus, bioproducts likealginate beads based on Bacillus pumilus OS.15and Bacillus subtilis 98a strains showed thegreatest percentage of inhibition (95%) to thegrowth of the pathogen followed by Bacillusamyloliquefaciens OS.17 bioproduct which hadan inhibitory effect on percentage of 87.5%(Photo 3).Microemulsion form of conditioned strains hasalso preserved the biological qualities ofantagonists, showing inhibitory effect on fungusgrowth with 85% (Bacillus subtilis 98a) and 70-75%, respectively (Photo 4).Table 2. In vitro antagonistic effect exerted by the bioproductson Sclerotium cepivorum growth (5 days of incubation at 22°C)Bioproducts vs Sclerotium cepivorumAlginate beads inhibition (%) Microemulsion inhibition (%)Bacillus pumilus OS.15 95 Bacillus pumilus OS.15 75Bacillus amyloliquefaciens OS.17 87.5 Bacillus amyloliquefaciens OS.17 75Bacillus subtilis Us.a2 75 Bacillus subtilis Us.a2 70Pseudomonas chlororaphis Sal.c2 75 Pseudomonas chlororaphis Sal.c2 70Bacillus subtilis 98a 95 Bacillus subtilis 98a 85bcadePhoto 3. In vitro antagonistic activity of alginate beads bioproducts againstSclerotium cepivorum pathogen (5 days incubation at 22°C): a – fungal control; b – Bacillus subtilis Us.a2;c – Bacillus amyloliquefaciens OS.17; d – Bacillus subtilis 98a; e - Bacillus pumilus OS.15)85

Sal.c2S.cepivorumOS.17Us.a298aOS.15Photo 4. In vitro antagonistic activity of microemulsion bioproducts vs. Sclerotium cepivorumpathogen (4 days of incubation at 22°C)In vivo preservation of the biologicalqualities of the bacterial bioproductsThe efficacy of the bioproducts againstSclerotium cepivorum in onion seedlings isrepresented graphically (figures 2, 3).Among the microemulsion bioproductsapplied as seed treatment the best efficacywas recorded in OS.15, 98a and OS.17treatment variants (80-90%) with similar orvery close values to the chemical control(90%). Also, Us.a2 and Sal.c2 bioproductsshowed a protective but moderate effect onseedlings (40-50%) compared to chemicalcontrol (figure 2).Administration of alginate beads showedreduced efficacy compared to the chemicalcontrol, mean values being recorded in OS.17,98a and Us.a2 treatment variants (50-60%).Disease characteristic symptoms were noticedonly in Salc2 treated variant which showed thelowest efficacy (10%) (figure 3).The differences between efficacy values recordedfor the two types of formulation of bioproductsmentioned above were significant because of theconditioning form. Thus, conditioning thebacterial strains as alginate beads allowed a slowrelease of microorganisms in soil, which led to aweaker colonization of seedling roots and,consequently, a lower protective effect fromchemical control.In the case of microemulsion, the bacteria wereapplied directly, as seed treatment causing abetter colonization of plant roots and significantprotection against Sclerotium cepivorum attack.Fig. 2. The efficacy of microemulsion bioproducts in controlling Sclerotium cepivorumfungus on onion crop (24°C, 70% humidity)86

Fig. 3. The efficacy of alginate beads bioproducts in controlling Sclerotium cepivorumfungus on onion crop (24°C, 70% humidity)abcdFig. 4. The influence of the biological treatments on onion seedlings in growth chamber conditions: a - Bacillusamyloliquefaciens OS.17; b – Chemical control TOPSIN 500 SC; c – positive control; d – Bacillus pumilus OS.15CONCLUSIONSFive bacterial strains with high antagonisticactivity in vitro against variousphytopathogenic fungi were selected andformulated as microemulsion and sodiumalginate beads.In vitro bioassay showed that our bacterialbioproducts preserved antagonistic capacityagainst Sclerotium cepivorum fungus.Conditioning methods used, allowed thedevelopment of high quality bioproductswhich showed the property to preserve verywell the biological qualities of themicroorganisms.In vivo results have shown the beneficialeffects of the microorganisms formulated andthe high efficacy in seedlings protectionagainst the Sclerotium cepivorum attack.REFERENCES[1] Coley-Smith, J.R. and King, J.E., 1969. Theproduction by species of Allium of alkyl sulphides andtheir effect on germination of sclerotia of Sclerotiumcepivorum. Berk. Ann. Appl. Biol. 64: 289-301.[2] Jesus Ricardo Sanchez Pale, Emma Zavaleta Mejia,Gustavo Mora Aguilera, Luis Perez Moreno, 2000.Viability of four mexican isolates of Sclerotiumcepivorum, Berk. Revista Mexicana de Fitopatologia,18( 002):103-110.[3] Leggett M., 1982. Potential for biological controlof onion white rot in the Fraser Valley, Proceedings of87

the 29 th annual meeting, Canadian Pest ManagementSociety; 12 – 15, July, 1982. Vancouver, BritishColumbia, (C.F. CAB abstracts 1984 – 1986).[4] Mahomed I. A. Ali, Ismail M.K. Ismail, Abdel -Aziz M.Salama, Salama A. Ouf, 1990. Effect of O-Cresol on Microbial Compozition in Soil andRhizosphere of Onion Plants. Cultivated in McleanK.L. and Stewart A., 2000. Infection sites ofSclerotium cepivorumon onion roots, New ZealandPlant Protection 53:118-121 presence of Sclerotiumcepivorum, Science, 3(1):5-21 (1411. 1991).[5] Mclean K.L. and Stewart A., 2000, Infection sitesof sclerotium cepivorumon onion roots. New ZealandPlant Protection 53:118-121.[6] Ozkan M, Dilek F.B., Yetis U., Ozcengiz G., 2003.Nutritional and cultural parameters influencingantidipteran delta-endotoxin production. Res.Microbiol.;154(1):49-53[7] Manka K, Manka M, 1992. A new method forevaluating interaction between soil inhabiting fungiand plant pathogens. New approaches in biologicalcontrol of soil-borne diseases. Buletin OILB/SORP.XV, 1: 73-75.88