Efficacy of rue extract, sodium bicarbonate and fungicides at ... - Inea

58Il Tabacco, 9industries and have broad-spectrum antimicrobialactivity. Moreover, they have been tested forcontrolling powdery mildew on several plantspecies (1,6,9,10,16).The objective of this study is to evaluate theefficacy of rue extract, sodium bicarbonate andfive fungicides at low rates in greenhouse andfield trials to control E. orontii on tobacco plants.Materials and MethodsFigure 1 - Conidiophores and conidia of Erysipheorontii.Fig. 1. Conidiofori e conidi di Erysiphe orontii.cells of the conidiophores and the conidia chainsare curved and with crenate edges in E. orontii(Fig. 1), while they are erect with sinuate edgesin E. cichoracearum (Fig. 2). Plotnikova et al.,(1998) identified E. orontii as the agent ofpowdery mildew even on Arabidopsis thaliana,where E. cichoracearum and E. cruciferarum hadbeen previously reported. Erysiphe isolatesrecovered from Italian tobacco crops correspondto the E. orontii description.Powdery mildew has spread to most countrieswhere tobacco is cropped, often occurring as adisease of major importance. When resistantcultivars are not available, fungicide applicationsare necessary to avoid yield losses, neverthelessthe reduction of agrochemicals use is one of themajor objectives to be achieved in Europeantobacco growing areas. Natural products,inorganic salts and/or fungicides at lower ratesthan those used on other crops, may limit bothresidues on cured tobacco leaves and theenvironmental impact of the crop. Biologicallyactive natural products (allelochemicals)isolated from higher plants are now beingemployed as fungicides and in biocontrol ofplant diseases (8,14).Oliva et al. (1999) demonstrated significantin vitro fungistatic activity of rue (Rutagraveolens L.) extract against six fungal species.Bicarbonate salts are widely used in the foodFungal isolate and inoculation: Plants withsymptoms of powdery mildew were harvested ina farm located in a typical «burley» tobaccogrowing area (Caserta) of southern Italy. Conidiafrom those plants were collected, then inoculatedon 2 month old plants using a suspension adjustedto 10-20 conidia/ml with the aid of anhemacytometer in 0.1% sterile water agar (SWA)to allow the development of spare colonies (Fig.3). At theoutbreak ofsymptoms,conidia from asingle spotwere re-inoculatedon theleaves of healthyplants. Thisprocedure wasrepeated until20 plants werewell colonizedas a source ofinoculum forthe greenhousetest.Suspensionconcentrationused for testingactive ingredientswas 10 5conidia/ml and10 ml/plantwas appliedusing a labo-Figure 2 - Conidiophores andconidia of Erisiphae cichoracearum.Fig. 2. Conidiofori e conidi diErisiphae cichoracearum.ue22ing.p65 5825/03/2005, 17.22

Lahoz et al. Efficacia dell’estratto di ruta, bicarbonato e....... 59ratory sprayer (Fig. 3). The inoculation was made1 day after the first application of the rue extract,sodium bicarbonate and fungicides.Greenhouse testing of active ingredients. Twotrials (A and B), planned as random completeblock design (RCBD) with four replicates, werecarried out at Tobacco Experimental Institute in1997 and 1998. Plants of S2/95 cultivar, whichis highly susceptible to Erysiphe orontii, weretransplanted in 35 cm plastic pots (1 plant/pot)after 30 days from sowing; treatments wereapplied to all leaves on 10, 3-month-old, plantsof each replicate. Temperature in the greenhousewas 22 ± 2°C with continuous illumination toallow sporulation (7) and 75 ± 10% relativehumidity. Rue extract, sodium bicarbonate andfive fungicides (Penconazole 10EC;Tebuconazole 25 WG; Benomyl 50WP; Dinocap25WP; Bupirimate 25 EC) at low rate ofapplication were compared. All treatments wereapplied with a laboratory hand sprayer to run off.Rates, intervals and number of applications foreach treatment are reported in Table 1.Phytotoxicity was evaluated using anempirical scale with values ranging from 1 (nophytotoxicity) to 9 (plant death).Disease severity (DS) was assessed using twomethods: a) starting from the outbreak ofsymptoms, the number of sporulating mildewcolonies on 8 basal leaves for each of 10 plants/replicate was recorded every 8 days; b) the leafarea covered with powdery mildew symptomswas evaluated 8 days after the last application,then percentage of disease control was calculated.Field trialTwo field trials planned as completely randomizedblock design with 4 replicates were carried out in1997 and 1998. Plants of cultivar S2/95 weretransplanted at 20/5 and 15/5 respectively for thetwo trials. Applications of the fungicides (the sameforeseen in the greenhouse test) started from thefirst ten days of July. The influence of the treatmentson the technological characteristics and on theefficacy were evaluated. Disease severity wasevaluated using Coresta scale ranging from 0 (nosymptoms) to 5 (> 50 % of leaf surface covered bythe fungus).Figure 3 – Leaves of inoculated tobacco plantsshowing spare colonies (top) and leaves inoculatedwith a higher concentration of conidia of Erysipheorontii.Fig. 3. Foglie di tabacco inoculate mostranti rare colonie(in alto) e foglie inoculate con elevate concentrazionidi conidi di Erysiphe orontii.Rue extract preparation. Fresh leaves of R.graveolens were collected from plants grown atthe Tobacco Research Institute in Scafati, Italy.250 g of fresh leaf material were soaked in 1 literdeionized water at 90 °C for 10 min, followedby filtration through Whatman No. 1 filter paper(17). The filtrate was lyophilized, yielding 10 g/l of powdered extract.Microscopic examination of conidia. Fourplants were inoculated with 10 ml (10 5 conidia/ml) of E. orontii conidial suspension, two of themwere then treated with rue extract and two withsterile distilled water (SDW). 24 h after theue22ing.p65 5925/03/2005, 17.22

Lahoz et al. Efficacia dell’estratto di ruta, bicarbonato e....... 61Figure 4 – Leaves of untreated control plants at theend of the trial showing severe powdery mildewsymptoms.Fig. 4.Foglie del controllo non trattato alla fine dellaprova con gravi sintomi di oidio.ResultsThe colonization of the untreated tobacco leaveswas complete 30 days after the inoculation, lateron spots became necrotic and basal leaves died.Inoculation methods using 10 5 conidia /mlsuspended in 0.1% SWA showed constant andreproducible results.Greenhouse testing of active ingredients. Nophytotoxicity was observed on plants for anycompound tested in the trials (Table 2).Rue extract was very effective forcontrolling powdery mildew in both trials.Sodium bicarbonate at 500 g/hl reduced diseaseover control, even if, in trial A, more than 9spots/leaf were observed, 8 days after the lastapplication. Penconazole, tebuconazole andbenomyl completely controlled diseasedevelopment (Table 2). On untreated controlplants were counted over 24 and 20 coloniesper leaf respectively in trial A and B,equivalent of about 60% damaged leaf surface(Fig.4), indicating that the disease pressure washigh.An increased disease severity was observedafter repeated applications of sodiumbicarbonate at 500 g/hl, while its efficacy washigher at 1000 g/hl, and a most satisfactorycontrol was reached after three applications.On benomyl treated plants, less than 1sporulating spot per leaf was observed, startingfrom the second applications. Bupirimatereduced disease severity to a level equivalentto that attained with rue extract, requiring twoapplications to obtain its maximumeffectiveness. A trend similar to sodiumbicarbonate at 1000 g/hl was observed fordinocap application (Fig. 5).Both trials gave similar results on percentof disease control. In trial B sodiumbicarbonate at 1000 g/hl, dinocap, bupirimateand rue extract showed statistically equivalentresults. In both trials the efficacy of sodiumbicarbonate at 500 g/hl was below 70%. Rueextract application reduced disease to a levelstatistically not different from bupirimate (Fig. 6).Table 3 – Disease severity as CORESTA index in thefield trials.Tab. 3. Gravità della malattia secondo gli indiciCORESTA per le prove in campo.ActiveingredientsratesDisease severityg/hl19971998Penconazole3.750 0Tebuconazole100 0Benomyl250 0Bupirimate37.5 0 0Rueextract 1000 0NaHCO10000 03Dinocap17.5 1 1NaHCO5000.5 0. 53C ontrol/ 2 2ue22ing.p65 6125/03/2005, 17.22

62Il Tabacco, 9Figure 5 – Dynamics of disease severity for the different active ingredients tested in greenhouse.Fig. 5. Dinamica della malattia per le differenti materie attive saggiate nelle prove di serra.Table 4 – Main technological characteristics of upper middle leaves.Tab. 4. Principali caratteristiche tecnologiche delle foglie mediane superiori.ActiveingredientsNa bicarbonate500 g/hlNa bicarbonate1000 g/hlyieldt d.m.*/haFillingpowercc/gBurningratesec.TotalAlcaloids% d.m.N tot.Chlorid easNH3 % d.m.% d.m.Ashes% d.m.Alcalinityof ashes% d.m.0,828,7 7 0,625,273,4422,8 7, 30,848,4 8 0,585,123,5323,9 8, 3Rueextract 0.858.3 8 0.615.203.6024.0 6. 3Penconazole0.878.2 8 0.645.233.5823.6 6. 4Tebuconazole0.868.2 8 0.665.273.6023.4 6. 3Dinocap0,878,1 8 0,615,263,4622,4 5, 8Bupirimate0,908,1 7 0,634,903,4624,2 6, 2Untreatedcontrol* d.m. = dry matter0,878,5 8 0,665,103,6624,1 5, 7ue22ing.p65 6225/03/2005, 17.22

Lahoz et al. Efficacia dell’estratto di ruta, bicarbonato e....... 63Figure 6 - Efficacy of rue extract, sodium bicarbonate and fungicides at reduced rates on Erysiphe orontii ontobacco.Fig. 6. Efficacia dell’estratto di ruta e del bicarbonato di sodio e di fungicidi a dosi ridotte su Erysipheorontii su tabacco.Table 5 – Main technological characteristics of middle leaves.Tab. 5. Principali caratteristiche tecnologiche delle foglie mediane.ActiveingredientsNa bicarbonate500 g/hlNa bicarbonate1000 g/hlyieldt d.m.*/haFillingpowercc/gBurningratesec.TotalAlcaloids% d.m.N tot.Chlorid easNH3 % d.m.% d.m.Ashes% d.m.Alcalinityof ashes% d.m.1,528,0 100,965,983,1718,7 6, 61,468,1 9 0,966,153,5721,1 7, 6Rueextract 1.457.3 8 0.985.9 3.5621.0 6. 3Penconazole1.507.6 8 1.015.963.4920.9 6. 4Tebuconazole1.487.6 8 0.985.963.5121.0 6. 3Dinocap1,487,6 8 1,045,933,4420,4 6, 1Bupirimate1,457,7 8 1,065,653,4921,3 5, 1Untreatedcontrol* d.m. = dry matter1,298,3 9 0,995,824,3321,9 4, 6ue22ing.p65 6325/03/2005, 17.22

Lahoz et al. Efficacia dell’estratto di ruta, bicarbonato e....... 65Table 6 - Effects of rue extract on the germinationand shape of conidia of Erysiphe orontii.Tab. 6. Effetto dell’estratto di ruta sulla germinazionee la forma di conidi di Erysiphe orontii.TreatmentsObservedconidiatarget spot and brown spot, avoiding the use offungicides.Rue extract and sodium bicarbonateapplications did not affect the technologicalproperties of cured tobacco leaves.Fungicide application at about half of thenormal rates suggests that there can be a reductionand optimization of active ingredients use intobacco cultivation. Penconazole, tebuconazoleand bupirimate can effectively control powderymildew at reduced doses.Dinocap needs a higher dose than that usedin the trial to increase its efficacy up to 80 %.A combined program of bicarbonate, naturalcompounds and fungicides at low rates shouldreduce the cost, may lessen the onset of fungicideresistance and limit both residues andenvironmental impact of culture.Further investigations on the mechanism of actionof rue extract and its possible combineduse with bicarbonate, are in progress.Literature CitedDistortedconidiaGerminatedconidiaControl1582835 wwRueextract 1961335wSignificant different p=0.011. Agapov, V., Reuveni, M., and Reuveni, R. 1993.Eradication of cucumber powdery mildew by posphateand potassium salts. (Abstr.) Int. Cong. Plant Path. 6 th214.2. Aliotta, G., Cafiero, G. Fiorentino and Strumia, S. 1993.Inhibition of radish germination and root growth bycoumarin and phenylpropanoids. J. Chem. Ecol. 19(2):175-183.3. Aliotta, G., Cafiero, G., De Feo, V., & Sacchi, R. 1994.Potential allelochemicals from Ruta graveolens L. andtheir action on radish seeds. J. Chem. Ecol. 20 (11):2761-2775.4. Aliotta, G., Cafiero, G., De Feo, V., Palumbo, M. D. andStrumia, S. 1996. Infusion of rue for control ofpurslane weed: Biological and chemical aspects.Allelopathy J. 3 (2): 207-2115. Braun, U., 1987. A monograph of the Erysiphales(powdery mildew). J. Cramer, Berlin, Stuttgart, 700 pp.6. Brunelli, A., Flori, P., Collina, M., Coatti, M., andCobelli, L. 1996. Attività di prodotti vegetali e mineralicontro l’oidio delle cucurbitacee (Sphaerothecafuliginea). Pr. Giornate Fitopat. 2:465-472.7. Cole, J.S. 1978. Powdery Mildew of Tobacco. In:Spencer, D.M. The Powdery Mildews. Acad. Press.447-457.8. Einhellig, F. A. 1984. Allelopathy – Natural protection,allelochemicals, in H. B. Mandaven (ed.). Handbook ofNatural Pesticides. I. Theory, Practice, and Detection,CRC Press, Florida, pp. 161-2009. Homma, Y., Arimoto, Y., and Misato, T. 1981. Effect ofsodium bicarbonate on each growth stage of cucumberpowdery mildew fungus (Sphaerotheca fuliginea) in itslife cycle. J. Pestic. Sci. 6:201-209.10. Horst, R. K., Kawamoto, S. O., and Porter, L.L. 1992.Effect of sodium bicarbonate and oils on the control ofpowdery mildew and black spot of roses. Plant Dis.76:247-251.11. Oliva, A., Lahoz, E., Contillo, R., and Aliotta, G. 1999.Fungistatic acticity of Ruta graveolens L. extract andits allelochemicals. J. Chem. Ecol. 25,(3):519-52612. Pizziol, P., Nicoletti, R., and Lahoz E. 1994. L’oidio deltabacco. Il Tabacco 2:31-37.13. Plotnikova, J. M., Reuber, T.L., and Ausubel, F. M.1998. Powdery mildew pathogenesis of Arabidopsisthaliana. Mycologia 90:1009-1016.14. Rice, E. L. 1985. Chemically mediated interactionsbetween plants and other organisms, in G. A. Cooper-Driver, T. Swain and E. E. Conn (eds.). Allelopathy –an overview, Plenum Press, New York, pp 81-105.15. Scopfer, P., and Plachy, C. 1993. Photoinhibition ofradish (Raphanus sativus L.) seed germination: Controlof growth potential by cell-wall yielding in theembryo. Plant, Cell Environ. 16: 223-229.16. Ziv, O., Zitter, A. 1992. Effects of bicarbonate and filmformingpolymers on cucurbit foliar diseases. PlantDis. 76:513-517.17. Zobel, A. M., and Brown, S. A. 1988. Determination offuranocoumarins on the leaf surface of Ruta graveolenswith an improved extraction technique. J. Nat. Prod.5:941-946.ue22ing.p65 6525/03/2005, 17.22