Inoculum production, the effect of vegetable extractsand fungicides and soybean genotypes reaction to Sclerotinia sclerotiorum
Stem white rot, caused by S. sclerotiorum, has increased in fields cultivated withsoybean due to the cropping of highly susceptible species during winter and the use ofseeds contaminated with S. sclerotiorum. Studies involving inoculum production,alternative and chemical control, inoculation methodology and soybean genotypesresistance to Sclerotinia sclerotiorum consisted on the objectives of this work. Theexperiments were done in the Laboratório de Micologia e Proteção de Plantas ? LAMIPat the Universidade Federal de Uberlândia. The isolates used were obtained formsoybean plants from Jataí-GO and Indianópolis-MG. Culture media amended withcornmeal were the most promising for sclerotium production both for yield and for thenumber of sclerotia. Culture media containing common beans and sunflower were thebest ones. The number of sclerotia and yield decreased as the doses of cornmeal, bulgurwheat and cassava crumbs increased. The concentration of 20% yielded the greatestproduction of sclerotia for all three amendments. In relation to the alternative control,the results indicate that the greatest inhibition of mycelial growth was directly related tothe increase of Azadirachta indica doses. The interaction A. indica with Pongamiaglabra was significant, and the dose of 1/3 P. glabra was the most effective, with 65%inhibition. Of all vegetable extracts, the fruit of Piper aduncum was the most effectivefor reducing mycelial growth, with 43% inhibition. The results of "in vitro" chemicalcontrol demonstrated that the fungicides flutriafol, fluazinam, propiconazole,epoxiconazole + piraclostrobin, tebuconazole + trifloxistrobin, tebuconazole,ciproconazole + propiconazole, cyproconazole, fluquinconazole, tetraconazole,procymidone, iprodione, ciproconazole + trifloxistrobin, epoxiconazole, myclobutaniland difenoconazole inhibited more than 98% of the mycelial growth for both isolates. Inthe ?in vivo? trial differences were noted for the fungicide effect, applied eitherpreventively or as a curative. The fungicide iprodione best controlled the disease forboth types of application. The results of inoculation of different soybean growth stagesdemonstrated that the smallest percentages of disease severity were directlyproportional to plant age, and the best stage for inoculating the plants were V2 (leavesand stems) e V3 (leaves). The most consistent inoculation method was the permanentdisk, for detached organs and for the whole plant, resulting in a significant correlation.Only 19 soybean genotypes performed as resistant or moderately resistant by thedetached leaf method. When whole plants were inoculated, only two of these weremoderately resistant, while all others were moderately susceptible or susceptible,generating a non significant correlation. CHAPTER 2: Sclerotia are fundamental for the life cycle of Sclerotinia sclerotiorum because they arethe resting structures that form apothecia and, consequently, ascospores, and thehyphae. Studies involving this pathogen require inoculum availability; thus, theobjective of this study was to evaluate vegetable growth media and the concentrationsof cornmeal, cassava crumbs and bulgur wheat for sclerotium production. The vegetableculture media analyzed were: carrot, cabbage, soybean, cauliflower, common beans,sunflower, snap beans, potato, sweet potato and pumpkin, with or without theamendment of cornmeal. The concentrations of cornmeal, wheat and cassava crumbsamended on the common beans medium were 0, 5, 20, 35, 50, 65, 85 and 100%. Theculture media of both tests were moistened with distilled water. The Erlenmeyers flaskscontaining the media were sterilized at 120ºC, for 20 minutes. Twelve hours after themedia had cooled to room temperature, 5 6-mm diameter mycelial plugs wereinoculated into each flask. The flasks were incubated at 22 ± 3ºC and 12 hours lighting,for 30 days (vegetable based media) or 45 days (concentrations of cornmeal, wheat andcassava crumbs). The sclerotia were separated from the original media after theincubation period by washing in tap water on a 2 mm screen. The sclerotia were driedon paper towel for 48 hours at room temperature. Subsequently, the weight and numberof sclerotia were determined. Culture media yield was determined based on the sclerotiaweight. The culture media amended with cornmeal were more effective, regardless ofthe vegetable used as a base, for both yield and number of sclerotia, and within thisgroup, the media based on common beans and sunflower were the best ones. Sclerotiayield and number decreased as the concentrations of cornmeal, wheat and cassavacrumbs increased. The concentration of 20% yielded the greatest production ofsclerotia, and cornmeal and wheat were more effective at that. CHAPTER 3: The constant use of pesticides in agriculture has brought an interest for the search ofnatural products and production systems that cause less impact on man and theenvironment. Plants present a great diversity of substances in their composition and,oftentimes, these can present fungicide or fungistatic potential, and are being studied forthe synthesis of new fungicides as well as for the induction of plant resistance, or, still,to be used directly by the farmer in the form of plant extracts on the crops. Consideringthat S. sclerotiorum is one of the most important pathogens in soybeans and other crops,this study analyzed the effect of vegetable oils and water extracts on the fungusmycelial growth. Concentrations of 25, 50, 75 and 100 ppm of the active ingredientazadiractine of Azadirachta indica oil were studied in association with the doses of 0,1/3, 1/6, 1/8 and 1/10 of Pongamia glabra oil. The doses of P. glabra were obtained inrelation to the volume of A. indica oil used to obtain the concentrations 25, 50, 75 and100 ppm of A. indica. The trial with vegetable water extracts used the following plants:Peruvian pepper tree (Schinus molle L.), goatweed (Ageratum conyzoides L.), holy basil(Ocimum spp. L.), absinth wormwood (Artemisia absinthium L.), jamun (Syzygiumcumini (L.) Skeels), common rue (Ruta graveolens L.), cassava (Manihot esculentaCrantz), white cedar (Melia azedarach L.) e matico (Piper aduncum L.). The vegetableextracts were incorporated to PDA at the concentration of 30%. The plant partsanalyzed were the leaves, with the exception of the pepper, which, besides the leaves,the fruit was also tested. The control treatments were non amended PDA as a negativecontrol and the fungicide procimidone at 10 ppm active ingredient as a positive control.The experimental design was completely randomized, with three repetitions (test ofvegetable oils) and 5 repetitions (test of vegetable water extracts). The treatments wereadded after autoclaving the medium PDA, at low temperature. Subsequent to mediasolidification 6-mm diameter mycelial disks were inoculated on the center of the Petriplates and incubated at 22 ± 3ºC and 12 hours lighting for 48 hours. Evaluations started24 hours after inoculation and lasted for 48 hours, when the negative control treatmenthad taken the whole plate surface. Mycelial growth inhibition percentage wascalculated. The results indicated that greater mycelial growth inhibition was directlyproportional to increasing concentration of A. indica and P. glabra. The interactionbetween A. indica and P. glabra was significant and the dose of 1/3 P. glabra was themost effective, with 65% inhibition. No dosage inhibited 100% of the mycelial growthas the fungicide. The water extract of the fruit of Piper aduncum was the mostpromising for the reduction of mycelial growth, with 43% inhibition. CHAPTER 4: Chemical control of soybean stem white rot is done based on previous studies aboutwhite rot on common beans. This study evaluated fungicides of different chemicalgroups on S. sclerotiorum ?in vitro?, selecting them for ?in vivo? trials. The fungicidesflutriafol, fluazinam, propiconazole, epoxiconazole + piraclostrobin, tebuconazole +trifloxistrobin, tebuconazole, cyproconazole + propiconazole, cyproconazole,fluquinconazole, tetraconazole, procymidone, iprodione, cyproconazole +trifloxistrobin, epoxiconazole, vinclozolin, prothioconazole, azoxistrobin +cyproconazole, chlorothalonil + thiophanate-methyl, flutriafol + thiophanate-methyl,myclobutanil, difenoconazole, carbendazim, benomyl, carboxin + thiram, methylthiophanate, quintozene, pencycuron, chlorothalonil and azoxistrobin were evaluated forcontrolling mycelial growth of two S. sclerotiorum isolates, from Jataí-GO andIndianópolis-MG, in a completely randomized design, with three repetitions. After themedia containing 100 ppm active ingridient solidified, 6-mm diameter mycelial plugswere inoculated in the center of the Petri plates. The plates were incubated at 22 ± 3ºCand 12 hours lighting. Growth evaluations started 24 hours after inoculation and lastedfor 48 hours for the isolate from Jataí and 72 for the one from Indianópolis. Mycelialgrowth percent inhibition was calculated. The ?in vivo? trial was done only with theisolate from Jataí with inoculation on cultivar FMT Tabarana at the stage V3. Thefungicides used in preventive and curative applications were fluazinam, epoxiconazole+ piraclostrobin, tebuconazole + trifloxistrobin, tebuconazole, cyproconazole +propiconazole, procymidone, iprodione, vinclozolin, prothioconazole, azoxistrobin +cyproconazole and thiophanate methyl. The experimental design was completelyrandomized, with 4 repetitions. Evaluations were done 72 hours after inoculation at 22 ±3ºC and 12 hours lighting, using a diagrammatic scale made with the program Quant.?In vitro? results demonstrated that the fungicides flutriafol, fluazinam, propiconazole,epoxiconazole + piraclostrobin, tebuconazole + trifloxistrobin, tebuconazole,cyproconazole + propiconazole, cyproconazole, fluquinconazole, tetraconazole,procymidone, iprodione, cyproconazole + trifloxistrobin, epoxiconazole, vinclozolin,prothioconazole, azoxistrobin + cyproconazole, chlorothalonil + thiophanate methyl,flutriafol + thiophanate methyl, myclobutanil and difenoconazole inhibited more than98% of the mycelial growth of both isolates. There were significant differences on thefungicide effects in the ?in vivo? trial, applied both preventively and curatively. Thefungicide iprodione had the best performance on disease control for both modes ofaction. CHAPTER 5: The reaction of soybean genotypes to Sclerotinia sclerotiorum, as well as thephenological stages and inoculation methods for the selection of genotypes resistant tostem white rot has not been well determined. Therefore, this study determined the bestphonological stage and inoculation method for S. sclerotiroum in soybean genotypesand studied partial resistance to stem white rot. The phenological stages analyzed forinoculation were Vc, V2, V3, V4 and R1 of cultivars MG/BR-46 (Conquista) and M-Soy8200. The inoculation methods tested were a permanent PDA mycelial plug, whichremained on the plant until evaluation, a plug for 24 hours, and a ?touch? plug, whichwas placed on the leaf and immediately removed. Inoculations were done both ondetached leaves and stems, as well as on intact plants of both cultivars. Soybeangenotype resistance reaction was analyzed on 90 soybean genotypes inoculated with apermanent PDA plug on detached leaves placed on gerboxes. The genotypes that wereresistant or moderately resistant were re-evaluated with inoculations on the wholeplants. Evaluations were done 72 hours after incubation at 22 ± 3ºC and 12 hourslighting, based on a diagrammatic scale prepared with the program Quant. Theexperimental design was completely randomized, with 3 repetitions. The resultsindicated that the smallest disease severity was directly proportional to plant age, andthe ideal stage for inoculation were V2 (leaves and stems) e V3 (leaves). The permanentmycelial plug was the best inoculation method for S. sclerotiorum for both soybeancultivars, and detached organs as well as the whole plant, in a significant correlation.Only 19 soybean genotypes performed as resistant to moderately resistant by thedetached leaf inoculation test. However, when these genotypes were tested as intactplants, only 2 were moderately resistant, while all others were moderately susceptible tosusceptible. In this case, the correlation was not significant, possibly due to the numberof genotypes used (only two susceptible), for the evaluation of inoculation methods, and19 with a different reaction for resistance evaluation.
Advisor:Jonas Jäger Fernandes; Francisco Xavier Ribeiro do Vale; Murillo Lobo Júnior; Fernando César Juliatti
School:Universidade Federal de Uberlândia
Source Type:Master's Thesis
Keywords:S. sclerotiorum Glycine max Sclerotium production Inoculation methods Resistance Water extracts and vegetable oils Mycelial growth Fungicides Stem white rot Phenological stages Soybeanresistance
Date of Publication:03/07/2008