Murillo Lobo Junior

Biochemical and metabolic profiles of Trichoderma strains isolated from common bean crops in the Brazilian Cerrado, and potential antagonism against Sclerotinia sclerotiorum

Some species of Trichoderma have successfully been used in the commercial biological control of fungal pathogens, e.g., Sclerotinia sclerotiorum, an economically important pathogen of common beans (Phaseolus vulgaris L.). The objectives of the present study were (1) to provide molecular characterization of Trichoderma strains isolated from the Brazilian Cerrado; (2) to assess the metabolic profile of each strain by means of Biolog FF Microplates; and (3) to evaluate the ability of each strain to antagonize S. sclerotiorum via the production of cell wall-degrading enzymes (CWDEs), volatile antibiotics, and dual-culture tests. Among 21 isolates, we identified 42.86% as Trichoderma asperellum, 33.33% as Trichoderma harzianum, 14.29% as Trichoderma tomentosum, 4.76% as Trichoderma koningiopsis, and 4.76% as Trichoderma erinaceum. Trichoderma asperellum showed the highest CWDE activity. However, no species secreted a specific group of CWDEs. Trichoderma asperellum 364/01, T. asperellum 483/02, and T. asperellum 356/02 exhibited high and medium specific activities for key enzymes in the mycoparasitic process, but a low capacity for antagonism. We observed no significant correlation between CWDE and antagonism, or between metabolic profile and antagonism. The diversity of Trichoderma species, and in particular of T. harzianum, was clearly reflected in their metabolic profiles. Our findings indicate that the selection of Trichoderma candidates for biological control should be based primarily on the environmental fitness of competitive isolates and the target pathogen.

Potencial antagônico de Trichoderma spp. originários de diferentes agroecossistemas contra Sclerotinia sclerotiorum e Fusarium solani

From 40 soil samples collected in the Rio Grande do Sul, Minas Gerais, Sao Paulo, Bahia, Goias and Tocantins states, Brazil, a collection of 230 monosporic isolates of Trichoderma spp. was established using TSM selective media. In order to select efficient isolates for biological control, dual culture tests were carried out on PDA media at 20 oC for Sclerotinia sclerotiorum and at 25 oC for Fusarium solani f.sp. phaseoli. From the whole collection, 50 isolates presented antagonism against F. solani and 111 isolates to S. sclerotiorum. The antagonism against both pathogens was found in only 10% of the isolates. Scanning electron microscopy assessments with seven in vitro selected isolates showed that not all promoted hyperparasitism on the pathogens, suggesting the existence of other mechanisms of antagonism, as antibiosis or competition.

Sistemas de cultivo, sucessões de culturas, densidade do solo e sobrevivência de patógenos de solo

The objective of this work was to evaluate the effect of cropping systems and crops cultivated previously to common beans (Phaseolus vulgaris L.) on soil density and soil populations of Rhizoctonia spp. and Fusarium spp. Previous crops included the following legumes: Cajanus cajan,Stylosanthes guianensis cv. Mineirao and Crotalaria spectabilis; and the following grasses: Pennisetum glaucum (cv. BN-2, millet), Sorghum bicolor (cv. BR 304), Panicum maximum cv. Mombaca, Brachiaria brizantha cv. Marandu and a consortium of corn (Zea mays) and B. brizantha. Previous crops were planted in Brazil summer seasons (wet) of 2002, 2003 e 2004, and bean crop (cv. BRS Valente) was planted in the correspondent subsequent winters (dry) of 2003, 2004 and 2005, irrigated by central pivot. Crop residues were incorporated to the soil, in conventional tillage, and kept on the surface, in no tillage management. In general, soil populations of Fusarium spp. and Rhizoctonia spp. were higher in no tillage cropping system. Higher populations of Rhizoctonia spp. were found in heavier soils. Legume crop residue increased soil populations of Rhizoctonia spp. and Fusarium spp. and, therefore, Leguminosae should be avoided as previous crops to common beans, in both cultivation systems. Generally, Gramineae previous crops are supressive to soil populations of Rhizoctonia spp. and Fusarium spp.The objective of this work was to evaluate the effect of cropping systems and crops cultivated previously to common beans (Phaseolus vulgaris L.) on soil density and soil populations of Rhizoctonia spp. and Fusarium spp. Previous crops included the following legumes: Cajanus cajan, Stylosanthes guianensis cv. Mineirao and Crotalaria spectabilis; and the following grasses: Pennisetum glaucum (cv. BN-2, millet), Sorghum bicolor (cv. BR 304), Panicum maximum cv. Mombaca, Brachiaria brizantha cv. Marandu and a consortium of corn (Zea mays) and B. brizantha. Previous crops were planted in Brazil summer seasons (wet) of 2002, 2003 e 2004, and bean crop (cv. BRS Valente) was planted in the correspondent subsequent winters (dry) of 2003, 2004 and 2005, irrigated by central pivot. Crop residues were incorporated to the soil, in conventional tillage, and kept on the surface, in no tillage management. In general, soil populations of Fusarium spp. and Rhizoctonia spp. were higher in no tillage cropping system. Higher populations of Rhizoctonia spp. were found in heavier soils. Legume crop residue increased soil populations of Rhizoctonia spp. and Fusarium spp. and, therefore, Leguminosae should be avoided as previous crops to common beans, in both cultivation systems. Generally, Gramineae previous crops are supressive to soil populations of Rhizoctonia spp. and Fusarium spp.

Controle de Fusarium oxysporum f.sp. phaseoli in vitro e em sementes, e promoção do crescimento inicial do feijoeiro comum por Trichoderma harzianum

Este trabalho objetivou avaliar seis isolados de Trichoderma harzianum no controle de Fusarium oxysporum f.sp. phaseoli em sementes, e seu efeito no crescimento inicial do feijoeiro comum. Os isolados foram inicialmente confrontados in vitro com o patogeno em testes de cultura pareada e antibiose a 25oC. Sementes sadias e artificialmente infectadas pelo patogeno foram microbiolizadas com 2 mL de suspensoes dos antagonistas (2,5 x 108 conidios mL-1) para cada 100 g de sementes, em rolo de papel germtest a 20 e 25°C. Os percentuais de incidencia do patogeno e de plântulas normais foram avaliados aos sete e nove dias, respectivamente. Em casa de vegetacao, os isolados foram aplicados a 5 x 109 conidios 500 g-1 de substrato autoclavado, com avaliacao do comprimento das raizes e parte aerea das plantas 11 dias apos o semeio (12 plantas tratamento-1). Todos os isolados apresentaram antagonismo in vitro contra o patogeno. Os isolados CEN202, CEN234, CEN238, CEN240 foram superiores a testemunha no controle de F. oxysporum em sementes, reduzindo entre 35 e 51% da incidencia do patogeno e proporcionando entre 73 e 81% de plântulas normais. O comprimento total das plantas com tratamento CEN239 (37,43 cm) foi superior aos demais, cujo comprimento variou entre 27,84 e 33,95 cm.

Biological control of Fusarium oxysporum f. sp. phaseoli by Trichoderma harzianum and its use for common bean seed treatment

Biological control of seed-borne pathogens has shown to enhance germination and physiological quality of seeds. The objectives of this study were to evaluate the in vitro antagonistic effect of five Trichoderma harzianum isolates (CEN287, CEN288, CEN289, CEN290, and CEN316) against Fusarium oxysporum f. sp. phaseoli (Foxy) and test its potential use in seed treatment. Initially, pathogen and antagonists were grown in paired cultures at 25oC, from which samples were assessed using scanning electron microscopy (SEM). Then, clean or Foxy-infected seeds were treated with conidial suspension of the antagonists. Percent of Foxy-infected seeds and normal seedlings were evaluated at seven and nine days of incubation, respectively. All but one Trichoderma isolate (CEN290) inhibited Foxy mycelial growth. SEM analysis revealed that only one Trichoderma isolate (CEN287) showed parasitic interaction with Foxy. Two isolates (CEN287 and CEN316) significantly reduced the Foxy incidence and enhanced seed germination, though less effective than the fungicide mixture (carboxin + thiram). A principal component analysis indicated the importance of volatile metabolites in reducing Foxy incidence on common bean seeds. CEN287 Trichoderma harzianum isolate formed a single group due to its increase in germination rate of Foxyinfected seeds.

Atributos do solo e produtividade do milho e do feijoeiro irrigado sob sistema integração lavoura-pecuária

The objective of this work was to evaluate changes in physical and chemical attributes of the soil, in the carrying capacity of pasture and on yield of corn and irrigated dry bean, in a crop-livestock system, under no-till. The field experiment was installed in an irrigated area with center pivot, at Santo Antonio de Goias, state of Goias, Brazil. The treatments, evaluated for three consecutive years, were: continuous pasture, annual succession of pasture/irrigated dry bean, annual succession of corn/pasture/irrigated dry bean and annual succession of corn/irrigated dry bean in the winter. Each plot, corresponding to a treatment, comprised an area of six hectares. The forage species used was Urochloa ruziziensis, grazed by halfbreed Gir x Holstein female cattle, with weight of approximately 450 kg. There was no significant change in the density and porosity of the soil under different treatments. Changes in the chemical attributes depended on the evaluated nutrients. All the successions with brachiaria grass increased the percentage of soil aggregates larger than 2 mm. The carrying capacity of pasture decreased as the number of grain crops increased in the annual succession. Productivity of dry bean was influenced by the annual succession involving pasture, which had no influence on corn yield.

Exogenous application of methyl jasmonate induces a defense response and resistance against Sclerotinia sclerotiorum in dry bean plants

Sclerotinia sclerotiorum (Lib.) de Bary is a necrotrophic fungal pathogen that causes a disease known as white mold, which is a major problem for dry bean (Phaseolus vulgaris L.) and other crops in many growing areas in Brazil. To investigate the role of methyl jasmonate (MeJA) in defending dry bean plants against S. sclerotiorum, we used suppression subtractive hybridization (SSH) of cDNA and identified genes that are differentially expressed during plant-pathogen interactions after treatment. Exogenous MeJA application enhanced resistance to the pathogen, and SSH analyses led to the identification of 94 unigenes, presumably involved in a variety of functions, which were classified into several functional categories, including metabolism, signal transduction, protein biogenesis and degradation, and cell defense and rescue. Using RT-qPCR, some unigenes were found to be differentially expressed in a time-dependent manner in dry bean plants during the interaction with S. sclerotiorum after MeJA treatment, including the pathogenesis-related protein PR3 (chitinase), PvCallose (callose synthase), PvNBS-LRR (NBS-LRR resistance-like protein), PvF-box (F-box family protein-like), and a polygalacturonase inhibitor protein (PGIP). Based on these expression data, the putative roles of differentially expressed genes were discussed in relation to the disease and MeJA resistance induction. Changes in the activity of the pathogenesis-related proteins β-1,3-glucanase, chitinase, phenylalanine ammonia-lyase, and peroxidase in plants after MeJA treatment and following inoculation of the pathogen were also investigated as molecular markers of induced resistance. Foliar application of MeJA induced partial resistance against S. sclerotiorum in plants as well as a consistent increase in pathogenesis-related protein activities. Our findings provide new insights into the physiological and molecular mechanisms of resistance induced by MeJA in the P. vulgaris-S. sclerotiorum pathosystem.

Epidemiologia da mela e produtividade do feijoeiro-comum tratado com fungicidas

Results of three years of field and greenhouse studies on the effects of the fungicides azoxystyrobin, carbendazin, mancozeb, tebuconazole, fentinhydroxide, pyraclostrobin, tryfloxistrobin + cyproconazole, tryfloxistrobin + propiconazol and chlorotalonil on the epidemiology of bean web blight and bean yields are reported. Field experiments were conducted during the rainy seasons of 2004/05, 2005/06 and 2006/07. Experimental design was a randomized complete block with four replicates. In 2004/05, fungicides were applied only once, 45 after planting (DAP); in 2005/06, there were two applications at 30 and 45 DAP, and in the 2006/07 field season, three applications were made, at 30, 45 and 60 DAP. Disease severity was estimated weekly, with aid of a 1 (healthy) to 9 (more than 90% diseased leaf area) disease scale. Disease progress rates were calculated following curve fitting to the logistic model. Results showed that one single fungicide application, 45 DAP, was inefficient for disease control (mean disease progress rate r = 0.2348). When number of applications increased, and started earlier (at 30 DAP), efficiency also improved for all active principles (mean r = 0.1988 and 0.1671 in 2005/06 and 2006/07, respectively). Overall, lowest disease severities were observed with fentinhydroxyde, tryfloxistrobin + propiconazole, tryfloxistrobin + cyproconazoleand. The protective and curative effects of all fungicides were assessed in the greenhouse, by applicationat pre- or post-inoculation, respectively. Efficiency was evaluated by measurement of lesion diameter. All fungicides presented curative and protective effects, whereas best results were observed in preventive application. A high negative correlation was found between web blight intensity and yield, and increase in grain yield with fungicide applications reached 304 % over control plots.

Acanthospermum australe is an alternate host of Thanatephorus cucumeris, the causal agent of web blight on dry beans

Acanthospermum australe, a frequently weed infesting bean crops in the State of Goias, Brazil, was found infected by T. cucumeris under natural field conditions. Plants were severely affected and may be an important reservoir of inoculum for infection of cultivated crops. Pathogenicity of T. cucumeris in both A. australe and Phaseolus vulgaris was confirmed by Koch´s postulates.

Comparison of inoculation methods for selecting common bean genotypes with physiological resistance to white mold

A bstractAlthough different methods of inoculation have been proposed to assess the reaction of common bean to white mold (WM) caused by Sclerotinia sclerotiorum, a thorough comparison among them is lacking. In this study, six approaches were tested to identify the most reproducible and efficient method for discriminating six common bean genotypes of carioca market class based on their resistance to white mold. These included: modified straw test (ST), cotton pad (CP), infected flower on intact plant (IFIP) or on detached leaf (IFDL), and mycelium disc on intact plant (MDIP) or on detached leaf (MDDL). All experiments were conducted in a greenhouse or laboratory in a completely randomized design with four replicates. Several statistics including coefficient of variation (CV), standard deviation, intraclass correlation coefficient (ICC), p value for Bartlett’s test for homoscedasticity and sensitivity ratio (SR) were used as criteria for discrimination. The Spearman’s correlation coefficient was used to test the association between the methods. Results showed ST as the most suitable for selecting WM-resistant genotypes, followed by the IFIP method.