Trichoderma spp no biocontrole de cylindrocladium candelabrum em mudas de eucalyptus saligna

Este trabalho teve como objetivo testar os efeitos in vitro e in vivo de bioprotetores à base de Trichoderma spp. no controle do fungo Cylindrocladium candelabrum Viegas. Os testes in vitro (confronto direto e inoculação em folhas destacadas) foram compostos pelos seguintes tratamentos: T1 - somente C. candelabrum; T2 - isolado 06006S x C. candelabrum; T3 - isolado 53RR x C. candelabrum; T4 - isolado 5D x C. candelabrum; T5 - Agrotrich® x C. candelabrum; e T6 - Trichodel® x C. candelabrum. Todos os tratamentos foram eficientes inibindo o crescimento do fungo C. candelabrum em confrontação direta, e os isolados de Trichoderma spp. 53RR e 06006S, bem como o produto comercial Trichodel®, controlaram a mancha-foliar em folhas destacadas. Para complementar os testes in vitro, os produtos comerciais Agrotrich® e Trichodel® foram testados em mudas de E. saligna cultivadas em casa de vegetação, com os seguintes tratamentos: T1 - Testemunha: sem inoculação; T2 - inoculação de C. candelabrum; T3 - inoculação de C. candelabrum x Agrotrich®; T4 - inoculação de C. candelabrum x Trichodel®; T5 - somente Agrotrich®; e T6 - somente Trichodel®. Este produto apresentou os melhores resultados na redução dos danos causados pelo patógeno em mudas de E. saligna.

Antagonismo de Trichoderma SPP. E Bacillus subtilis (UFV3918) a Fusarium sambucinum em Pinus elliottii engelm

Pinus elliottii é uma espécie de importância no setor florestal e apresenta vulnerabilidade na qualidade sanitária de suas sementes, especialmente pela associação de Fusarium spp., responsável por perdas de plântulas no viveiro. Este trabalho teve como objetivo avaliar a ação antagonista in vitro e in vivo dos agentes Trichoderma spp. e Bacillus subtilis (UFV3918) no controle de Fusarium sambucinum, responsável por danos em plântulas de Pinus elliottii. O controle in vitro foi avaliado através da inibição do crescimento micelial (confronto pareado de culturas), após a incubação a 25±2 ºC e fotoperíodo de 12 h. Para os testes in vivo (desenvolvidos em condições de viveiro), as sementes inicialmente foram inoculadas com o patógeno e, na sequência, microbiolizadas com os agentes antagônicos, para posterior semeadura. Utilizaram-se as técnicas de contato com o biocontrolador em meio BDA por 48 h e peliculização, como formas de microbiolização. Tanto Trichoderma spp. quanto Bacillus subtilis (UFV3918) foram eficientes no controle in vitro de F. sambucinum, e no teste de biocontrole in vivo o produto Bacillus subtilis (UFV3918) destacou-se, reduzindo as perdas de plântulas causadas pelo patógeno, assim como potencializando as variáveis de comprimento de plântula, massa verde e massa seca.

Trichoderma spp. NA EMERGÊNCIA E CRESCIMENTO DE MUDAS DE CAMBARÁ (Gochnatia polymorpha (Less.) Cabrera)1

Neste trabalho, o objetivo foi avaliar o efeito de isolados de Trichoderma spp. na emergência de plântulas e no crescimento de mudas de cambará (Gochnatia polymorpha). Utilizou-se, em casa de vegetação, substrato esterilizado e não esterilizado, sendo avaliados os efeitos de quatro isolados de trichoderma: TSM1 e TSM2 de Trichoderma viride, 2B2 e 2B22 de Trichoderma harzianum mais um mix preparado com a mistura desses quatro isolados, além de dois produtos comerciais à base de trichoderma. A análise dos dados permitiu concluir que os isolados de trichoderma testados não interferem na emergência das plântulas, mas os isolados 2B2 e 2B22 de T. harzianum apresentam potenciais como promotores de crescimento de mudas de cambará.

Impacto de herbicidas em isolados de Trichoderma spp.

O uso de microrganismos é uma alternativa para o controle de doenças em plantas. Todavia, é prudente verificar a interação desse com os demais métodos de controle empregados em determinada cultura. Dessa forma, objetivou-se avaliar a fungitoxicidade dos herbicidas sobre o crescimento e desenvolvimento dos isolados de Trichoderma spp. Utilizou-se o delineamento inteiramente casualizado, em esquema fatorial 6 x 6 x 4, com quatro repetições. O fator A correspondeu aos herbicidas pendimethalin, clomazone, carfentrazone-ethyl, oxadiazon, thiobencarb + propanil e byspiribac-sodium; o fator B, às doses dos herbicidas - 0, 25, 50, 75, 100 e 200% da dose recomendada; e o fator C, aos isolados de Trichoderma spp. AJAM 18, CE 66, TRI 01 e TRI 02. O ensaio foi realizado em condições in vitro; avaliaram-se o crescimento micelial radial (CMR) e a esporulação dos isolados após aplicação dos herbicidas. Observaram-se diferenças de sensibilidade dos isolados para o mesmo produto testado. O oxadiazon reduziu o CMR dos isolados AJAM 18 e TRI 01 em 66 e 35%, respectivamente. No entanto, reduziu apenas 16% do CMR do isolado TRI 02 e não alterou o CMR do isolado CE 66 mesmo em 200% da dose recomendada. Verificaram-se diferentes efeitos dos produtos em cada isolado. A mistura comercial de thiobencarb+propanil foi altamente tóxica aos isolados de Trichoderma spp., com reduções em torno de 85% no CMR e no número de esporos. Por outro lado, o byspiribac-sodium pouco afetou os isolados, apresentando reduções inferiores a 10% no CMR e na esporulação. O carfentrazone-ethyl e byspiribac-sodium demonstraram ser compatíveis com os isolados de Trichoderma spp. estudados.

Atividade da celulase de fungos isolados do solo da Estação Ecológica de Juréia-Itatins, São Paulo, Brasil

Oitenta fungos filamentosos isolados do solo da Mata Atlântica da região conhecida como Banhado Grande, Estação Ecológica de Juréia-Itatins, São Paulo, Brasil, foram analisados para avaliar seus potenciais quanto a produção de celulases em resposta à presença de celulose, como única fonte de carbono, em meio de cultura. Foi utilizada a técnica de coloração com vermelho congo e determinada a atividades da celulase em papel de filtro (FPase) e em carboximetilcelulose (CMCase). Os fungos foram diferenciados quanto à atividade dessas enzimas, pois tais atividades variaram em relação ao tipo de substrato e à metodologia aqui utilizados. A melhor atividade CMCase (1,64 U) foi obtida com o cultivo de Trichoderma harzianum (V) em meio de farelo de trigo após cultivo por 4 dias, a 25 ºC. Os resultados obtidos não forneceram evidências para diferenciar qualquer linhagem que tivesse melhor atividade da celulase em relação às demais. Contudo, sugerem que estudos mais detalhados com as linhagens de Trichoderma: T. harzianum III e V, T. inhamatum I, T. longibrachiatum, T. pseudokoningii II e T. viride I, serão necessários para avaliar se estas são potencialmente boas produtoras de celulase, sob condições adequadas de cultivo.

Hydrolysis of xylans by enzyme systems from solid cultures of Trichoderma harzianum strains

Xylanase activity was isolated from crude extracts of Trichoderma harzianum strains C and 4 grown at 28oC in a solid medium containing wheat bran as the carbon source. Enzyme activity was demonstrable in the permeate after ultrafiltration of the crude extracts using an Amicon system. The hydrolysis patterns of different xylans and paper pulps by xylanase activity ranged from xylose, xylobiose and xylotriose to higher xylooligosaccharides. A purified ß-xylosidase from the Trichoderma harzianum strain released xylose, xylobiose and xylotriose from seaweed, deacetylated, oat spelt and birchwood xylans. The purified enzyme was not active against acetylated xylan and catalyzed the hydrolysis of xylooligosaccharides, including xylotriose, xylotetraose and xylopentaose. However, the enzyme was not able to degrade xylohexaose. Xylanase pretreatment was effective for hardwood kraft pulp bleaching. Hardwood kraft pulp bleached in the XEOP sequence had its kappa number reduced from 13.2 to 8.9 and a viscosity of 20.45 cp. The efficiency of delignification was 33%.

Characterization of the chitinolytic system during the mycoparasitic interaction between Trichoderma aggressivum f. aggressivum and different host strains of Agaricus bisporus /

Green mould is a serious disease of commercially grown mushrooms, the causal agent being attributed to the filamentous soil fungus Triclzodenna aggressivum f. aggressivu11l and T. aggressivum f. ellropaellm. Found worldwide, and capable of devastating crops, this disease has caused millions of dollars in lost revenue within the mushroom industry. One mechanism used by TricllOdenlla spp. in the antagonism of other fungi, is the secretion of lytic enzymes such as chitinases, which actively degrade a host's cell wall. Therefore, the intent of this study was to examine the production of chitinase enzymes during the host-parasite interaction of Agaricus bisporus (commercial mushroom) and Triclzodemza aggressivum, focusing specifically on chitinase involvement in the differential resistance of white, off-white, and brown commercial mushroom strains. Chitinases isolated from cultures of A. bisporus and T. aggressivu11l grown together and separately, were identified following native PAGE, and analysis of fluorescence based on specific enzymatic cleavage of 4-methylumbelliferyl glucoside substrates. Results indicate that the interaction between T. aggressivulll and A. bisporus involves a complex enzyme battle. It was determined that T. aggressivum produces a number of chitinases that appear to correlate to those isolated in previous studies using biocontrol strains of T. Izarziallilm. A 122 kDa N-acetylglucosaminidase of T. aggressivu11l revealed the highest and most variable activity, and is therefore believed to be an important predictor of antifungal activity. Furthermore, results indicate that brown strain resistance of mushrooms may be related to high levels of a 96 kDa N-acetylglucosaminidase, which showed elevated activity in both solitary and dual cultures with T. aggressivum. Overall, each host-parasite combination produced unique enzyme profiles, with the majority of the differences seen between day 0 and day 6 for the extracellular chitinases. Therefore, it was concluded that the antagonistic behaviour of T. aggressivli1ll does not involve a typical response, always producing the same types and levels of enzymes, but that mycoparasitism, specifically in the form of chitinase production, may be induced and regulated based on the host presented.

Cell wall degrading enzymes and interaction between Trichoderma Aggressivum and Agaricus Bisporus

Agaricus bisporus is the most commonly cultivated mushroom in North America and has a great economic value. Green mould is a serious disease of A. bisporus and causes major reductions in mushroom crop production. The causative agent of green mould disease in North America was identified as Trichoderma aggressivum f. aggressivum. Variations in the disease resistance have been shown in the different commercial mushroom strains. The purpose of this study is to continue investigations of the interactions between T. aggressivum and A. bisporus during the development of green mould disease. The main focus of the research was to study the roles of cell wall degrading enzymes in green mould disease resistance and pathogenesis. First, we tried to isolate and sequence the N-acetylglucosaminidase from A. bisporus to understand the defensive mechanism of mushroom against the disease. However, the lack of genomic and proteomic information of A. bisporus limited our efforts. Next, T. aggressivum cell wall degrading enzymes that are thought to attack Agaricus and mediate the disease development were examined. The three cell wall degrading enzymes genes, encoding endochitinase (ech42), glucanase (fJ-1,3 glucanase) and protease (prb 1), were isolated and sequenced from T. aggressivum f. aggressivum. The sequence data showed significant homology with the corresponding genes from other fungi including Trichoderma species. The transcription levels of the three T. aggressivum cell wall degrading enzymes were studied during the in vitro co-cultivation with A. bisporus using R T -qPCR. The transcription levels of the three genes were significantly upregulated compared to the solitary culture levels but were upregulated to a lesser extent in co-cultivation with a resistant strain of A. bisporus than with a sensitive strain. An Agrobacterium tumefaciens transformation system was developed for T. aggressivum and was used to transform three silencing plasmids to construct three new T. aggressivum phenotypes, each with a silenced cell wall degrading enzyme. The silencing efficiency was determined by RT-qPCR during the individual in vitro cocultivation of each of the new phenotypes with A. bisporus. The results showed that the expression of the three enzymes was significantly decreased during the in vitro cocultivation when compared with the wild type. The phenotypes were co-cultivated with A. bisporus on compost with monitoring the green mould disease progression. The data indicated that prbi and ech42 genes is more important in disease progression than the p- 1,3 glucanase gene. Finally, the present study emphasises the role of the three cell wall degrading enzymes in green mould disease infection and may provide a promising tool for disease management.

Defence of Agaricus bisporus against toxic secondary metabolites from Trichoderma aggressivum.

Trichoderma spp are effective competitors against other fungi because they are mycoparasitic and produce hydrolytic enzymes and secondary metabolites that inhibit the growth of their competitors. Inhibitory compounds produced by Trichoderma aggressivum, the causative agent of green mold disease, are more toxic to the hybrid off-white strains of Agaricus bisporus than the commercial brown strains, consistent with the commercial brown strain’s increased resistance to the disease. This project looked at the response of hybrid off-white and commercial brown strains of A. bisporus to the presence of T. aggressivum metabolites with regard to three A. bisporus genes: laccase 1, laccase 2, and manganese peroxidase. The addition of T. aggressivum toxic metabolites had no significant effect on MnP or lcc1 transcript abundance. Alternatively, laccase 2 appears to be involved in resistance to T. aggressivum because the presence of T. aggressivum metabolites results in higher lcc2 transcript abundance and laccase activity, especially in the commercial brown strain. The difference in laccase expression and activity between A. bisporus strains was not a result of regulatory or coding sequence differences. Alteration of laccase transcription by RNAi resulted in transformants with variable levels of laccase transcript abundance. Transformants with a low number of lcc transcripts were very sensitive to T. aggressivum toxins, while those with a high number of lcc transcripts had increased resistance. These results indicated that laccase activity, in particular that encoded by lcc2, serves as a defense response of A. bisporus to T. aggressivum toxins and contributes to green mold disease resistance in commercial brown strains.

Role of temperature, moisture and Trichoderma species on the survival of Fusarium oxysporum ciceri in the rainfed areas of Pakistan

It has been observed in the present study that when spores of Trichoderma harzianum (Th-2) isolate were applied in the sandy clay loam soil and continuously incubated for 4 months at 25 degrees C and 35 degrees C and at three water potentials, -0.03 MPa, -0.3 MPa and <-50 MPa, it has resulted in significantly reduced (P<0.05), growth of Fusarium oxysporum ciceri (Foc) on branches of chickpea plant. The pathogen population was greatly reduced in the moist soil (43 MPa) when compared with the wet soil (-0.03 MPa) at both temperatures which was indicated by greater colonization and growth of T. harzanum-2 on the branch pieces of chickpea plants. The pathogen was completely eradicated from the chickpea branch pieces, after 6 months at 35 degrees C in the moist soil. In air-dry soil (<-50 MPa), Foc survived in 100% of the branch pieces even after 6 months at both temperatures. When chickpea plant branch pieces having pathogen was sprayed with Th-2 antagonistic isolates of Trichoderma spp., the Th-2 isolate killed the pathogen up to minimum level (10-12%) after 5 months at 35 degrees C in the sandy clay loam soil. It can be concluded that in chickpea growing rainfed areas of Pakistan having sandy clay loam soil, Foc can be controlled by using specific Trichoderma spp., especially in the summer season as after harvest of the crop the temperature increased up and there is rainfall during this period which makes the soil moist. This practice will be able to reduce the inoculum of Foc during this hot period as field remain fallow till next crop is sown in most of the chickpea growing rainfed areas of Pakistan.