Selection and characterization of novel zinc-tolerant Trichoderma strains obtained by protoplast fusion

Aim: To develop new Trichoderma strains, capable of removing toxic heavy metal ions from polluted environments, via protoplast fusion.Methodology: Trichoderma parental strains (T. viride and T. koningii) along with their ten fusants (Tk+Tv 1, Tk+Tv 2, Tk+Tv 3, Tk+Tv 4, Tk+Tv 5, Tk+Tv 6, Tk+Tv 7, Tk+Tv 8, Tk+Tv 9 and Tk+Tv 10) were obtained from the Department of Plant Pathology, Junagadh Agricultural University, Junagadh. The strains obtained by protoplast fusion were examined for their ability to remove toxic heavy metal ions, especially zinc ion. Fourier-transform infrared spectroscopy (FTIR) was conducted to detect the zinc uptake mechanism of Trichoderma parental and their fusant strains. Results: FTIR results demonstrated the Zn ion uptake capacity of fusant strains was found to be higher than that of the parental strains (12.8 to 10.7 mg g-1 on a dry weight basis at 1300 ppm). The highest Zn ion mobility observed was 62.1 mg. kg-1 and the highest Zn ion mobility observed per strain was 12.4% in Tk+Tv 3, followed by 11.86 % in Tk + Tv 7, 11.84% in Tk + Tv 9 and 11.28% in Tk + Tv 10. Parental and fusant strains Tk + Tv 3, Tk + Tv 8 and Tk + Tv 10 confirmed the involvement of different functional groups for different concentrations of zinc during adsorption by the fungus. Interpretation: FTIR results identified greater metal removal capacity in the fusant strains, particularly for soil Zn ion. Zinc tolerance was higher in the fusant strains than in the parental strains. Thus, protoplast fusion is an effective and feasible method for constructing new strains that can be used for bioremediation of contaminated environments.

The Effectiveness of Trichoderma spp. against Downy Mildew Disease of Corn

Introduction: One of environmentally friendly method for controlling plant diseases is the use of Trichoderma spp. as a natural controlling agent. Objectives: The objective of this research was to find out the effectiveness of Trichoderma spp. against downy mildew disease. Methodology: This research was conducted in the Plant Pest and Disease Laboratory in the Plant Protection Department of Faculty of Agriculture in Lampung University. This research used completely randomized design consisting without treatment (0), Trichoderma spp. Gading Rejo Region (GDR) isolate (1) Trichoderma spp. Nusantara Tropical Farm (NTF) isolate (2), and Trichoderma spp. Trimurjo (TRJ) isolate (3) treatments which were applied to the plant growing points as fungicide (B) and as inducer of plant resistance to be applied in the plant roots (P). Results: The research results showed that the Trichoderma spp. treatments could reduce the disease occurrence at 4 and 5 days after inoculation, but they could not reduce the disease severity and improve stover dry weight of corn plant. Conclusion: The Trichoderma spp. Treatment as biofungicide and plant resistance inducer are effective against the incubation period and suppress the disease occurrence of downy mildew disease significantly at the early course of the disease.

Development and multiplication of Eisinea andrei in the manure of cattle subjected to high Trichoderma doses / Desenvolvimento e multiplicação de Eisenia andrei em esterco bovino em altas doses de Trichoderma

The efficacy of the vermicomposting and products based on the antagonistic fungus and plant growth promoter trichoderma (Trichoderma spp) is well known and studied in organic agriculture. However, for a better methodological efficiency are necessary studies to evaluate the effect of high doses of these bioproducts in the biology and development of earthworms. Thus, the present work aims to test the use of high commercial biocontrol product (ICB Nutrisolo Trichoderma) doses by evaluating the multiplication and development of Eisenia andrei. Changes in the chemical features of the substrate produced by the vermicomposting process using in natura and sterilized organic cattle manure were also assessed. Each experimental unit consisted of 6 kg of substrate (in multipurpose polypropylene box ­ 20 x 40 x 50 cm) containing 48 clitelate adult Eisenia andrei earthworms. ICB Nutrisolo Trichoderma was used as biological agent along with eight strains of the following species: T. koningiopsis, T. asperellum and T. harzianum. The following treatments were applied at doses of 1011 CFU kg-1 of ICB Nutrisolo Trichoderma in the presence of earthworms: T1 (0.5); T2 (1.0); T3 (2.0); T4 (4.0); T5 (8.0) and T6 (0.0). The T7 treatment was herein used in order to evaluate the chemical features of the vermicompost. It was a completely randomized design with four replications per treatment. The temperature was kept at 28°C and humidity ranged between 60 and 70%. After 60 days, the number of young and adult earthworms, and cocoons was counted; then, their dry biomass was assessed. The results found in the lethality test showed decrease in the number of earthworms treated with 4.0x1011 CFU kg-1 of ICB. The biological product doses up to 1.0x1011 CFU kg-1 did not alter the number of adult earthworms and cocoons, or the multiplication index of E. andrei in cattle waste vermicomposts. There was no influence of the tested doses on earthworms' individual development. However, doses above 2.0x1011 CFU kg-1 decreased their total biomass. The C/N ratio for all treatments indicates maturity within acceptable results for organic compounds.

A eficácia da vermicompostagem e de bioprodutos à base do fungo antagonista e promotor de crescimento vegetal trichoderma (Trichoderma spp) é bem conhecida e estudada na agricultura orgânica. Entretanto, para uma melhor eficiência metodológica, são necessários estudos que possam avaliar a interferência de altas doses desses bioprodutos na biologia e desenvolvimento das minhocas. Baseado nesse contexto, o objetivo deste trabalho foi testar altas doses do produto comercial biológico ICB Nutrisolo Trichoderma (ICB), avaliando-se a multiplicação e desenvolvimento de Eisenia andrei, bem como alterações nas características químicas do substrato produzido no processo de vermicompostagem, a partir do resíduo orgânico esterco bovino. O esterco bovino in natura foi autoclavado a 121°C, por duas vezes, em um intervalo de 24 h. A unidade experimental constituiu-se de 6 kg de substrato condicionados em caixa multiuso de polipropileno, com dimensões 20 x 40 x 50 cm, contendo 48 minhocas adultas e cliteladas da espécie E. andrei. Como agente biológico, utilizou-se o produto comercial ICB na forma de fluído, composto por oito cepas das espécies T. koningiopsis, T. asperellum e T. harzianum, com as seguintes doses nos tratamentos a seguir: T1 (0.5); T2 (1.0); T3 (2.0); T4 (4.0); T5 (8.0); e T6 (0.0), sendo todas as concentrações em 1011 UFC kg-1 do produto em vermicomposto, e, para a avaliação das características químicas do vermicomposto em altas doses do produto ICB, foi utilizado também o T7 (somente substrato). O delineamento foi inteiramente casualizado com quatro repetições por tratamento. A temperatura foi mantida a 28ºC e a umidade entre 60 e 70%. Após 60 dias do início da instalação, fez-se a contagem do número de minhocas adultas, jovens e casulos; posteriormente, avaliou-se o seu peso seco total. Os resultados observados no teste de letalidade mostram que, somente a partir de 4.0x1011 UFC kg-1 de ICB, há decréscimo do número de minhocas. Doses altas até 1.0x1011 UFC kg-1 do produto não alteram o número de minhocas adultas e de casulos de E. andrei em vermicompostagem com esterco bovino, entretanto, o índice de multiplicação foi inferior em todos os tratamentos com o produto. Doses acima de 2.0x1011 UFC kg-1 diminuíram o peso seco total. A relação C/N em todos os tratamentos indica maturidade dentro de resultados aceitáveis para compostos orgânicos.

Manejo biológico del tamo de arroz bajo diferentes relaciones C:N empleando consorcios microbianos y promotores de crecimiento vegetal / Bio-based management of rice straw under different C:N ratios using microbial consortia and plant growth promoters

Resumen El tamo de arroz es uno de los residuos agrícolas lignocelulósicos más abundantes en el planeta, luego de los residuos producidos por los cultivos de maíz y trigo, con una producción mundial estimada de 1000 millones de toneladas según estadísticas de la FAO. En el contexto de la agricultura moderna es determinante lograr un manejo ambientalmente sostenible de este recurso mediante su incorporación al suelo, de tal forma que se logre el reciclaje de nutrientes, evitando la incorporación de patógenos al sistema y la inmovilización de elementos como el nitrógeno por la comunidad microbiana. El objetivo de este trabajo consistió en la evaluación de un inoculo microbiano mixto a partir de productos comerciales basados en hongos del género Trichoderma y bacterias aerobias formadoras de endosporas, con potencial degradador del tamo de arroz, así como el empleo de bacterias promotoras de crecimiento vegetal al momento de la siembra, que pudieran aprovechar los nutrientes del proceso de descomposición del tamo de arroz, potenciando su actividad biológica. Los tratamientos fueron evaluados bajo diferentes relaciones C:N del tamo de arroz que favorecieran el proceso de degradación, mediante la adición de nitrógeno inorgánico. Los resultados del trabajo permitieron identificar que la aplicación de una enmienda de nitrógeno a una relación C:N 35 más una dosis adicional de urea al momento de la siembra de las semillas de arroz, fue el tratamiento más adecuado para potenciar el efecto de los microorganismos e incrementar las variables agronómicas obtenidas mediante un esquema de fertilización convencional del cultivo.

Abstract Rice straw is one of the most abundant lignocellulosic agricultural residues on the world, after residues produced by maize and wheat crops, with an estimated global production of 1000 million tones according to FAO statistics. In the context of modern agriculture, it is essential to achieve an environmentally sustainable management of this resource, through the incorporation of rice straw into the soil, in order to achieve nutrient recycling avoiding the incorporation of pathogens into the system and the losses of nitrogen due to soil microbial biomass immobilization. In this context, the objective of this work consisted in the evaluation of a mixed microbial inoculum with degrading potential of rice straw from commercial products based on the fungus Trichoderma spp. and aerobic endospore forming bacteria. It was also used a plant growth promoting bacteria at the time of planting, in order to take advantage of nutrients released from RS decomposition and improve its biological activity. These microbial treatments, were evaluated at different concentrations of inorganic nitrogen amendments that allowed different levels of rice straw´s C:N ratio, favoring the degradation process. Overall results allowed to identify that the application of a nitrogen amendment to rice straw up to C:N 35, plus an additional dose of urea at the time of rice seeds planting, was the most adequate treatment to potentiate the effect of the microorganisms and to increase or maintain the agronomic variables obtained through a conventional fertilization management of the crop.

Efficacy of Trichoderma asperellum, T. harzianum, T. longibrachiatum and T. reesei against Sclerotium rolfsii / Eficácia de Trichoderma asperellum, T. harzianum, T. longibrachiatum E T. reesei contra Sclerotium rolfsii

This work was carried out to select and to evaluate efficacy of Trichoderma isolates to control sclerotium wilt (Sclerotium rolfsii) of common-bean (Phaseolus vulgaris). For the experiments were used two isolates of S. rolfsii UB 193 and UB 228. Sixty-five Trichoderma spp. isolates were tested and the following ones were selected in vitro for the in vivo tests: 5, 11, 12, 15, 102, 103, 127, 136, 137, 1525 (T. longibrachiatum), 1637 (T. reesei), 1642, 1643 (T. harzianum), 1649 (T. harzianum), 1700 (T. asperellum) and EST 5. The most promising isolates were identified by Sequencing of the internal transcribed spacer regions ITS1, ITS2, and the 5.8s rRNA genomic region, using the ITS5 and ITS4 primers and compared with sequences in the National Center for Biotechnology Information (NCBI) database. These selected isolates 1649 (T. harzianum), 1525 (T. longibrachiatum) and 1637 (T. reesei) were tested for evaluation of sclerotial germination inhibition under laboratory conditions, and to evaluate the effects of these on disease of bean plants under greenhouse conditions. The Trichoderma isolates 1649, 1525 and 1637 were more efficient in reducing sclerotial germination. In addition to 1649, 1525 and 1637, the isolates 5, 12, 102 and 1525 (T. longibrachiatum) significantly reduced de amount of diseased bean plants under greenhouse conditions.

O estudo teve como objetivo selecionar e testar isolados de Trichoderma spp. para o controle da murcha-por-esclerócio (Sclerotium rolfsii) em feijoeiro (Phaseolus vulgaris) em casa-de-vegetação. Nos ensaios realizados foram utilizados dois isolados de S. rolfsii (UB 193 e UB 228). Dos 65 isolados Trichoderma testados in vitro selecionaram-se os seguintes: 5, 11, 12, 15, 102, 103, 127, 136, 137, 1525 (T. longibrachiatum), 1637 (T. reesei), 1642 (T. longibrachiatum), 1643 (T. harzianum), 1649 (T. harzianum), 1700 (T. asperellum) e EST 5. Os isolados mais promissores foram identificados por sequenciamento das regiões genômicas ITS1, ITS2 e 5.8s rRNA, usando iniciadores ITS5 e ITS4, e então, tais sequencias foram comparadas com as sequências do banco de dados do "National Center for Biotechnology Information" (NCBI). Esses isolados foram avaliados quanto ao efeito sobre a germinação de esclerócios do patógeno em laboratório e sobre a doença em casa de vegetação. Os isolados 1649 (T. harzianum), 1525 (T. longibrachiatum) e 1637 (T. reesei) foram os mais eficientes na inibição da germinação de esclerócios de S. rolfsii em laboratório. Além dos isolado 1649, 1525 e 1637, os isolados 5, 12, 102 e 1525 (T. longibrachiatum) foram eficientes na redução da doença em plantas de feijoeiro em casa de vegetação.

Efeito de metabólitos produzidos por Trichoderma spp. sobre o índice mitótico em células das pontas de raízes de Allium cepa / Effect of metabolites produced by Trichoderma spp. on mitotic index in cells of Allium cepa root tips

Microrganismos do solo produzem metabólitos importantes para a produção de compostos biologicamente ativos. Dentre estes microrganismos, as espécies de trichoderma (Trichoderma spp.) possuem a capacidade de produzir metabólitos. A promoção do desenvolvimento de plantas por trichoderma pode estar relacionada, entre outros fatores, ao estimulo à multiplicação celular, através da produção de hormônios, entre outros. O método de avaliação do índice mitótico em raízes de Allium cepa é validado pelo Programa Internacional de Segurança Química (IPCS, OMS) e o Programa Ambiental das Nações Unidas (UNEP) como um eficiente teste para análise e monitoramento in situ. O objetivo do estudo foi selecionar cepas e produtos biológicos comerciais de trichoderma, utilizados no controle biológico de fitopatógenos que promovam o aumento do índice mitótico em células de pontas de raízes pelo sistema teste de A. cepa. Foram utilizadas cepas de Trichoderma harzianum (2B2, 2B22, 2B12); Trichoderma viride (TSM1, TSM2, C1) e os produtos biológicos comerciais de trichoderma, Agrotrich® e Trichodermil®. Os bulbos de Allium cepa foram arranjados em dez tratamentos (quatro repetições por tratamento), oito com metabólitos de cepas de Trichoderma spp. e dois controles na ausência de metabólitos. A contagem das células ocorreu na região meristemática, onde foram contadas 500 células por bulbo em cada uma das lâminas. Foram observadas as células em interfase, prófase, metáfase, anáfase e telófase, com auxílio de microscópio ótico com a objetiva de 40X. Foram contadas as células de cada uma das fases do ciclo celular estudadas, após calculou-se os índices mitóticos. A análise estatística dos dados foi realizada pelo teste c 2 (Qui-quadrado), com probabilidade de 5%, pelo programa estatístico BioEstat 4.0. Os tratamentos T1, T3 e T4 (metabólitos das cepas 2B2, 2B12 e C1 de trichoderma, respectivamente) apresentaram os maiores números de células para as fases prófase, metáfase, anáfase e telófase e alcançaram os maiores IM (c 2= 5.45 a= 0.05), (c 2= 5.0 a= 0.05) e (c 2= 3.92 a= 0.05), respectivamente. O tratamento controle T10 (meio líquido BD), apresentou o maior número de células em interfase, e o menor nas fases da divisão celular. O teste com raízes de Allium cepa possibilita selecionar cepas de trichoderma que induzem o aumento do índice mitótico em pontas de raízes pela ação de metabólitos e apresenta variabilidade entre as cepas estudadas.

Soil microorganisms produce metabolites important for the production of biologically active compounds. In relation to these organisms, species of trichoderma (Trichoderma spp.) have the ability to produce metabolites. Promoting the development of plants by trichoderma may be related, among other factors, to stimulate cell proliferation through the production of hormones, among others. The method of evaluation of mitotic index in Allium cepa roots is validated by the International Programme on Chemical Safety (IPCS, WHO) and United Nations Environment Programme (UNEP) as an effective test for in situ analysis and monitoring. The aim of the study was to select strains and commercial biological products trichoderma used in biological control of plant pathogens that promote increased mitotic index in cells of the root tips of A. cepa test system. Strains of Trichoderma harzianum (2B2, 2B22, 2B12) and Trichoderma viride (TSM1, TSM2, C1) and the biological products commercials Agrotrich® and Trichodermil® of trichoderma were used. The bulbs of Allium cepa were arranged in ten treatment (four replicates per treatment), eight with metabolite of Trichoderma spp. and two controls in the absence of metabolites. Cell counts occurred on the meristematic region where 500 cells were counted for each bulb on the blades. Cells were observed in interphase, prophase, metaphase, anaphase and telophase, with the aid of an optical microscope with a 40X objective. The cells were counted in each phase of the cell cycle analyzed and after it was calculated mitotic rates. Statistical analysis of data was performed by c 2 test (chi-square), with a probability of 5%, the statistical program BioEstat 4.0. Treatments T1, T3 and T4 (metabolites of isolates 2B2, 2B12 and C1 of trichoderma, respectively) showed higher cell numbers for the phases prophase, metaphase, anaphase and telophase and reached the highest IM (c 2= 5.45 a= 0.05), (c 2= 5.0 a= 0.05) e (c 2= 3.92 a= 0.05, respectively. The T10 control (liquid medium BD), had the highest number of interphase cells, and the lower phases of cell division. The test with Allium cepa roots enables selecting trichoderma isolates that induce increased mitotic index in root tips by the action of metabolites and shows variability among the isolates studied.

Evolution of disease and potential biocontrol activity of Trichoderma SP. against Rhizoctonia solani on potato / Potencial do biocontrole por Trichoderma em relação à evolução da doença causada por Rhizoctonia solani em batata

Black scurf and stem canker disease cause by the fungal pathogen of Rhizoctonia solani and it is an economical important disease of potatoes in Bangladesh and throughout the world. This study evaluated the black scurf and stem canker disease development in potato and antagonistic activity of Trichoderma spp. against R. solani. The artificial infections were carried out using the inoculums of R. solani. The treatments (%inoculum) were: T1 (0% inoculum), T2 (5% inoculum), T3 (10% inoculum), T4 (20% inoculum), T5 (50% inoculum), and T6 (100% inoculum). The infection of stem canker and black scurf on progeny tubers increased with increase in inoculum levels. The highest disease incidence and severity was found in T6 (100% inocula). T6 showed the maximum black scurf infected tubers (russet, deformed and sclerotia). The lowest germination percentage, plant height and tuber yield were also obtained in the same treatment (100% inocula). Trichoderma spp reduced the growth of R. solani and the highest growth suppression was noted in isolate TM12. According to antagonistic activity, Trichoderma spp. reduced the growth of R. solani but was not able to stop the pathogen development. This finding showed management of this disease or R. solani invasion requires an integrated approach compared to Trichoderma single approach.

A rizoctoniose ou crosta negra causada por Rhizoctonia solani é a mais importante doença nos campos de batata em Bangladesh, bem como em várias regiões do mundo. Este trabalho avaliou o potencial do biocontrole com Trichoderma spp. e sua ação antagonista contra R. solani em batateira. Realizou-se as avaliações do potencial antagonosta usando inoculação artificial de R. solani. Os tratamentos (% de inóculo) foram: T1 (0% de inóculo), T2 (5% ), T3 (10%), T4 (20%), T5 (50%) , e T6 (100% de inóculo). A infecção de rizoctoniose na haste e crosta negra nos tubérculos aumentou proporcionalmente com o aumento do nível de inóculo. A maior incidência e severidade da doença ocorreu no tratamento 6 (100 % de inóculo), o qual apresentou maior quantidade de tubérculos infectados e deformados com escleródios em sua superfície. A menor porcentagem de germinação e produção de tubérculos também foi encontrada no tratamento 6, o qual também apresentou menor altura de planta. Trichoderma spp reduziu o crescimento de R. solani e a maior atividade de supressão do crescimento foi encontrada pelo isolado TM12. Foi detectada a atividade antagonista de Trichoderma spp. em reduzir o crescimento de R. solani, mas este não inibiu o crescimento total do patógeno. Conclui-se que o manejo da rizoctoniose da batateira por colonização de R. solani necessita táticas de manejo integrado em detrimento do uso isolado do manejo ou biocontrole com Trichoderma spp.

From the ancient times of the agriculture to the biological control in plants: a little of the history / Dos primórdios da agricultura ao controle biológico em plantas: um pouco de história

The advancement of technology in agriculture driven by the need to increase efficiency in production transformed agriculture in an eminently anti-ecological activity, with large-scale use of industrialized products. Human beans still live the paradigm of agriculture based on the use of chemical inputs, which often brings harmful consequences to the environment. As an alternative to u the use of pesticides to control pests and diseases, biological control is a practice that has been increasingly encouraged. With it, it is possible to think on changes that promote the conservation of natural resources and of the planet humans live in, contributing to strength the new era in biotechnology education.

O avanço da tecnologia na área agrícola, induzido pela necessidade de aumento da eficiência na produção, transformou a agricultura numa atividade eminentemente antiecológica, com uso em larga escala de insumos industrializados. Vivemos ainda o paradigma da agricultura baseada no uso de insumos químicos, a qual muitas vezes traz consequências danosas ao meio ambiente. Como alternativa ao uso de agrotóxicos para o controle de pragas e doenças, o controle biológico é uma prática que vem sendo cada vez mais incentivada. Com ele, pode-se pensar em transformações que, visando a conservação de recursos naturais e do planeta em que vivemos, contribuam para o fortalecimento da nova era biotecnológica em formação.

Studies on strains of Trichoderma spp. from high altitude of Garhwal Himalayan region.

Two species of Trichoderma i.e. T. harzianum and T. viride have been isolated from the soil samples collected from the higher altitude (2000-3500 m) of Garhwal Himalayan region in India. The two species were grown in Petri plates on TSM agar media and it was also observed that the optimum temperature and pH for Trichoderma growth was 300 C and 5.5 respectively. When incubated on TSM agar medium at 40 C, the fungus grew normally with heavy induced sporulation within three weeks of incubation. Induction of sporulation on exposure to low temperature appeared to be strategies for survival of these species in extreme cold environment temperature 4 to 50 C. Antifungal activities of the two species of Trichoderma were demonstrated with phytopathogenic fungi in dual cultures. The antifungal metabolites produced by Trichoderma spp., diffusible as well as volatile, caused abnormalities in pathogenic fungi. Plant growth promotion of Trichoderma spp. was also shown through plant analysis in greenhouse.

Potential strain of trichoderma spp. to control damping off of disease in chilli.

The present investigation deals with the population of soil fungi in chilli field of Thiruvarur District. The antibiotic ability and potential strain of Trichoderma spp.isolated from chilli field soil were used against damping off disease and its tolerance to antibiotics. Among the soil fungi, tested Trichoderma spp. showed maximum activity in controlling the disease with influence of soil physico-chemical parameters. The antibiotic interactions of some soil fungi against Pythium debaryanum were studied in vitro dual culture experiments. All the Trichoderma spp. Showed the ability to inhibit the pathogen, some of the species showed variability in the percentage of inhibition were studied.

Isolation and Identification of Mushroom Pathogens from Agrocybe aegerita

Agrocybe aegerita is an important mushroom cultivated in Korea, with good feel and a peculiar fragrance. A. aegerita can be cultivated throughout the year using culture bottles but is more susceptible to contamination than other mushrooms. Twenty-two pathogens were isolated from the fruiting bodies and compost of A. aegerita, and seven isolates were isolated from Pleurotus ostreatus to compare with the A. aegerita isolates, collected from Gimje, Iksan, Gunsan of Chonbuk, and Chilgok of Gyeongbuk Province in 2009. These isolates were identified based on morphological and molecular characteristics. Of the 29 isolates, 26 were identified as Trichoderma spp. and the remaining three were Aspergillus spp., Mucor spp., and Penicillium spp. A phylogenetic analysis revealed that the 26 isolates of Trichoderma were divided into four taxa, namely T. harzianum, T. pleuroticola, T. longibrachiatum, and T. atroviride. Among the Trichoderma spp., 16 isolates (55.2%) were identified as T. harzianum, six as T. pleuroticola (20.7%), two as T. longibrachiatum, and the remaining two were T. atroviride.

DINÁMICA MICROBIANA EN VERMICOMPOSTAS COMERCIALES CON Y SIN INOCULACIÓN DEL HONGO Trichoderma spp / Microbial dynmiccs in commercial vermicomposts with and withot trichoderma sspp. fungus inoculation

En el presente trabajo se presentan los resultados de un ensayo en el cual se evaluó la efectividad de la inoculación del hongo Trichoderma spp, en una vermicomposta comercial, comparándose ésta con la población del hongo presente en un vermicompost sin inocular. El experimento se realizó en el laboratorio de postcosecha de la Universidad de Costa Rica. Se desarrolló en tres fases (cultivo, aislamiento e inoculación de cepas del hongo) y se utilizaron tres vermicompostas comerciales elaboradas a partir de un sustrato de broza de café, procedentes de diferentes composteras. Los resultados muestran que no hubo aumento ni disminución de la población de Trichoderma como producto de la inoculación de cepas del mismo.

In tThis paper, presents the results are presented onof the growthefectiveness of the Trichoderma fungus in a inoculation in a commercial vermicompost, in comparison with the fungus from a vermicompostand without inoculation. In a three-phase laboratory process (cultivation, isolation and inoculation of fungal strains), three different commercial vermicompost, prepared from coffee pulp, have beenwere used, coming from three different commercial vermicomposts. The results of experiments carried out in the Post-Harvest Laboratory of the Universidad de Costa Rica, don’t show any increase or decrease of the Trichoderma population after inoculation. The experiment was carried out in the Post -harvest Laboratory of University of Costa Rica. It was developed following three phases, using three commercial vermicompost prepared with broza of coffee. Results showed no differences in Trichoderma population as product of inoculation of it into the compost.

In Vitro Antagonistic Characteristics of Bacilli Isolates against Trichoderma spp. and Three Species of Mushrooms

Twenty isolates of Bacillus species obtained from livestock manure composts and cotton-waste composts were tested for their antagonistic effects in vitro against three green mold pathogens of mushrooms (Trichoderma harzianum, T. koningii, and T. viridescens). However, there exists a possibility Bacillus species may have antagonistic effects against mushrooms themselves, and thus the same 20 isolates were tested in vitro against three species of mushrooms (Flammulina velutipes, Lentinus edodes, and Pleurotus ostreatus). Of the 20 Bacillus species isolates tested, two inhibited mycelial growth of T. harzianum, seven that of T. koningii, and eight that of T. viridescens. Importantly, the bacterial isolates M27 and RM29 strongly inhibited mycelial growth of all the Trichoderma spp. isolates tested. The isolate M27 was subsequently identified as the most effective in inhibiting mycelial growth of all the Trichoderma species. Interesting results of the effect Bacillus isolates had upon the mushroom species followed. It was found that most Bacillus isolates except 5T33 at least somewhat inhibited mycelial growth of the three mushroom species or some of the mushrooms. Furhermore, the antagonistic effects of the bacterial isolates against the three species of mushrooms varied depending on the mushroom species, suggesting a role for mushroom type in the mechanism of inhibition. The bacterial isolates M27 and RM29 were identified as having the most antagonistic activity, inhibiting mycelial growth of all the Trichoderma spp. as well as mycelial growth of the three species of mushrooms. These results suggest that the bacterial isolates and their antagonistic effects on green mold pathogens should be further studied for their practical use for biological control of green mold in the growing room of the mushrooms.

Molecular and Morphological Characterization of Green Mold, Trichoderma spp. isolated from Oyster Mushrooms

Isolates of Trichoderma spp. collected from Pleurotus ostreatus and P. eryngii beds, which included loosened substrate compactness and development of green colour, were grouped into three species. The occurrence of different species of Trichoderma was as T. cf. virens (70.8%), T. longibrachiatum (16.7%) and T. harzianum (12.5%). The conidia of Trichoderma spp. were ellipsoidal, obovoid and phialides were bowling pins, lageniform and the length of phialides was 3.5~10.0 x 1.3~3.3 micromm. Phialides of T. cf. virens and T. harzianum were tending clustered, but it was solitary disposition in T. longibrachiatum. T. cf. virens was characterized by predominantly effuse conidiation, sparingly branched, and fertile to the apex and it was penicillate type. RAPD analysis could detect variability amongst three different species of Trichoderma using two newly designed URP-primers. However, intra-specific variation could not be detected in all the isolates except for rDNA sequence data classified Trichoderma isolates into three distinct groups representing three species. The profiles of rDNA sequences of isolates representing a species showed high similarity in T. cf. virens and T. harzianum. However, there was a variation in rDNA sequences of isolates representing T. longibrachiatum. The results of present study reveals that molecular techniques of RAPD and rDNA sequencing can greatly aid in classification based on morphology and precise identification of fast evolving species of Trichoderma.

Physiological Characteristics of Green Mold (Trichoderma spp.) Isolated from Oyster Mushroom (Pleurotus spp.)

This study was conducted to investigate physiological characteristics of Trichoderma spp. isolated from Pleurotus spp. Damage tests of Pleurotus spp. and mycotoxins tests of Trichoderma spp. were also done. The optimal growth temperature of Trichoderma spp. was 27~30degrees C. Although, T. longibrachiatum was able to grow at 37degrees C and grew 30~40 times faster than Pleurotus. The colony colour on PDA medium of T. cf. virens was yellowish green, T. longibrachiatum was yellow, and T. harzianum was turning to bright green. In damage tests of Pleurotus by Trichoderma, T. cf. virens caused the most severe damage to Pleurotus. T. longibrachiatum and T. harzianum caused less damage on Pleurotus but were able to cause greater damage to P. eryngii. One of the mushroom cultivars, P. ostreatus 8 was the most resistant to all Trichoderma spp.. Chitinolytic mycotoxin released by Trichoderma spp. caused 52.7% damage to Pleurotus. Mycotoxins released by T. longibrachiatum caused the greatest damaged (78.6%) on P. eryngii.

Effect of Sclerotial Distribution Pattern of Sclerotinia sclerotiorum on Colonizing Ability of Trichoderma harzianum

Field studies were conducted over two seasons during the summers of 1997 and 1998 to investigate the effects of different spatial arrangements(random or highly aggregated) of sclerotia of Sclerotinia sclerotiorum and alginate pellet types(bran or polyethylene glycol) on colonization of sclerotia by Trichoderma spp. Treatment with alginate pellets increased the mean percentages of sclerotia colonized by Trichoderma spp. in both years. Distribution patterns of sclerotia affected the mean percentage of sclerotia colonized by Trichoderma spp. in both years, indicating that a highly aggregated distribution of sclerotia was more favorable to colonization by Trichoderma spp. The effects of the different pellet types(bran or PEG) were not siginificant in both years(P>0.05). The application of higher densities(200 pellets per 1 m2) of alginate pellets resulted in higher mean percentages of sclerotia colonized by Trichoderma spp. in 1998(p<0.05), but did not in 1997.