Trichoderma harzianum MTCC 5179 impacts the population and functional dynamics of microbial community in the rhizosphere of black pepper (Piper nigrum L )

Abstract Employing Illumina Hiseq whole genome metagenome sequencing approach, we studied the impact of Trichoderma harzianum on altering the microbial community and its functional dynamics in the rhizhosphere soil of black pepper (Piper nigrum L.). The metagenomic datasets from the rhizosphere with (treatment) and without (control) T. harzianum inoculation were annotated using dual approach, i.e., stand alone and MG-RAST. The probiotic application of T. harzianum in the rhizhosphere soil of black pepper impacted the population dynamics of rhizosphere bacteria, archae, eukaryote as reflected through the selective recruitment of bacteria [Acidobacteriaceae bacterium (p = 1.24e-12), Candidatus koribacter versatilis (p = 2.66e-10)] and fungi [(Fusarium oxysporum (p = 0.013), Talaromyces stipitatus (p = 0.219) and Pestalotiopsis fici (p = 0.443)] in terms of abundance in population and bacterial chemotaxis (p = 0.012), iron metabolism (p = 2.97e-5) with the reduction in abundance for pathogenicity islands (p = 7.30e-3), phages and prophages (p = 7.30e-3) with regard to functional abundance. Interestingly, it was found that the enriched functional metagenomic signatures on phytoremediation such as benzoate transport and degradation (p = 2.34e-4), and degradation of heterocyclic aromatic compounds (p = 3.59e-13) in the treatment influenced the rhizosphere micro ecosystem favoring growth and health of pepper plant. The population dynamics and functional richness of rhizosphere ecosystem in black pepper influenced by the treatment with T. harzianum provides the ecological importance of T. harzianum in the cultivation of black pepper.

Screening of Trichoderma isolates for their potential of biosorption of nickel and cadmium

Abstract Fourteen Trichoderma isolates were evaluated for their tolerance to two heavy metals, nickel and cadmium. Three isolates, MT-4, UBT-18, and IBT-I, showed high levels of nickel tolerance, whereas MT-4, UBT-18, and IBT-II showed better tolerance of cadmium than the other isolates. Under nickel stress, biomass production increased up to a Ni concentration of 60 ppm in all strains but then decreased as the concentrations of nickel were further increased. Among the nickel-tolerant isolates, UBT-18 produced significantly higher biomass upon exposure to nickel (up to 150 ppm); however, the minimum concentration of nickel required to inhibit 50% of growth (MIC50) was highest in IBT-I. Among the cadmium-tolerant isolates, IBT-II showed both maximum biomass production and a maximum MIC50 value in cadmium stress. As the biomass of the Trichoderma isolates increased, a higher percentage of nickel removal was observed up to a concentration of 40 ppm, followed by an increase in residual nickel and a decrease in biomass production at higher nickel concentrations in the medium. The increase in cadmium concentrations resulted in a decrease in biomass production and positively correlated with an increase in residual cadmium in the culture broth. Nickel and cadmium stress also influenced the sensitivity of the Trichoderma isolates to soil fungistasis. Isolates IBT-I and UBT-18 were most tolerant to fungistasis under nickel and cadmium stress, respectively.

Cultural conditions on the production of extracellular enzymes by Trichoderma isolates from tobacco rhizosphere

Abstract Twelve isolates of Trichoderma spp. isolated from tobacco rhizosphere were evaluated for their ability to produce chitinase and β-1,3-glucanase extracellular hydrolytic enzymes. Isolates ThJt1 and TvHt2, out of 12 isolates, produced maximum activities of chitinase and β-1,3-glucanase, respectively. In vitro production of chitinase and β-1,3-glucanase by isolates ThJt1 and TvHt2 was tested under different cultural conditions. The enzyme activities were significantly influenced by acidic pH and the optimum temperature was 30 °C. The chitin and cell walls of Sclerotium rolfsii, as carbon sources, supported the maximum and significantly higher chitinase activity by both isolates. The chitinase activity of isolate ThJt1 was suppressed significantly by fructose (80.28%), followed by glucose (77.42%), whereas the β-1,3-glucanase activity of ThJt1 and both enzymes of isolate TvHt2 were significantly suppressed by fructose, followed by sucrose. Ammonium nitrate as nitrogen source supported the maximum activity of chitinase in both isolates, whereas urea was a poor nitrogen source. Production of both enzymes by the isolates was significantly influenced by the cultural conditions. Thus, the isolates ThJt1 and TvHt2 showed higher levels of chitinase and β-1,3-glucanase activities and were capable of hydrolyzing the mycelium of S. rolfsii infecting tobacco. These organisms can be used therefore for assessment of their synergism in biomass production and biocontrol efficacy and for their field biocontrol ability against S. rolfsii and Pythium aphanidermatum infecting tobacco.

Molecular characterization of a Xylanase-producing fungus isolated from fouled soil

Xylanase (EC 3. 2. 1. 8), hydrolyzes xylo-oligosaccharides into D-xylose and required for complete hydrolysis of native cellulose and biomass conversion. It has broad range of applications in the pulp and paper, pharmaceutical and Agri-food industries. Fifty fungal species were isolated from the fouled soil around an oil refinery and screened for the production of xylanase enzyme by enrichment culture techniques. The isolated fungal strain was identified as Hypocrea lixii SS1 based on the results of biochemical tests and 18s rRNA sequencing. The phylogenetic tree was constructed using the MEGA 5 software. Further, Hypocrea lixii SS1 was tested for the ability to utilize the sunflower oil sludge (waste from the oil industry) as the sole carbon source for xylanase production. The growth characteristics of Hypocrea lixii SS1 were also studied and maximum growth was found on the 7th day of incubation. The fungus showed a remarkable xylanase production of 38.9 U/mL. Xylanase was purified using a combination of 0-50% NH4SO2 precipitation, DEAE-sepharose and Sephacryl S-200 chromatography. Single peak obtained in RP-HPLC confirms the purity of xylanase. Further the enzyme produced was affirmed as xylanase with its molecular weight (29 kDa) using SDS-PAGE.

Antifungal activity of metabolites of the endophytic fungus Trichoderma brevicompactum from garlic

The endophytic fungus strain 0248, isolated from garlic, was identified as Trichoderma brevicompactum based on morphological characteristics and the nucleotide sequences of ITS1-5.8SITS2 and tef1. The bioactive compound T2 was isolated from the culture extracts of this fungus by bioactivity-guided fractionation and identified as 4β-acetoxy-12,13-epoxy-Δ9-trichothecene (trichodermin) by spectral analysis and mass spectrometry. Trichodermin has a marked inhibitory activity on Rhizoctonia solani, with an EC50 of 0.25 µgmL-1. Strong inhibition by trichodermin was also found for Botrytis cinerea, with an EC50 of 2.02 µgmL-1. However, a relatively poor inhibitory effect was observed for trichodermin against Colletotrichum lindemuthianum (EC50 = 25.60 µgmL-1). Compared with the positive control Carbendazim, trichodermin showed a strong antifungal activity on the above phytopathogens. There is little known about endophytes from garlic. This paper studied in detail the identification of endophytic T. brevicompactum from garlic and the characterization of its active metabolite trichodermin.

Bioethanol production from rice straw residues

A rice straw -cellulose utilizing mold was isolated from rotted rice straw residues. The efficient rice straw degrading microorganism was identified as Trichoderma reesei. The results showed that different carbon sources in liquid culture such as rice straw, carboxymethyl cellulose, filter paper, sugar cane bagasse, cotton stalk and banana stalk induced T. reesei cellulase production whereas glucose or Potato Dextrose repressed the synthesis of cellulase. T. reesei cellulase was produced by the solid state culture on rice straw medium. The optimal pH and temperature for T. reesei cellulase production were 6 and 25 ºC, respectively. Rice straw exhibited different susceptibilities towards cellulase to their conversion to reducing sugars. The present study showed also that, the general trend of rice straw bioconversion with cellulase was more than the general trend by T. reesei. This enzyme effectively led to enzymatic conversion of acid, alkali and ultrasonic pretreated cellulose from rice straw into glucose, followed by fermentation into ethanol. The combined method of acid pretreatment with ultrasound and subsequent enzyme treatment resulted the highest conversion of lignocellulose in rice straw to sugar and consequently, highest ethanol concentration after 7 days fermentation with S. cerevisae yeast. The ethanol yield in this study was about 10 and 11 g.L-¹.

Characteristics of a â-1, 4-D endoglucanase from Trichoderma virens wholly applied in a palm-fruit husk-based diet for poultry layers

The characteristics of an endoglucanase produced by a Trichoderma virens strain T9 newly isolated from a palm-fruit husk dump site, its physiological characteristics and enzyme production were studied. Whole cells of the depolymerizing-enzyme producing T. virens were applied to palm-fruit husk and bird performance characteristics when employed as poultry diet additive were considered. Endoglucanase activity in submerged fermentation was 1.6 nkat. Optimum activity was recorded at pH 6.0 and 55ºC. The enzyme retained 50% residual glucanase activity at 70ºC for 10 minutes. 1.0% Tween-80 and SDS yielded endoglucanase activity 2.15 times higher than the control. Activity wasboosted by 20mM Ca2+ (115.0%); 10mM K+ (106.5%); and was totally inhibited by 1mM Hg2+. The addition of T. virens -fermented palm-fruit husk with other layer feed components on the bird characteristics showed that change in bird weight between the control and test birds were not significantly different (p>0.05) but differed in terms of daily feed ingested (p<0.05). The feed to weight-gain ratio was best with the unmodified palm-fruit husk based diet (8.59). There was no significant difference in the egg weights from modified palm-fruit husk based diet and control (p>0.05). The shell thickness (0.64mm) and yolk content (23.61%) were highest in the microbially-modified husk diet. The alternative to maize based diets proffered by the application of T. virens -modified palm-fruit husk in poultry nutrition in terms of bird weight and feed to weight-gain ratio affords the poultry farmer an economic advantage and allows for a greater utilization of the maize in human diets.

Effect of acid hydrolysis and fungal biotreatment on agro-industrial wastes for obtainment of free sugars for bioethanol production

This study was designed to evaluate selected chemical and microbiological treatments for the conversion of certain local agro-industrial wastes (rice straw, corn stalks, sawdust, sugar beet waste and sugarcane bagasse) to ethanol. The chemical composition of these feedstocks was determined. Conversion of wastes to free sugars by acid hydrolysis varied from one treatment to another. In single-stage dilute acid hydrolysis, increasing acid concentration from 1 % (v/v) to 5 % (v/v) decreased the conversion percentage of almost all treated agro-industrial wastes. Lower conversion percentages for some treatments were obtained when increasing the residence time from 90 to 120min. The two-stage dilute acid hydrolysis by phosphoric acid (1.0 % v/v) followed by sulphuric acid (1.0 % v/v) resulted in the highest conversion percentage (41.3 % w/w) on treated sugar beet waste. This treatment when neutralized, amended with some nutrients and inoculated with baker's yeast, achieved the highest ethanol concentration (1.0 % v/v). Formation of furfural and hydroxymethylfurfural (HMF) were functions of type of acid hydrolysis, acid concentration, residence time and feedstock type. The highest bioconversion of 5 % wastes (37.8 % w/w) was recorded on sugar beet waste by Trichoderma viride EMCC 107. This treatment when followed by baker's yeast fermentation, 0.41 % (v/v) ethanol and 8.2 % (v/w) conversion coefficient were obtained.

The hydrolysis of agro-industrial residues by holocellulose-degrading enzymes

Holocellulose structures from agro-industrial residues rely on main and side chain attacking enzymes with different specificities for complete hydrolysis. Combinations of crude enzymatic extracts from different fungal species, including Aspergillus terreus, Aspergillus oryzae, Aspergillus niger and Trichoderma longibrachiatum, were applied to sugar cane bagasse, banana stem and dirty cotton residue to investigate the hydrolysis of holocellulose structures. A. terreus and A. oryzae were the best producers of FPase and xylanase activities. A combination of A. terreus and A. oryzae extracts in a 50% proportion provided optimal hydrolysis of dirty cotton residue and banana stem. For the hydrolysis of sugar cane bagasse, the best results were obtained with samples only containing A. terreus crude extract.

Production of lytic enzymes by Trichoderma isolates during in vitro antagonism with Aspergillus niger, the causal agent of collar rot of peanut

Twelve isolates of Trichoderma (six of T. harzianum, five of T. viride, one of T. virens), which reduced variably the incidence of collar rot disease caused in peanut by Aspergillus niger Van Tieghem, were evaluated for their potential to produce lytic enzymes during in vitro antagonism. T. viride 60 inhibited highest (86.2%) growth of test fungus followed by T. harzianum 2J (80.4%) at 6 days after inoculation (DAI) on PDA media. The specific activities of chitinase, â-1,3-glucanase and protease were 11, 3.46 and 9 folds higher in T6 antagonist (T. viride 60 and A. niger interactions) followed by 8.72, 2.85 and 9 folds in T8 antagonist (T. harzianum 2J and A. niger interactions), respectively, compared to the activity produced by control petri plate T13 (A. niger alone) at 6 DAI. Activity of these lytic enzymes induced in antagonists' plates comprises the growth of Trichoderma isolates. However, cellulase and poly galacturonase were found least amount in these antagonists treatment. A significant positive correlation (p=0.01) between percentage growth inhibition of test fungus and lytic enzymes - (chitinase, â-1,3-glucanase and protease) in the culture medium of antagonist treatment established a relationship to inhibit growth of fungal pathogen by increasing the levels of these enzymes. Among the Trichoderma isolates, T. viride 60 was found best strain to be used in biological control of plant pathogen A. niger.

Biological control of rice brown spot with native isolates of three Trichoderma species

Brown spot caused by Bipolaris oryzae is an important rice disease in Southern coast of Caspian Sea, the major rice growing region in Iran. A total of 45 Trichoderma isolates were obtained from rice paddy fields in Golestan and Mazandaran provinces which belonged to Trichoderma harzianum, T. virens and T. atroviride species. Initially, they were screened against B. oryzae by antagonism tests including dual culture, volatile and nonvolatile metabolites and hyperparasitism. Results showed that Trichoderma isolates can significantly inhibit mycelium growth of pathogen in vitro by producing volatile and nonvolatile metabolites Light microscopic observations showed no evidence of mycoparasitic behaviour of the tested isolates of Trichoderma spp. such as coiling around the B. oryzae. According to in vitro experiments, Trichoderma isolates were selected in order to evaluate their efficacy in controlling brown spot in glasshouse using seed treatment and foliar spray methods. Concerning the glasshouse tests, two strains of T. harzianum significantly controlled the disease and one strain of T. atroviride increased the seedling growth. It is the first time that the biological control of rice brown spot and increase of seedling growth with Trichoderma species have been studied in Iran.

Exploitation of trichoderma species on the growth of Pythium Aphanidermatum in Chilli

Damping-off of chilli caused by Pythium aphanidermatum is a major nursery disease in vegetables. In vitro experiments evaluated the effect of eight isolates of Trichoderma species (from chilli rhizosphere) were tested against P. aphanidermatum. All the Trichoderma species had varied antagonistic effects against the pathogen. Among them, TVC3 recorded maximum growth inhibition of P. aphanidermatum and produced more amounts of volatile and non-volatile metabolites. The culture filtrate of the Trichoderma isolate TVC3 recorded complete inhibition on the mycelial growth of pathogen at 15 percent concentration. Moreover, chilli seeds treated with culture filtrate of the isolate TVC3 recorded maximum germination percentage, shoot length, root length and vigour index of chilli. The study identified the Trichoderma isolate (TVC3) performed well in inhibiting the mycelial growth of pathogen as well as increased the plant growth in chilli.

Efeito da aplicação de maravilha (Mirabilis jalapa L. ), primavera (Bougainvillea spectabilis L. ) e isolados de Trichoderma na produção de alface / Effect of four o'clock flowers (Mirabilis jalapa L. ), bougainvillea (Bougainvillea spectabilis L. ) and Trichoderma spp. isolates in production of lettuce

Objetivou-se com esse trabalho avaliar o efeito de extratos foliares de primavera (Bougainvillea spetabilis L.) e maravilha (Mirabilis jalapa L.) e de Trichoderma spp., isoladamente ou em combinação, no manejo de viroses e na produção de alface. Os ensaios foram conduzidos em área de produção de hortaliças folhosas no município de Pinhalzinho/SP. O delineamento experimental foi de blocos casualizados, com dez tratamentos comparados com controle químico convencional. Foram observadas reduções de 18 por cento e 32 por cento na população de bactérias da rizosfera nas plantas de alface tratadas com o isolado IB18/22 e extrato de primavera + isolados de Trichoderma spp., respectivamente. Verificou-se que o extrato de maravilha estimulou o aumento do número de UFC/g da população de fungos. Entretanto, não houve diferença significativa na massa fresca da parte aérea e, apesar da pressão de inóculo e da alta densidade populacional de insetos vetores de vírus no local, não foi constatada a ocorrência de viroses, com exceção do espessamento de nervuras, que ocorreu em menor número em plantas tratadas, comparadas as do controle. Economicamente, houve 27,8 por cento de lucratividade e margem bruta de 38,5 por cento, indicando a viabilidade do uso de ferramentas de base ecológica no cultivo de alface. Além disso, os produtos usados são de fácil aquisição, seguros em termos de aplicação, meio ambiente e para o consumidor.

The aim of the work was to evaluate the effect of leaf extracts of bougainvillea (Bougainvillea spetabilis L.) and of four o'clock flowers (Mirabilis jalapa L.) plants and Trichoderma spp., alone or in combination, on viruses management and lettuce production. The tests were conducted in a producing area in Pinhalzinho-SP. The experimental design was randomized blocks with ten treatments in comparison with conventional chemical control. Reductions of 18 percent and 32 percent in the bacterial population of the lettuce rhizosphere treated with IB18/22 isolate and bougainvillea extract + Trichoderma spp. isolates, respectivelly, were observed. Four o'clock plant leaf extract stimulated growth of CFU / g number of fungi population. However, no significant change in fresh mass and size of lettuce was observed, and despite the inoculum pressure and high population density of virus vector insects in place, the occurrence of viruses was not found, except for big-vein syndrome which was higher in control than in treated plants. Economically, 27.8 percent net profit and 38.5 percent gross profit margin were verified, indicating that the use of ecological tools in lettuce crops is feasible. Moreover, the products are easy to obtain, safe to apply and safe for environment and human consumption.

Phosphate solubilization potential and phosphatase activity of rhizospheric Trichoderma spp

Trichoderma sp., a well known biological control agent against several phytopathogens, was tested for its phosphate (P) solubilizing potential. Fourteen strains of Trichoderma sp. were isolated from the forest tree rhizospheres of pinus, deodar, bamboo, guava and oak on Trichoderma selective medium. The isolates were tested for their in-vitro P-solubilizing potential using National Botanical Research Institute Phosphate (NBRIP) broth containing tricalcium phosphate (TCP) as the sole P source, and compared with a standard culture of T. harzianum. All the cultures were found to solubilize TCP but with varying potential. The isolate DRT-1 showed maximum amount of soluble phosphate (404.07 µg.ml-1), followed by the standard culture of T. harzianum (386.42 µg.ml-1) after 96 h of incubation at 30+1(0)C. Extra-cellular acid and alkaline phosphatases of the fungus were induced only in the presence of insoluble phosphorus source (TCP). High extra-cellular alkaline phosphatase activity was recorded for the isolate DRT-1 (14.50 U.ml-1) followed by the standard culture (13.41 U.ml-1) at 72h. The cultures showed much lesser acid phosphatase activities. Under glasshouse conditions, Trichoderma sp. inoculation increased chickpea (Cicer arietinum) growth parameters including shoot length, root length, fresh and dry weight of shoot as well as roots, in P-deficient soil containing only bound phosphate (TCP). Shoot weight was increased by 23 percent and 33 percent by inoculation with the isolate DRT-1 in the soil amended with 100 and 200 mg TCP kg-1 soil, respectively, after 60 d of sowing. The study explores high P-solubilizing potential of Trichoderma sp., which can be exploited for the solubilization of fixed phosphates present in the soil, thereby enhancing soil fertility and plant growth.

Molecular cloning of chitinase 33 (chit33) gene from Trichoderma atroviride / Clonagem molecular do gene quitinase 33 (chit33) em Trichoderma atroviride

In this study Trichoderma atroviride was selected as over producer of chitinase enzyme among 30 different isolates of Trichoderma sp. on the basis of chitinase specific activity. From this isolate the genomic and cDNA clones encoding chit33 have been isolated and sequenced. Comparison of genomic and cDNA sequences for defining gene structure indicates that this gene contains three short introns and also an open reading frame coding for a protein of 321 amino acids. The deduced amino acid sequence includes a 19 aa putative signal peptide. Homology between this sequence and other reported Trichoderma Chit33 proteins are discussed. The coding sequence of chit33 gene was cloned in pEt26b(+) expression vector and expressed in E. coli.

Neste estudo Trichoderma atroviride foi escolhido como superprodutor da enzima quitinase dentre 30 isolados de Trichoderma sp. com base na atividade específica de quitinase. Clones de cDNA e genômico codificando chit33 foram obtidos deste isolado e seqüenciados. A comparação das seqüências genômica e de cDNA para definir a estrutura do gene indicou que este contém três pequenos introns e uma fase aberta de leitura codificando uma proteína de 321 aminoácidos. A seqüência de aminoácidos deduzida inclui um possível peptídio sinal de 19 aminoácidos. Homologia entre esta seqüência e outras proteínas Chit33 descritas de Trichoderma é discutida. A seqüência codificadora do gene chit33 foi clonada no vetor de expressão pET26b(+) e expressa em E. coli.

Xylanase production with xylan rich lignocellulosic wastes by a local soil isolate of Trichoderma viride / Produção de xilanase com resíduos lignocelulósicos ricos em xilana por uma cepa local de Trichoderma viride isolada de solo

In the present study, cultural and nutritional conditions for enhanced production of xylanase by a local soil isolate of Trichoderma viride, using various lignocellulosic substrates in submerged culture fermentation have been optimized. Of the lignocellulosics used, maize straw was the best inducer followed by jowar straw for xylanase production. The highest activity achieved was between 14 to 17 days of fermentation. A continuous increase in xylanase production was observed with increasing level of lignocellulosics in the medium and highest activity was observed with maize straw at 5 percent level. Xylanase production with higher levels of lignocellulosics (3 to 5 percent) of maize, jowar and barseem was found to be higher as compared to that with commercial xylan as carbon source. Sodium nitrate was the best nitrogen source among the six sources used. Maximum xylanase production was achieved with initial medium pH of 3.5-4.0 and incubation temperature of 25ºC.The enzyme preparation was effective in bringing about saccharification of different lignocellulosics. The xylanase production could be further improved by using alkali treated straw as carbon source.

Neste estudo, otimizou-se as condições culturais e nutricionais para produção aumentada de xilanase por uma cepa local de Trichoderma viride isolada de solo, empregando-se vários substratos lignocelulósicos, em fermentação submersa. Entre os substratos utilizados, o melhor indutor de produção de xilanase foi palha de milho, seguido de palha de sorgo. A atividade mais alta foi obtida entre 14 e 17 dias de fermentação. Com palha de milho observou-se um aumento contínuo na produção de xilanase com o aumento da concentração dos substratos lignocelulósicos no meio, sendo que a melhor atividade foi obtida com 5 por cento de palha de milho. A produção de xilanase com níveis mais altos de (3 a 5 por cento) de milho, sorgo e forragem verde (barseem) foi mais levada do que com xilana comercial como fonte de carbono. Entre as fontes de nitrogênio testadas, a melhor foi nitrato de sódio. Produção máxima de xilanase foi obtida quando o pH inicial do meio foi 3,5 4,0 e a temperatura de incubação 25ºC. A enzima foi eficiente na sacarificação de diferentes substratos lignocelulósicos. A produção de xilanase poderia ser aumentada empregando-se álcali ao invés de palha tratada como fonte de carbono.

Greenhouse evaluation of Bacillus subtilis AP-01 and Trichoderma harzianum AP-001 in controlling tobacco diseases / Avaliação em estufa de dois agentes biológicos de controle de doenças do tabaco

Two biological control agents, Bacillus subtilis AP-01 (LarminarTM) and Trichoderma harzianum AP-001 (TrisanTM) alone or/in combination were investigated in controlling three tobacco diseases, including bacterial wilt (Ralstonia solanacearum), damping-off (Pythium aphanidermatum), and frogeye leaf spot (Cercospora nicotiana). Tests were performed in greenhouse by soil sterilization prior to inoculation of the pathogens. Bacterial-wilt and damping off pathogens were drenched first and followed with the biological control agents and for comparison purposes, two chemical fungicides. But for frogeye leaf spot, which is an airborne fungus, a spraying procedure for every treatment including a chemical fungicide was applied instead of drenching. Results showed that neither B. subtilis AP-01 nor T. harzianum AP-001 alone could control the bacterial wilt, but when combined, their controlling capabilities were as effective as a chemical treatment. These results were also similar for damping-off disease when used in combination. In addition, the combined B. subtilis AP-01 and T. harzianum AP-001 resulted in a good frogeye leaf spot control, which was not significantly different from the chemical treatment.

Dois agentes de controle biológico, Bacillus subtilis AP-01 (Larminar®) e Trichoderma harzianum AP-001 (Trisan®) foram avaliados separadamente ou em combinação quanto à capacidade de controlar três doenças do tabaco: murcha bacteriana (bacterial wilt, Ralstonia solanacearum), tombamento de mudas (damping-off, Pythium aphanidermatum), e mancha olho-de-rã (frogeye leaf spot, Cercospora nicotiana). Os testes foram realizados em estufa, esterilizando-se o solo antes da inoculação dos patógenos. Os patógenos causadores da murcha bacteriana e tombamento de mudas foram inicialmente encharcados e acompanhados com os agentes de controle biológico e, para comparação, com um fungicida químico. Para a mancha olho-de-rã, causada por um fungo anemófilo, utilizou-se um processo de spray ao invés do encharcamento. Os resultados indicaram que nenhum dos dois agentes de controle biológico, aplicado isoladamente, foi capaz de controlar a murcha bacteriana, mas quando em combinação a capacidade de controle foi similar ao do tratamento químico. Resultados semelhantes foram obtidos para o tombamento de mudas. Além disso, a combinação de Bacillus subtilis AP-01 e Trichoderma harzianum AP-001 resultou em um controle muito eficiente da mancha olho-de-rã, que não diferiu significativamente daquele obtido com o tratamento químico.

Sensibilidad in vitro de cepas de Trichoderma aisladas de semillas de soja frente al fungicida Maxim® XL / In vitro sensitivity to Maxim® XL fungicide in strains of Trichoderma isolated from soybean seeds

La utilización combinada de fungicidas a bajas dosis y cepas de Trichoderma tolerantes a los agroquímicos sería una estrategia aplicable para el manejo integrado del cultivo de soja. El objetivo del presente trabajo fue evaluar la sensibilidad in vitro de 3 cepas de Trichoderma aisladas de semillas de soja, frente a curasemilla Fludioxonil 2.5 por ciento + Metalaxil-M 1 por ciento(Maxim® XL). Se ensayaron 8 concentraciones de los ingredientes activos Fludioxonil 2.5 por ciento y Metalaxil-M 1 por ciento entre los rangos 10-5 a 102 ug/mL y 4.10-6 a 40 ug/mL, respectivamente. El fungicida se agregó al medio Agar Papa Glucosa 2 por ciento fundido, sembrándose un disco de agar con micelio en crecimiento activo en el centro de las placas de Petri. Se efectuaron mediciones del diámetro de las colonias cada 24 horas en 4 oportunidades. La concentración efectiva capaz de inhibir el crecimiento micelial en un 50 por ciento (CE50) fue calculada mediante la función de regresión de los valores de inhibición micelial en respuesta a las diferentes concentraciones expresadas logarítmicamente. Las cepas ensayadas fueron altamente sensibles al curasemilla estudiado, con valores de CE50 que variaron entre 8. 10-5 y 1.10-2 ug/mL, los que resultaron muy inferiores a la dosis agronómica del fungicida recomendada. Estos resultados indican que no sería compatible el uso conjunto del fungicida estudiado con los aislamientos de Trichoderma evaluados.

The combined use of low fungicide doses and Trichoderma strains that are resistant to agrochemical products would constitute a viable strategy for integrated soybean cultivation management. The aim of this work was to evaluate in vitro sensitivity to Fludioxonil 2.5 percent + Metalaxil-M 1 percent (MaximR XL) in 3 strains of Trichoderma isolated from soybean seeds. Eight concentrations of the active ingredient Fludioxonil 2.5 percent and Metalaxil-M 1 percent were tested, with a range of 10-5 to 102 ug/mL and 4.10-6 to 40 ug/mL, respectively. The fungicide was added to melted 2 percent Potate Glucose Agar medium, and sowing was carried out at the center of the Petri plates of an agar disk that contained fungus mycelium in active growth. Colony diameters were measured every 24 hours, on four successive occasions. The concentration capable of inhibiting mycelia growth by 50 percent (CE50) was calculated by means of the regression function of mycelial inhibition values obtained as response to different concentrations, which were expressed logarithmically. The three strains tested were highly sensitive to the seed treatment studied, with CE50 values that varied between 8.10-5 and 1.10-2 ug/mL, which are inferior to the agronomic dose recommended. These results indicated that the joint use of the fungicide and the Trichoderma isolates evaluated would not be compatible.

Hongos saprofitos con actividad biológica frente a los fitopatógenos Botrytis cinerea y Alternaria solani / Fungi saprophytes with activity biologica front to the phytopathogenic botrytis cinerea and Alternaria terrace

De un tamizaje realizado entre las cepas fúngicas que conforman el cepario del IIFB, como parte del proyecto “Biodiversidad Microbiana” del Programa UMSA-Asdi/ Sarec que se desarrolla en forma conjunta con eldepartamento de Biotecnología Ambiental de la Universidad de Lund, se seleccionaron 6 cepas con actividad biológica contra Botrytis cinerea y Alternaria solani, estas fueron clasificadas, a nivel de género, comoTrichoderma sp., (12-QD), Cephalosporium sp., (8-QD),Aspergillus sp., (24-QD), Rizophus sp., (291-QD),— Wardomyces sp., (178-QD), Streptomyces sp., (22-QD).Estos microorganismos saprofiticos fueron sometidos a condiciones de fermentación, en estanco y agitado. Losfiltrados de los fermentos que presentaron mayor actividad fueron los obtenidos de Trichoderma sp con un 79,9% de actividad frente a Botrytis cinerea y 61% frente a Alternaria solani. Se observó que la actividadbiológica esta relacionada con la génesis de pigmentos amarillos

Characterization of pectinases from Acrophialophora nainiana and Trichoderma harzianum strain T6

Duas preparações de pectinases foram isoladas de culturas em estado líquido de Acrophialophora nainiana e Trichoderma harzianum linhagem T6, contendo pectina como a fonte de carbono. As atividades de pectina liase e pectina metilesterase foram somente detectadas nas preparações enzimáticas de A. nainiana. A exo-polymethylgalacturonase de A. nainiana foi mais ativa a pH 7,0 e 60oC, enquanto que a atividade ótima da exo-polymethylgalacturonase de T. harzianum linhagem T6 foi obtida a pH 4,3 e 40oC. As suas estabilidades a 40, 50 e 65oC diferiram, sendo que a atividade de exo-polymethylgalacturonase de A. nainiana apresentou maior estabilidade. Ambas preparações enzimáticas mostraram boa estabilidade em pH ácido. Os extratos brutos foram parcialmente parcialmente purificados por ultrafiltração e cromatografia de filtração em gel em coluna de Sephacryl S-100. Estudos de parâmetros cinéticos mostraram que os valores de Km mais baixos foram obtidos com exo-polimetilgalacturonase de T. harzianum linhagem T6. O tratamento de sucos de frutas com amostras de pectinase de A. nainiana e T. harzianumlinhagem T6 resultou em um decréscimo na viscosidade. A pectinase de T. harzianum linhagem T6 parece ser mais eficiente na redução da turbidez de suco de maça. Somente a pectinase de T. harzianum linhagem T6 foi capaz de extrair suco da banana.