Isolation and expression of two polyketide synthase genes from Trichoderma harzianum 88 during mycoparasitism

Abstract Metabolites of mycoparasitic fungal species such as Trichoderma harzianum 88 have important biological roles. In this study, two new ketoacyl synthase (KS) fragments were isolated from cultured Trichoderma harzianum 88 mycelia using degenerate primers and analysed using a phylogenetic tree. The gene fragments were determined to be present as single copies in Trichoderma harzianum 88 through southern blot analysis using digoxigenin-labelled KS gene fragments as probes. The complete sequence analysis in formation of pksT-1 (5669 bp) and pksT-2 (7901 bp) suggests that pksT-1 exhibited features of a non-reducing type I fungal PKS, whereas pksT-2 exhibited features of a highly reducing type I fungal PKS. Reverse transcription polymerase chain reaction indicated that the isolated genes are differentially regulated in Trichoderma harzianum 88 during challenge with three fungal plant pathogens, which suggests that they participate in the response of Trichoderma harzianum 88 to fungal plant pathogens. Furthermore, disruption of the pksT-2 encoding ketosynthase–acyltransferase domains through Agrobacterium -mediated gene transformation indicated that pksT-2 is a key factor for conidial pigmentation in Trichoderma harzianum 88.

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.

Extracts of Caesalpinia ferrea and Trichoderma sp. on the control of Colletotrichum sp. transmission in Sideroxylon obtusifolium seeds / Extratos de Caesalpinia ferrea e Trichoderma spp. no controle da tramissibilidade de Colletotrichum sp. em sementes de Sideroxylon obtusifolium (Sapotaceae)

ABSTRACT Recent research reports the importance of preserving plants in Brazilian semiarid regions, in this context, the scientific literature has reported different pharmacological studies from plant extracts with an antifungal potential, coming from forest species that can contribute as a control and management strategy in the transmission of phytopathogens. This study aimed to evaluate the effect of biotech treatments in controlling the transmission of Colletotrichum sp. in seeds of S. obtusifolium. In this study, 100 seeds were subjected to the following preventive treatments: fungicide Captan®, extract of Caesalpinia ferrea Mart. Ex. Tul., and biological control with Trichoderma spp. The biological control with Trichoderma spp. and the alternative control using C. ferrea extract provided a greater protection to seeds and seedlings of S. obtusifolium facing the transmissibility of Colletotrichum sp.The treatment based on plant extract is more efficient for this purpose only in large seeds and does not interfere on the germination percentage and speed. Therefore it is necessary to perform other studies with Trichoderma spp. and C. ferrea extract to test different doses of these products.

RESUMO Recentes pesquisas relatam a importância da preservação de plantas do semiárido brasileiro. Neste contexto, a literatura científica tem relatado diferentes estudos farmacológicos com extratos vegetais com potencial antifúngico proveniente de espécies florestais que podem contribuir como estratégia de controle e gerenciamento na transmissão de fitopatógenos. No presente estudo o objetivo foi avaliar o efeito de tratamentos biotecnológicos no controle da transmissibilidade de Colletotrichum sp. em sementes de S. obtusifolium. Neste estudo foram utilizadas 100 sementes submetidas aos seguintes tratamentos preventivos: fungicida Captan®, extrato de Caesalpinia ferrea Mart. Ex. Tul. e controle biológico com Trichoderma spp. O controle biológico com Trichoderma spp. e o alternativo com extrato de C. ferrea proporcionam maior proteção às sementes e plântulas S. obtusifolium quanto a transmissibilidade do Colletotrichum sp. O tratamento à base de extrato vegetal foi o mais eficiente para este fim, apenas em sementes de maior tamanho, por não interferir na porcentagem e velocidade de germinação. Portanto, faz-se necessário à realização de outros trabalhos com Trichoderma spp. e extrato de C. ferrea para testar doses diferentes desses produtos.

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.

Biosynthesis of silver nanoparticles from Trichoderma species.

A total of 75 isolates belonging to five different species of Trichoderma viz., T. asperellum, T. harzianum, T. longibrachiatum, T. pseudokoningii and T. virens were screened for the production of silver nanoparticles. Although all the isolates produced nanoparticles, T. virens VN-11 could produce maximum nanoparticles as evident from the UV-Vis study. The highest Plasmon band was observed at 420 nm at every 24 h that attained maximum intensity at 120 h (0.543). The high resolution transmission electron microscopy (HRTEM) further provided the morphology of the nanoparticles. These nanoparticles were found single or aggregated with round and uniform in shape and 8-60 nm in size. The nitrate reductase activity of VN-11 was found to be 150 nmol/h/mL which confirmed the production of silver nanoparticles through reduction of Ag+ to Ag0.