Trichoderma biodiversity in major ecological systems of China.

An investigation of Trichoderma biodiversity involving a large-scale environmental gradient was conducted to understand the Trichoderma distribution in China. A total of 3,999 isolates were isolated from forestry, grassland, wetland and agriculture ecosystems, and 50 species were identified based on morphological characteristics and sequence analysis of genetic markers. Trichoderma harzianum showed the largest proportion of isolates and the most extensive distribution. Hypocrea semiorbis, T. epimyces, T. konilangbra, T. piluliferum, T. pleurotum, T. pubescens, T. strictipilis, T. hunua, T. oblongisporum and an unidentified species, Trichoderma sp. MA 3642, were first reported in China. Most Trichoderma species were distributed in Jilin and Heilongjiang Provinces in northeast China and the fewest were distributed in Qinghai Province. Based on the division of ecological and geographic factors, forestry ecosystems and low-altitude regions have the greatest species biodiversity of Trichoderma.

Cloning and functional analysis of a novel chitinase gene Trchi1 from Trichothecium roseum.

Chitinases produced by mycoparasites play an important role in disease control in plants. To explore the functions of chitinases in Trichothecium roseum, we cloned a new chitinase gene named Trchi1 from T. roseum by RT (reverse transcription)-PCR techniques. The T. roseum gene, Trchi1, contains an 1278-bp ORF that shares 76 % similarity with chitinase from Bionectria ochroleuca (ABV57861 3G6L_A). A plant expression vector, containing the Trchi1 gene driven by the CaMV35S promoter, was constructed and transformed into tobacco via Agrobacterium tumefaciens. Southern blot analysis showed that Trchi1 was integrated into the tobacco genome. Total chitinase activity in Trchi1-transgenic tobacco leaves was enhanced 2.2- to 5.8- times with respect to non-transgenic leaves. Transgenic tobacco plants transformed with the Trchi1 gene had increased resistance to Alternaria alternata and Colletotrichum nicotianae.

[Characterization and production optimization of a chitinase (Tachil) from Trichoderma asperellum in recombinant Pichia pastoris expression system].

OBJECTIVE: We characterized a chitinase (Tachi1) from Trichoderma asperellum and optimized its production conditions, by methanol induction of the recombinant strain Pichia pastoris GS-tachi1-K transformed with the gene tachi1 (GenBank accession: GU457411). METHODS: We purified Tachi1 from the fermentation broth to analyze enzymatic properties after it was secreted by GS-tachi1-K. The production conditions of GS-tachi1-K were optimized by single-factor experiment and orthogonal experiment. RESULTS: The molecular weight of Tachi1 was about 44 kDa. Tachil had a broad range of temperature and pH adaption with the optimal reaction temperature at 50 degrees C and pH 5.5. It was stable at the temperature below 50 degrees C, yet less stable under alkaline conditions. Its activity was significantly reduced by 0.05 mol/L of Ag+, Hg2+, Cu2+, Fe2+, 1% of Sodium dodecyl sulfate (SDS) and 10 mmol/L of beta-mercaptoethanol. The optimum conditions obtained were: initial cell density with an OD600 equal to 2, 0.5% of methanol, pH 6.5, induction time 180 h. Under the optimized condition, the activity of Tachi1 reached 17.93 U/mL and the expression of tachi1 was 6.19 g/L. CONCLUSION: The recombinant strain GS-tachi1-K showed high expression of tachi1 and the protein secreted by GS-tachi1-K had high chitinase activity. It will provide theoretical basis for further research and application in this chitinase.

[Isolation and identification of dominant microorganisms in rhizosphere of continuous cropping with peanut].

OBJECTIVE: We isolated and identified dominant microorganisms from the rhizosphere of continuous cropping with peanut, to study the relationship between dominant microorganisms and peanut continuous cropping. METHODS: By using dilution-plate method we isolated dominant bacteria, dominant fungi and actinomycetes from the rhizosphere of continuous cropping with peanut. Morphological specificity, culture shape, physiological-biochemical characteristic and partial 16S rDNA sequences were used to identify bacteria and actinomycetes. Morpholog, growth on various media, and Internal Transcribed Spacer (ITS) rDNA sequences homology analysis were performed to identify dominant fungi. RESULTS: We isolated seven dominant bacteria strains, seven dominant fungi and seven dominant actinomycetes. Dominant bacteria were identified as Leifsonia xyli, Arthrobacter chlorophenolicus, Microbacterium flavescens, Sphingomonas sp., Pasteurella sp., Bacillus simplex and Bacillus megaterium. Dominant fungi were identified as Cladosporium cladosporioides, Penicillium purpurogenum , Hypocrea lixii, Exophiala pisciphila, Penicillium janthinellum, Aspergillus sp. and Verticillium dahliae. Dominant actinomycetes were identified as Streptomyces violaceoruber, Streptomyces flaveus, Streptomyces panaciterrae, Streptomyces achromogenes, Streptomyces pseudogriseolus, Streptomyces cellulosae and Streptomyces aureus. CONCLUSION: This study was the first time to isolate and identify dominant microorganisms from the rhizosphere of continuous cropping with peanut. The type of dominant microorganisms changed obviously after planting peanut, although the change was without regularity.

Expression of a novel thermostable Cu, Zn-superoxide dismutase from Chaetomium thermophilum in Pichia pastoris and its antioxidant properties.

A new superoxide dismutase (SOD) gene from the thermophilic fungus Chaetomium thermophilum (Ctsod) was cloned and expressed in Pichia pastoris and its gene product was characterized. The specific activity of the purified CtSOD was 2,170 U/mg protein. The enzyme was inactivated by KCN and H(2)O(2) but not by NaN(3), confirming that it belonged to the type of Cu, ZnSOD. The amino acid residues involved in coordinating copper and zinc were conserved. The recombinant CtSOD exhibited optimum activity at pH 6.5 and 60°C. The enzyme retained 65% of the maximum activity at 70°C for 60 min and the half-life was 22 and 7 min at 80 and 90°C, respectively. The recombinant yeast exhibited higher stress resistance than the control yeast cells to salt and superoxide-generating agents, such as paraquat and menadione.