Biolistic transformation of Trichoderma harzianum and Gliocladium virens using plasmid and genomic DNA

Biolistic (biological ballistic) and protoplast-mediated procedures were compared as methods for transforming strains of Gliocladium virens and Trichoderma harzianum. For biolistic transformation, conidia were bombarded using a helium-driven biolistic device to accelerate M5 tungsten particles coated with plasmid or genomic DNA. DNA from either source contained a bacterial hygromycin B resistance gene (hygB) as a dominant selectable marker. The same sources of DNA were also used to transform protoplasts using a standard polyethylene glycol-CaCl₂ protoplast fusion protocol. Hygromycin B-resistant (HygB^R) transformants were recovered from all strains, methods, and DNA sources except for genomic DNA used with the protoplast method. The biolistic procedure was technically simpler, and increased transformation frequency and genetic stability in the progeny as compared with the protoplast-mediated transformation. Southern analysis of homokaryotic HygB^R progenies showed that the transforming sequences were integrated into the genome of the recipient strains, and apparently were methylated. This is the first study presenting detailed results on biolistic transformation of a filamentous fungus.     

卡伍尔链霉菌TJ430的分离鉴定及抗菌活性研究

目的 从土壤中分离、筛选稀有放线菌,对其产生的抗生素进行评价,以期发现新型农用抗生素.方法 采用高温烘烤法对土壤中稀有放线菌进行富集,采用改良的HV培养基进行分离.对分离获得的编号为TJ430的菌株采用形态学观察法、细胞化学组分分析法、生理生化及酶学特性分析、16S rDNA序列分析和DNA杂交法进行鉴定.菌株TJ430产生的抗生素水溶液在温室内进行生物防效试验.结果 共计分离获得570株稀有放线菌,其中TJ430表现出优良的广谱抗真菌活性.鉴定表明T J430是一株卡伍尔链霉菌.生物防效试验结果表明,TJ430产生的抗生素水溶液对番茄晚疫病、番茄细菌性斑点病、黄瓜菌核病及黄瓜炭疽病的防效分别为100％、79.36％,98.11％和75.79％.结论 菌株TJ430产生的抗生素具有宽泛的抗菌谱和良好的抗菌活性,具备较好的开发应用前景.     

The heterologous overexpression of <Emphasis Type="Italic">hsp23</Emphasis>, a small heat-shock protein gene from <Emphasis Type="Italic">Trichoderma virens</Emphasis>, confers thermotolerance to <Emphasis Type="Italic">T. harzianum</Emphasis>

An EST showing high values of identity with genes coding for small heat shock proteins (sHSPs) was selected from an EST library collection of Trichoderma virens T59. The cDNA gene (hsp23) with a sequence size of 645 bp long was amplified by PCR. The expression of this gene was evaluated in cultures grown at temperatures ranging from 4 to 41°C. An increased level of expression was detected when the fungus was grown at extreme temperatures (4, 10 or 41°C). A high-expression level was also observed when the fungus was grown in 10% ethanol for 4 h. The hsp23 gene was present as a unique copy in the T. virens genome, and a homologous gene was also present in other five investigated Trichoderma species. Strain T. harzianum T34 was transformed with the hsp23 gene from T. virens T59 under the control of the pki (pyruvate kinase) promoter from T. reesei and the ble (phleomycin resistance) gene as selection marker. Statistically significant differences were detected between the strains T34 and two selected transformants in the biomass quantities obtained after heat shock treatment and in the colony diameters after incubation at 4°C for 2 months.     

安汶巨蚊生物学的实验室进一步观察

在恒温恒湿条件下,观察并研究了安汶巨蚊(Toxorhynchites amboinensis)的某些生物学特性。该蚊发育历期:卵2天幼虫11—12天、蛹期5天。该蚊幼虫每天捕食35条白纹伊蚊(Ae.albopictus)幼虫;随食量减少该蚊幼虫期延长,最长可达80天:成蚊羽化后第八天产卵,平均每天产11粒;雌蚊寿命40天,雄蛇95天;雌雄性比例为1:1.5(♀:♂1:1.5)。     

Characterization of an antifungal compound produced by Bacillus sp. strain A5F that inhibits Sclerotinia sclerotiorum

A potential antagonist, Bacillus sp. strain A₅F was isolated from soybean rhizosphere following in vitro dual plate screening. The bacterium displayed strong inhibitory activity in vitro against soybean stem rot pathogen, Sclerotinia sclerotiorum. The culture supernatant of strain A₅F completely suppressed the mycelial growth of the pathogen, indicating that suppression was due to the presence of antifungal compounds in the culture filtrate. The culture filtrate also suppressed other phytopathogenic fungi including Fusarium oxysporum and Macrophomina phaseolina, in vitro suggesting a broad spectrum antagonistic activity against fungal pathogens. Chemical extraction followed by chromatographic analysis resulted in two antifungal fractions. The high resolution‐electron spin ionization‐mass spectrometry (HR‐ESI‐MS) and Nuclear Magnetic Resonance (1D and 2D¹H) spectra of these antifungal fractions revealed the presence of antifungal compounds, one of which showed similarity to bacillomycin D. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Development of a strain-specific SCAR marker for the detection of Trichoderma atroviride 11, a biological control agent against soilborne fungal plant pathogens

The genus Trichoderma includes biocontrol agents (BCAs) effective against soilborne plant pathogenic fungi. Several potentially useful strains for biological control are difficult to distinguish from other strains of Trichoderma found in the field. So, there is a need to find ways to monitor these strains when applied to natural pathosystems. We have used random amplified polymorphic DNA (RAPD) markers to estimate genetic variation among sixteen strains of the species T. asperellum, T. atroviride, T. harzianum, T. inhamatum and T. longibrachiatum previously selected as BCAs, and to obtain fingerprinting patterns. Analysis of these polymorphisms revealed four distinct groups, in agreement with previous studies. Some of the RAPD products generated were used to design specific primers. Diagnostic PCR performed using these primers specifically identify the strain T. atroviride 11, showing that DNA markers may be successfully used for identification purposes. This SCAR (sequence-characterised amplified region) marker can clearly distinguish strain 11 from other closely related Trichoderma strains. Received: 9 June 2000 / Accepted: 5 October 2000