内生真菌Trichoderma harzianum次生代谢产物研究

目的:对格木Erythrophleum fordii Oliver的内生真菌菌株Trichoderma harzianum次生代谢产物进行研究。方法:将此内生真菌进行发酵培养,对其发酵液利用色谱方法进行分离纯化,利用波谱方法鉴定得到化合物结构。结果:从其发酵液中分离得到了10个化合物,包括7个环二肽类、2个酚酸和1个嘧啶类化合物,其结构分别为环(R-脯氨酸-甘氨酸)(1)、环-(R-脯氨酸-R-丙氨酸)(2)、环-(S-脯氨酸-R-缬氨酸)(3)、环-(4-甲基-R-脯氨酸-S-正缬氨酸)(4)、环-(R-脯氨酸-R-亮氨酸)(5)、环-(R-脯氨酸-R-苯丙氨酸)(6)、环-(4-羟基-S-脯氨酸-S-亮氨酸)(7)、尿嘧啶(8)、对羟基苯乙醇(9)、间羟基苯乙酸(10)。结论:这些化合物均是首次从该菌株中分离得到。

哈茨木霉(Trichoderma harzianum)多种胞壁降解酶基因表达载体的构建及转化水稻

为提高水稻的抗病性,利用哈茨木霉(Trichodermahazianum)P1菌株的3个胞壁降解酶基因ech42、nag70与gluc78构建了7个植物表达载体,每个基因受独立的Act1启动子调控.构建的7个载体不仅包含3个外源基因的所有组合(A,B,C,A+B,A+C,B+C,A+B+C),而且具有双元载体本身携带的HPT基因与Gus基因,为研究不同T-DNA长度、不同基因组合与不同基因排列方向对植物遗传转化效率以及外源基因在转基因植株中表达的影响提供了一套比较完整的材料.利用本实验室的农杆菌高效转化体系,将所有组合的7个载体分别转入粳稻品种石狩白毛(OryzasativaLssp.Japonicacv.Ishikari-shiroge)中,共获得再生植株1800余株.对部分再生植株进行了PCR检测,证明96%的植株至少携带有外源基因中的一个,80%以上的植株整合有完整的外源基因片断.

Trichoderma harzianum及其近缘种的分子系统学研究

Trichodermaharzianum是木霉属内最常见的一个“集合种”。本研究对来源不同的T .harzianum及其相似种的 4 6个菌株进行了ITS序列测定 ,将其ITS1 5 .8S ITS2序列与来自EMBL的参考菌株的序列进行比较 ,并进行系统发育分析 ,此外对其中的 18个菌株进行了RAPD多态性分析 ,试图明确T .harzianum的多样性以及与其相似种之间的关系。ITS结果表明 ,T .harzianum及其相似种可分成 2个群 (A、B) :A群由T .hamatum、T .asperellum、T .atroviride、T .koningii和T .viride组成 ,并形成 2个分支 ,表明T .viride和T .koningii、T .atro viride的亲缘关系较近 ,而与T .hamatum、T .asperellum较远 ;B群由T .spirale、T .hamatum、T .inhamatum、T .harzianum和T .anam .Hypocreavinosa组成 ,并形成 6个分支。T .inhamatum可分成 2个群 (Ti1、Ti2 )、T .harzianum至少可分成 5个群 (Th1、Th2、Th4、Th5、Th6 )。结果还表明T .hamatum的遗传差异较大 ,T .hama tum的模式菌株归属于A群 ,而其他的T .hamatum的菌株归属于B群。

降解聚乙烯真菌哈茨木霉(Trichoderma harzianum)的筛选和鉴定

从长期覆膜农田土壤中筛选出一种能够降解聚乙烯的菌株——哈茨木霉(Trichoderma harzianum).将筛选出的哈茨木霉接种到以聚乙烯为唯一碳源的培养基中培育30天,聚乙烯失重率约为10%,聚乙烯薄膜表面出现明显的孔洞和裂痕.

哈茨木霉(Trichoerma harzianum)T23生防菌筛选及防治中药材根腐病的研究

研究表明，引起麦冬、丹参、川芎等中药材根腐病的主要病原种群是尖孢镰刀菌及腐皮镰刀菌．为了有效防治中药材根腐病，保护环境，提高品质和产量，通过不同生态区的土样采集，采取病原菌诱饵法及稀释平扳法分离菌株，经筛选、纯化得到抑菌拮抗效果达80％以上的哈茨木霉生防菌T23；不同剂量试验结果表明，施用哈茨木霉T23生物制剂7．5kg／hm^2效果最佳，田间防治麦冬、丹参、川芎等中药材根腐病效果达70％以上，显著优于50％多菌灵粉剂，比对照增产14．2％～24．5％．大面积防治中药材根腐病的防病增产效果与田间小区试验的一致．

哈茨木霉菌(Trichoderma harzianum)TR409诱导辣椒防御酶活性变化及防治疫病效果

采用组织分离法获得辣椒疫霉菌(Phytophthora capsici)Pc-2,分析哈茨木霉菌(Trichoderma harzianum)TR409诱导辣椒茎基部防御酶活性变化及对辣椒疫病防治效果。结果表明:哈茨木霉菌TR409分生孢子和厚垣孢子处理后辣椒体内防御反应酶活性发生显著变化,厚垣孢子比分生孢子处理的酶活性高峰峰值更高,且出现的时间更晚,其中厚垣孢子灌根24h后接种辣椒疫霉菌的处理中辣椒体内苯丙氨酸解氨酶(PAL)、过氧化物酶(POD)、多酚氧化酶(PPO)和超氧化物歧化酶(SOD)的活性分别比对照提高6.26,2.33,6.41和4.28倍。分生孢子和厚垣孢子处理后辣椒疫病的病情指数分别由对照的59.5降低至29.4和27.6。

Increased resistance of drought by Trichoderma harzianum fungal treatment correlates with increased secondary metabolites and proline content

Plant secondary metabolites play vital role in plant stress response. In this study we investigated whether root colonization of tomato （Solanum lycopersicum） infected by Trichoderma harzianum leads to alterations in the biosynthesis of secondary plant metabolites including phytohormones and osmolyte proline under drought stress. Exposure of tomato to drought caused a drastic decline in plant growth and physiological parameters. Tomato inoculated with T. harzianum showed increased root and shoot growth and chlorophyll pigments as compared to uninoculated controls as well as drought stressed plants. Proline and total soluble protein content was increased in plants inoculated with T. harzianum under both normal as well as drought conditions. An obvious increase in phenol and flavonoid content was observed due to T. haczianum. In addition, T. hat-zianum inoculated plants maintained higher levels of growth regulators indole acetic acid, indole butyric acid, and gibberellic acid under drought stress. Improved secondary metabolites which play an important role in plant stress tolerance by T. hat-zianum may have coordinately worked for bringing the growth regulation by protecting membranes from reactive oxygen species （ROS） and enhance plant growth through accessing more nutrients by root system.

哈茨木霉菌 (Trichoderma harzianum)和终极腐霉菌 (Pythium ultimum)对玉米蛋白质组的影响(I)(英文)

哈茨木霉 (Trichodermaharzianum)T2 2菌株已普遍用于防治包括由终极腐霉 (Pythiumultimum)引起的苗病或根腐病在内的各种病害。玉米自交系Mo17种子经T2 2处理后播种在接种腐霉或未接种的田间土壤内 ,5d后取幼苗的根系或幼茎提取蛋白。结果表明 :在接种腐霉菌的土壤内 ,未进行T2 2处理的 5d龄幼苗长势明显比对照差 ,而经T2 2处理的幼苗长势明显比对照好。T2 2和腐霉菌复合处理及T2 2单独处理对幼苗生长影响基本相同。本研究建立了蛋白质提取和双向电泳分离技术。通过双向电泳及相应的分析软件 (PDQuestTM 2 Dsoftware)可将不同处理的幼苗自交系蛋白进行分离。T2 2菌株处理的根系产生 10 4种上游调控蛋白和 16 4种下游调控蛋白 ,T2 2与腐霉菌复合处理可产生 97种上游调控蛋白和 15 0种下游调控蛋白 ,而用腐霉菌单一处理诱导的上游或下游蛋白的数量明显少于上述 2个处理。T2 2或腐霉菌单一或复合处理的根系蛋白质组图谱与空白对照相比差异显著 ,它们与对照的蛋白质组图谱相似系数分别为 0 .72、0 .5 1和0 .4 9;T2 2与腐霉菌分别处理的蛋白质组图谱间也相差明显 ,两者的相似系数仅为 0 .6 5。进一步研究发现 ,T2 2菌体蛋白质组图谱与上述各种处理的蛋白质组图谱相似系数均很低 ,说明各种处理诱导后的幼苗?

Combined application of Trichoderma harzianum SH2303 and difenoconazole-propiconazolein controlling Southern corn leaf blight disease caused by Cochliobolus heterostrophus in maize

Southern corn leaf blight(SCLB)disease caused by Cochliobolus heterostrophus is one of the major threats to corn production worldwide.The synergistic application of low toxic chemical fungicide and biocontrol agents could improve biocontrol stability and efficiency against plant diseases,which ultimately reduce use of chemical fungicide.Trichoderma spp.,well-known biocontrol fungi have been used to control some foliar diseases.However,few works have been reported on synergistic application of chemical fungicide and Trichoderma against foliar diseases.This study was aimed to investigate the control effect on the synergistic application of Trichoderma harzianum SH2303 and difenoconazole-propiconazole(DP)against SCLB.Results showed that the synergistic application of DP and SH2303 reduced the leaf spot area compared to the control.The efficacy of synergistic application of DP+SH2303 against SCLB could last for 15–20 d in pot trial under the greenhouse condition.Under the natural field condition,maize treated with DP+DP and DP+SH2303 showed 60%control,which was higher than that of SH2303+DP(45%)and SH2303+SH2303(35%).All these treatments induced the synthesis of defense-related enzymes(phenylalanine ammonia lyase(PAL),catalase(CAT),and superoxide dismutase(SOD))and the defence-related gene expression of SA pathway(PR1).Taken together the in-vitro leaf test and field trial,the control of SCLB by synergistic application of DP+SH2303 was similar to that of DP+DP.Among synergistic application,the sequential application of DP+SH2303 showed better control than the sequential application of SH2303+DP.It was concluded that the synergistic application of chemical fungicide(DP)and biocontrol agent(T.harzianum SH2303)could be used to reduce the chemical fungicide and to reduce the SCLB diseases in maize,which provided alternative approach to realize an eco-friendly controlling of the foliar disease.

Ttrichoderma harzianum对Rhizoctonia solani的抗生现象

ＴｒｉｃｈｏｄｅｒｍａｈａｒｚｉａｎｕｍＦＯ６０菌株产生的代谢物质能抑制Ｒｈｉｚｏｃｔｏｎｉａｓｏｌａｎｉ的菌落生长和降低其菌丝干重，其中非挥发性代谢物具热稳定性。光学显微镜和扫描电镜下，发现ＰＯ６０菌丝在Ｒ·Ｓｏｌａｎｉ菌丝上平行或波浪式生长，并在其上形成附着胞或钩状分枝，有溶解和穿入Ｒ·ｓｏｌａｎｉ菌丝，Ｒ·ｓｏｌａｎｉ菌丝断裂和原生质发生颗粒状变化的现象。

Effect of <i>Trichoderma harzianum</i>in Combination with Fungicides in Controlling Gray Mould Disease (<i>Botrytis cinerea</i>) of Strawberry

The effects of biofungicide formula containing the biocontrol agent Trichoderma harzianum (Jn14) as wettable powder in combination with the fungicides pyrimethanil and a mixture of cyprodinil and flydioxonil on Botrytis cinerea of strawberry in vitro, in vivo and in greenhouse were studied. The pathogen B. cinerea was more affected in vitro by low concentrations of the fungicides tested above 0.4 μg&middot;ml-1 than the bioagent T. harzianum (Jn14). The later was almost insensitive to pyrimethanil. In the same direction, gray mould disease severity on strawberry detached leaves and whole plants was reduced up to 89% by the tested fungicides, compared to the control, while the application of T. harzianum (Jn14) alone reduced disease severity up to 45% on strawberry detached leaves, compared to the control. In the integrated control approach, the combination of T. harzianum (Jn14) with higher concentrations of the tested fungicides (ED50) completely inhibited strawberry gray mould disease severity in pots and the greenhouse.

The RACE amplifying and sequence analyzing of secreted aspartic proteinase gene SA76 of Trichoderma harzianum

Total RNA was isolated from mycelium of T. harzianum by Total RNA extraction kit, and two clear bands of rRNA （28S and 18S） were observed in agarose electrophoresis. By joining the 3＇end sequence with the known SA76 EST from cDNA library of T. harzianum, a full-length cDNA sequence of 2019bp was obtained, whose open reading frame contained 1593bp, a stop codon TAA, a 5＇untranslated region （5＇UTR） of 266bp, a 3＇untranslated region （3＇UTR） of 201bp, and poly （A） 29 encoded a protein of 530 amino acids, had a signal peptide. T. harzianum shared 53% identity of secreted aspartic proteinase gene with G. zeae, 37% with N. crassa and 36% with C. globosum. The full-length cDNA sequence of secreted aspartic proteinase gene from T. harzianum was cloned for the first time by using BD SMART RACE technique, which provides a foundation to obtain and validate functional genes of T. harzianum.

Laboratory efficacy of mycoparasitic fungi(Aspergillus tubingensis and Trichoderma harzianum) against tropical bed bugs(Cimex hemipterus)(Hemiptera:Cimicidae)

Objective: To test the effectiveness of conidial spore formulations [Aspergillus tubingensis(A.tubingensis) and Trichoderma harzianum(T.harzianum)] against tropical bed bugs, Cimex hemipterus.Methods: Spore formulations were made from two fungal strains, T.harzianum and A.tubingensis.The bed bugs were exposed to the conidial spores placed soaked onto a fabric cloth for 1 h and the mortality counts were recorded daily until 14 days.Results: Mean survival times based on Kaplan–Meier survival analysis showed no significant differences between all the concentrations in both the fungal isolates:T.harzianum and A.tubingensis.However, the evaluation of both the isolates in terms of virulence resulted in low lethal hours in all the concentrations except for the high concentration of A.tubingensis(LT_(50)= 44.629 h) at the conidial exposure of 1 × 10~6 spores/mL.Rapid mortality of the bed bugs was observed from Day 6 to Day 12, ranging from 13% to 90% in all three concentrations of A.tubingensis.With reference to the T.harzianum exposure, the concentration of 1 × 10~4 spores/mL displayed a gradual increase in the percentage mortality of 90 on Day 14.Conclusions: Approaches to the bed bugs treatment should be explored in-depth using a natural biological agent like fungus especially A.tubingensis to reduce this pest population, in order to replace chemical methods.

Transcriptomic analysis reveals biocontrol mechanisms of Trichoderma harzianum ACCC30371 under eight culture conditions

A transcriptomic database was constructed to study the biocontrol mechanisms of Trichoderma harzianum ACCC30371 using high quality UniGenes following growth in eight culture media[(1/2PD,minimal medium MM(containing dextrose 10 g L^-1),C starvation medium(derived from MM without dextrose),N starvation medium(derived from MM without ammonium sulphate),and four kinds of phytopathogenic fungi cell wall media].A 4 Gbp transcriptome was generated and 96.7%of the database had a sequencing error rate less than 1%.A total of 25,013 UniGene sequences were obtained with a mean length of 1135 nt.There were 2571 sequences longer than 3000 nt.The National Center for Biotechnology Information Accession number of this transcriptome is SRR8382572.There were 16,360 Unigenes annotated to the Nr protein database,9875 to the SwissProt database,10,266 to the KEGG database,7164 to the COG database,and 1508 to the GO database along with their protein functional annotations.There were 16,723 functional genes identified.We identified 402 bio-control genes,including 14 related to competition,311 to mycoparasitism,76 to antibiosis,and one related to eliciting a plant response.This shows that T.harzianum ACCC30371 has integrated biocontrol mechanisms,and of these mechanisms,mycoparasitism is the most prevalent.Antibiosis and induced systemic resistance also play important roles.These results provide a foundation for further research into the biocontrol mechanisms of Trichoderma,as well as the development and utilization of biological fungicides.

Expression of the SOD gene from Trichoderma harzianum in Saccharomyces cerevisiae

Superoxide dismutases are metalloproteins which play a major role in defense against oxygen radicalmediated toxicity in aerobic organisms. Such proteins are important endogeneity cytoprotection factor involving defence. A 751-bp full-length cDNA sequence of an SOD gene was isolated from the Trichoderma harzianum. The full-length cDNA of the SOD gene consists of one 465-bp open reading frame nucleotide, which encodes a 15.7-kDa polypeptide consisting of 154 amino acid residues. Sequence analysis revealed that SOD gene has more than 72%-86% amino acid sequence homology with those of other fungi. The SOD gene was integrated into the genomic DNA of pYES2 by insertion into a single site for recombination, yielding the recombinant pYES2-SOD. SOD expressed by pYES2-SOD was induced by galactose. We test whether SOD could offer abiotic stress resistance when it was introduced into yeast ceils. A transgenic yeast harboring T. harzianum SOD was generated under the control of a constitutively expressed GAL promoter. The results indicated that SOD yeast transformants had significantly higher resistance to salt and drought stress.

Evaluation of Plant Growth Promoting Ability of Bacillus amyloliquefaciens Bc2 and Trichoderma harzianum TR In Vivo

Bacillus amyloliquefaciens Bc2 and Trichderma harzianum TR were used to evaluate their growth-promoting activity on cultivated strawberries, under laboratory and field conditions, and we have noticed that the percentage of achene germination is important for ones treated with TR (=97%) followed by those treated with Bc2 strain (=90%) and the control (=84%). Inoculations on field showed that on untreated soil with insecticide, TR is effective and allows the development of plants and extends the duration of flowering and fruiting. On treated soil, Bc2 clearly promotes the growth and development of strawberry seedlings and its role as plant growth promoting microorganisms has been proved.

Biocontrol Potential of Bacillus amyloliquefaciens Bc2 and Trichoderma harzianum TR against Strawberry Anthracnose under Laboratory and Field Conditions

The increasingly strict regulation of the use of phytosanitary products and the will of the populations to move towards sustainable development allow bio-sourced products and more particularly for biocontrol in the field of pesticides to progress. The study carried out here concerns the evaluation of the potency of Bacillus amyloliquefaciens Bc2 and Trichoderma harzianum to fight against strawberry cultures infected with anthracnose (Fragaria × ananassa Duch.). The studies were carried out in the laboratory and in the field. The results indicate that B. amyloliquefaciens Bc2 and Trichoderma harzianum TR are effective for the biological control of anthracnose, gray mold and powdery mildew on strawberries grown in field conditions. The increase in plant size and the number of fruits produced with these control agents has also been observed.

In Vitro Antagonistic Activity of Trichoderma harzianum and Bacillus amyloliquefaciens against Colletotrichum acutatum

Biological control of plant diseases makes extensive use of the antagonists. The effectiveness of the control depends on the choice of an effective antagonist strains from criteria of implying a good knowledge of biological peculiarities of the material used. In this study, the antagonistic activities of nine isolates of Bacillus amyloliquefaciens and one isolate of Trichoderma harzianum were tested in vitro against seven strains of Colletotrichum acutatum, the causal agent of strawberry anthracnose. It was found that T. harzianum and B. amyloliquefaciens strains showed the ability to inhibit the mycelial growth of the pathogen by dual culture technique by more than 50 percent. The nonvolatile substances produced by the antagonists showed high inhibition percentages that are more than 99 percent, meanwhile their volatile compounds inhibited the mycelial growth of C. acutatum strains with low inhibition percentages starting from 30 percent except the substances produced by B. amyloliquefaciens Bc2 that showed a high inhibition percentage of more than 70 percent.

Carbendazim resistance and calculation effective concentration of carbendazim for Trichoderma harzianum

There is a method for investigating the transformation of resistance gene of carbendazim into Trichoderma harzianum. In order to introduce the resistance to benzimidazole fungicide into bio-control microorganism Trichoderma harzianum was transformed with the resistance gene. In this study, we investigate resistance level and calculate EC 50 (effective concentration of carbendazim that can survive 50% of Trichoderma harzianum in that concentration) and stability of the resistance for the transformant isolate of Trichoderma harzianum. Results show the transformants can growth on the medium containing more than 1 000 μg/ml carbendazim and the resistance is stabled after 10 times transfer on non-selective medium and have EC 50 average about, 1 200 μg/ml.

Primary Study on Biological Control Potential of Trichoderma harzianum TL-1

Trichoderma harzianum is a widely used biocontrol fungus. The growth promoting effect of strain Trichoderma harzianum TL-1 on tomato and pepper and its biological control effects against tomato seedling damping-off and pepper blight were investigated through pot experiments. The results showed that the stain TL-1 had significant promotion effect on growth of pepper and tomato in sterilized and natural soils. With the application dose of 3. 0 and 0. 5g/ pot,their dry weight were increased up to 46% and 150% compared with control,respectively. In addition,TL-1 had good control effects against tomato seedling damping-off and pepper blight. Compared with fungicide treatment,TL-1 treatment could control diseases for long term,without repeat occurrence of diseases.