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(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.

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?

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.

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.

Molecular Similarity between Gibberellic Acid and Gliotoxin： Unravelling the Mechanism of Action for Plant Growth Promotion by Trichoderma harzianum

Besides control of the fungal plant pathogens, another interesting aspect observed when plants are treated with Trichoderma harzianum include effects such as complete and even stand of plants, improved seed germination, increases in plant height and overall enhanced plant growth. No research has yet been conducted to elucidate the mechanism by which these effects occur. Improved seed germination, in particular, suggest that Trichoderma harzianum produces a metabolite that may mimic the plant growth hormone gibberellic acid. The metabolite gliotoxin, produced by Trichoderma harzianum appear to be structurally most similar to gibberellic acid. In this study, common pharmacophore generation and molecular ligand docking simulations were used to evaluate the molecular similarity between gibberellic acid, specifically GA3, and gliotoxin. For the common pharmacophore evaluation, the structure of various gibberellic acids were used to construct a pharmacophore space to which gliotoxin was aligned, and during the molecular docking simulations the gibberellic acid receptor, GID1, served as ligand target for GA3 and gliotoxin. During the common pharmacophore evaluation, gliotoxin was successfully aligned to the common pharmacophore model constructed from various gibberellic acids. Furthermore, molecular docking simulations of gliotoxin and GA3 into the gibberellic acid receptor （GIDI） yielded docking scores of-10.78 kcal/mol for the GA3 molecule from Corina and a docking score of-10.17 kcal/mol for glioto^in. The docking scores suggest that gliotoxin may be able to competitively occupy the receptor space for gibberellic acid, and as such elicit the similar physiological responses observed in literature.

Control Effect of Trichoderma harzianum against Potato Early Dying in China

[Objective] The paper was to explore the optimum dosage and application pattern of Trichoderma harzianum for controlling Potato Verticillium wilt. [ Method] Two trials, sowing seed tubers in bacterial manure, sowing seed tubers in bacterial manure ＋ root irrigation after seedling emergence, were conducted in the paper. [Result] T.harzianum could effectively control potato early dying and promote potato growth, thereby increasing potato yield. The control effects of sowing tuber in 300 times of bacterial manure ＋ root irrigation with 300 times of conidia suspension after seedling emergence were ideal ; the highest control effect and the highest saved yield loss were 72.60% and 33.33%, respectively. [ Conclusion] The paper provides reference for field application of T. harzianum.

Trichoderma harzianum TUV-13对鱼腥草白绢病的防治及其促生长作用

应用抗腈菌唑标记的Trichoderma harzianum TUV-13的分生孢子制剂对鱼腥草Houttuynia cordata进行种茎浸泡和土壤处理。结果表明,两种处理均可促进鱼腥草种茎提早发芽、发根,并提高植株生物量。种茎浸泡处理对芽根萌发、地上部分生长的促进效果较好,而土壤处理则对提高地下茎产量效果更明显;两种方法当年对由Sclemtium rolfsii引起的白绢病的田间防效均可达70%以上,两年后在田间自然状态下,对该病仍有较好的防治作用。其中土壤处理两年后,对白绢病的防效为68.2%。木霉回收试验结果表明,TUV-13菌株可在土壤中较好地定殖,2年后其种群数量为2.31×105cfu.g-1,是土著木霉菌数量的289倍。

Purification and Some Properties of Endo-1,4-β-Glucanases of Trichoderma harzianum UzCF-28

This work aimed at isolation, purification and study of biochemical features of cellulolytic enzymes synthesized by Trichoderma harzianum UzCF-28 strain. Strain UzCF-28 revealed a high cellulolytic activity during submerged cultivation in the liquid culture on modified Mandels nutrient medium, where wheat straw was used as a source of carbon. As a result of purification by precipitation with ammonium sulfate and further ion exchange chromatography, two isoforms of endo- 1,4-β-glucanase-EG II and EG III with molecular weight of 135 and 75 kDa respectively were revealed. The pH optimum for EG I and EG III was 4.5, while for EG II—4.7, irrespective of the applied substrates—either CMC or “Whatman filter” paper. Heating up to 40°C of EG III did not lead to its inactivation, and on the contrary, its activity increased by more than three times comparing to the initial activity of the enzyme, i.e. thermostability of EG III among tested enzymes significantly varied.

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.

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.

Application of Trichoderma harzianum in the control of basal stem rot of oil palms

The palm, Elaeis guineensis, has its origins in Africa but is planted on a commercial basis in several countries. Statistics for 2002 showed that in the lead for land mass under oil palm cultivation is Indonesia, at 3,769,000. ha, followed by Malaysia at 3,376,000. ha; however, the world’s leading producer of palm oil is still Malaysia, since the 1970’s. Both countries are predicted to produce 82.4% of the world’s palm oil production by the year 2005. However, the palm is susceptible to basal stem rot, a devastating disease which results in direct loss of field stands and to which no effective chemical control is yet available. Caused by Ganoderma boninense, infected palms appear symptomless, at the first sign of disease, at least 50% of the internal trunk tissue stem would have actually rotted. This study investigated the efficacy of Trichoderma harzianum (isolate FA 1132) as a biological control agent, using 6-month old oil palm seedlings as models and the experiment performed in a greenhouse at 29- 30 ℃. ambient conditions. The plants were artificially infected with G. boninense and a conidial suspension of 1×109-9×109 spores/mL was applied as a soil drench at 1L/plant every 2 weeks for 20 weeks. The parameters examined were efficacy of the biocontrol agent and the effect of Trichoderma- incorporated mulch in addition to the soil drench. Efficacy was assessed in terms disease severity index (DSI) where a higher percentage indicates a higher severity. Results showed that infection first sets in on untreated plants at week 12 and got worse progressively. The completely untreated plants were all infected and the DSI at 20 weeks after infection (wa.i.) was 92.5%. Plants given only a Trichoderma -infused food base supplement without conidial suspension gave a DSI of 70% whereas those given a conidial soil drench without supplemental food base gave a DSI of 85% at 20 w.a.i. Infected plants given a conidial treatment together with a food base supplement gave a DSI of 5% at 20 w.a.i. This investigation showed that there is potential in the use of T. harzianum (isolate FA 1132) as a biological control agent of basal stem rot and warrant further studies in its mass propagation for field trials.

Colonization and degradation of senescent flowers of zucchini squash by Trichoderma harzianum YC459, a biocontrol agent of gray mold, Botrytis cinerea

In commercial greenhouses, senescent flower petals or flowers of vegetables such as tomato, strawberry, hot pepper and zucchini squash were blighted to be removed from fruits within five days after spraying of Trichoderma harzianum YC459 (TORY, JGreen Inc.), a biocontrol agent with good and consistent efficacy as chemical fungicides for the control of gray mold rot caused by B. cinerea. The mechanism for selective colonization of senescent floral tissues by T. harzianum YC459 was elucidated using fresh and senescent (4 days and 14 days after pollination, respectively) floral tissues of zucchini squash (Cucurbita moschata Duchesne) . The spores of T. harzianum YC459 were produced much more on water agar and liquid culture media supplemented with 5% dry powder of senescent floral tissues than with fresh tissues during 15 days incubation. Mycelial growth was also much better in the media with senescent tissues than with fresh tissues. Enzyme activities of carboxymethyl cellulase, amylase and polygalacturonase in the liquid media, which might be involved in the colonization and degradation of tissues by T. harzianum YC459 were compared. The activities of three enzymes were significantly higher in the media with senescent floral tissues than with fresh floral tissues reaching to the maximum during 9 to 12 days of incubation. Especially, the activities of carboxymethyl cellulase and polygalacturonase of T. harzianum YC459 were much higher than those of other Trichoderma species, T. asperellum, T. viride and T. koningii in the liquid media with senescent floral tissues. Based on the results, the selective colonization and degradation of senescent floral tissues, an important habitat for B. cinerea, may be another mechanism for the biocontrol of gray mold rot of vegetables by T. harzianum YC459.