哈茨木霉Th-33厚垣孢子形成过程的转录组变化分析

应用高通量转录组测序技术对哈茨木霉产厚垣孢子前期和中期两个不同转录本进行测序,获得12186个unigenes,平均长度为1483 bp。被注释到GO(gene ontology)数据库的unigenes有6042个。通过与KEGG(kyoto encyclopedia of genes and genomes pathway database)数据库比对,有5151个unigenes被注释到239条KEGG代谢途径中。比较两个样本,共有差异表达基因(DEG)6329个,包括3602个上调基因,2727个下调基因。其中几丁质酶基因16个,13个上调、3个下调;几丁质合成酶基因4个,均上调;葡聚糖酶基因21个,11个上调、10个下调。KEGG富集分析显示,氨基糖和核苷酸的糖代谢途径涉及的差异表达基因最多,有23个;其次是淀粉和糖代谢途径,有14个差异表达基因参与。推测这两条代谢途径以及相关的差异表达基因可能与厚垣孢子形成有关。本研究为深入研究哈茨木霉Th-33厚垣孢子的形成机制奠定了基础。     

哈茨木霉发酵产木霉素的培养条件优化

采用快速有效的数学统计方法对一株枸骨内生真菌哈茨木霉生产木霉素的培养条件进行了优化。首先利用Plackett-Burrman设计在影响木霉素产量的6个因素中筛选出主效因素：葡萄糖浓度、发酵时间、接种量。在此基础上,再利用响应面分析法对以上三个显著因子的最佳水平范围进行研究,建立并分析了各因子与木霉素产量关系的回归模型。通过模型确定出最佳的试验条件：葡萄糖浓度42.8g/L,接种量1.39ml和发酵时间102.88h,该条件下木霉素的产量可达147.44mg/L。经五批培养验证,预测值与验证试验平均值接近。     

木霉Tr-92菌株厚垣孢子发酵条件的优化

利用单因素试验、正交试验方法对木霉Tr-92厚垣孢子发酵培养基及培养条件进行优化,筛选获得了适合此菌株厚垣孢子产生的最佳培养基组成为:草炭2.5%,玉米浆4.5%,葡萄糖2%,酵母膏0.5%;最佳培养条件为:接种量3%,装液量75mL/250mL,转速为180r/min,初始pH 5.0,温度28℃,培养时间7d。在此培养条件下,木霉Tr-92菌株厚垣孢子产量达到3.01×108个/mL。与优化前相比,厚垣孢子产量增长130.80%。通过培养条件优化确定了适合木霉Tr-92菌株产生厚垣孢子的条件,为高效木霉厚垣孢子生防菌剂的研制奠定了基础。     

利用虫草头孢菌发酵废液培养哈茨木霉

木霉（Ｔｒｉｃｈｏｄｅｒｍａｓｐｐ．）对多种病原真菌均有较好的拮抗作用，培养该菌一般以麦麸为原料。由于麦麸供应较紧张，成本也较高，影响了木霉的推广应用。虫草头孢菌（ＣｅｐｈａｌｏｓｐｏｒｉｕｍｓｉｎｅｎｓｉｓＣｈｅｎ）为名贵中药材冬虫夏草的产生菌，生...     

木霉Tr-92厚垣孢子可湿性粉剂的储存性能及应用效果

为评价木霉Trichoderma spp.厚垣孢子可湿性粉剂的储存性能及应用效果,采用稀释涂平板法测定了保存过程中木霉Tr-92分生孢子和厚垣孢子可湿性粉剂的存活率以及在黄瓜叶片上的定殖能力,并采用盆栽试验法比较了2种木霉制剂对黄瓜灰霉病的防效。结果表明,木霉厚垣孢子制剂贮存180 d时孢子存活率达到72.8%,显著高于分生孢子制剂的42.2%;喷施后7 d,木霉厚垣孢子制剂和分生孢子制剂在黄瓜叶片上的定殖数量分别为1 580个/cm~2和1 490个/cm~2,二者间存在显著差异,且厚垣孢子制剂定殖时间大于分生孢子制剂;1 000倍稀释的木霉厚垣孢子制剂对黄瓜灰霉病的防效达99.20%,与嘧菌酯的防效无显著差异,在喷施后7 d和12 d的防效均显著高于分生孢子制剂。研究表明,木霉厚垣孢子制剂的储存性能和对黄瓜灰霉病的防效均显著优于木霉分生孢子制剂。     

木霉的孢子与土壤抑菌作用

本文从木霉孢子特点、产生、萌发及土壤对孢子的抑制作用方面,对木霉厚垣孢子及分生孢子的研究进展进行综述,以期为合理开发木霉孢子制剂提供理论依据.     

哈茨木霉防治茉莉白绢病效果试验

1977年从水稻叶面分离获得木霉82(T—82)经鉴定为哈茨木霉Trichoderma harzianum Rifai。用这种木霉的分生孢子浓度0.5％和0.7％,防治茉莉白绢病Sclerotium rolfsii,效果分别为97.62％和100％。本文也讨论了防治机理,以及对其它病原真菌的拮抗作用。     

木霉厚垣孢子可湿性粉剂的研制

通过测定不同载体、润湿剂、分散剂、紫外保护剂对木霉厚垣孢子萌发率和菌丝生长以及不同含量助剂对可湿性粉剂性能的影响,确定了木霉厚垣孢子可湿性粉剂的组成成分和比例,其中厚垣孢子粉为25%(厚垣孢子粉由硅藻土吸附木霉发酵液制备而成),润湿剂十二烷基硫酸钠为4%,分散剂CMC为5%,紫外保护剂糊精为1%,以硅藻土补齐100%。该厚垣孢子可湿性粉剂的活孢子数为2.5×109 cfu/g,润湿时间为58s,总悬浮率为78%,孢子悬浮率为85.27%,pH为6.92,含水量为2.16%,98%通过200目标准筛,该制剂的各项指标均符合可湿性粉剂及微生物制剂的相关标准。该研究的进行为农业生产提供一种新型的木霉制剂,对稳定木霉制剂的田间防效具有重要的意义。     

紫外光诱导哈茨木霉产生腐霉利抗性菌株的研究

通过紫外光照射结合使用含药培养基培养得到了 3个哈茨木霉腐霉利抗性突变菌株。抗性菌株与亲本菌株相比较 ,其抗药性提高 1 0 0倍以上 ,与异菌脲、菌核净和甲基立枯磷存在交互抗性。抗性菌株在无药培养基上连续转移培养 8次 ,抗性程度未见下降。在适合度上 ,抗性菌株的菌丝生长速率有所下降 ,而在产孢能力、活体抑菌能力上有的抗性菌株要高于亲本菌株。 3个抗性菌株均保持了对灰霉病菌的拮抗能力     

不同发酵条件对木霉产孢类型的影响

木霉T2 3固体发酵以产生分生孢子为主。液体发酵可以通过控制培养条件来生产不同类型的孢子。马铃薯葡萄糖培养液、马铃薯蔗糖培养液等可作为生产厚垣孢子的培养基 ;木霉T2 3的孢子类型和产孢速度受温度影响。在适于木霉生长的温度范围内 ,2 4℃以下产生厚垣孢子 ,最大产孢量为 1 0 6× 1 0 8个 /ml。 2 8℃以上形成分生孢子 ;30℃时产孢量最大 ,为 1 99×1 0 8个 /ml,温度越高产孢速度越快。初始pH值低易产生厚垣孢子 ,pH值高易产生分生孢子。适当降低液体发酵的供氧量 ,有利于厚垣孢子的形成     

哈茨木霉发酵液中peptaibols抗茵肽的鉴定及活性研究

通过大孔吸附树脂、葡聚糖凝胶、薄层制备硅胶板（TLC）及制备高效液相色谱（HPLC）从哈茨木霉菌Trichoderma harzianum发酵液中分离、纯化出 4 种由 20 个氨基酸组成的线性peptaibols 抗菌肽FD1、FD2、FD3、FD4.经ESI-IT-MS串联质谱鉴定,其分别为抗菌肽Alamethicin F-50、Atroviridin B、Alamethicin II和Atroviridin C.菌丝生长及孢子萌发实验表明其对水稻纹枯病菌Rhizoctonia solani AG4、黄瓜立枯病菌R. solani、黄瓜枯萎病菌Fusarium oxysporum f. sp. cucumerinum、西瓜枯萎病菌F. oxysporum f. sp. niveum、水稻稻瘟病菌Magnaporthe oryzae具有较好的抑制作用,同时发现木霉素Trichodermin与peptaibols抗菌肽Alamethicin F-50联合作用时对菌丝生长具有协同抑制作用.     

Integrated control of Rhizoctonia solani by methyl bromide and Trichoderma harzianum

Under laboratory conditions, isolate TH–203 of Trichoderma harzianum was found to be tolerant of up to 20 000 ppm methyl bromide (MB) (v/v), whereas the plant pathogen Rhizoctonia solani was susceptible to a dose of less than 9000 ppm (v/v). Exposure to sub–lethal concentrations of MB had no effect on the in vitro antagonistic ability of T. harzianum . Soil fumigation with MB at the equivalent of a commercial dose of 500 kg/ha did not reduce the population of Trichoderma in soil and allowed rapid colonization of Trichoderma to develop in the soil.
Under greenhouse conditions a combination of T. harzianum and a reduced dose of MB (equivalent to 200 kg/ha) completely controlled disease incidence of R. solani in bean seedlings compared with controls in untreated soils. Similar disease control was achieved with the recommended dose of MB. Under field conditions, the combination of 200 kg/ha MB and T. harzianum gave a significant synergistic effect on damping–off of carrot seedlings caused by R. solani , and had a similar effect on growth, yield and disease control to that of the recommended dose.
T. harzianum was also able to prevent reinfestation by R. solani in fumigated soils.     

Induced resistance to foliar diseases by soil solarization and Trichoderma harzianum

The effect of soil solarization and Trichoderma harzianum on induced resistance to grey mould (Botrytis cinerea) and powdery mildew (Podosphaera xanthii) was studied. Plants were grown in soils pretreated by solarization, T. harzianum T39 amendment or both, and then their leaves were inoculated with the pathogens. There was a significant reduction in grey mould in cucumber, strawberry, bean and tomato, and of powdery mildew in cucumber, with a stronger reduction when treatments were combined. Bacillus, pseudomonad and actinobacterial communities in the strawberry rhizosphere were affected by the treatments, as revealed by denaturing gradient gel electrophoresis fingerprinting. In tomato, treatments affected the expression of salicylic acid (SA)‐, ethylene (ET)‐ and jasmonic acid (JA)‐responsive genes. With both soil treatments, genes related to SA and ET – PR1a, GluB, CHI9 and Erf1 – were downregulated whereas the JA marker PI2 was upregulated. Following soil treatments and B. cinerea infection, SA‐, ET‐, and JA‐related genes were globally upregulated, except for the LOX genes which were downregulated. Upregulation of the PR genes PR1a, GluB and CHI9 in plants grown in solarized soil revealed a priming effect of this treatment on these genes' expression. The present study demonstrates the capacity of solarization and T. harzianum to systemically induce resistance to foliar diseases in various plants. This may be due to either a direct effect on the plant or an indirect one, via stimulation of beneficial microorganisms in the rhizosphere.     

Biological Control of the Root-Knot Nematode Meloidogyne javanica by Trichoderma harzianum

ABSTRACT The fungal biocontrol agent, Trichoderma harzianum, was evaluated for its potential to control the root-knot nematode Meloidogyne javanica. In greenhouse experiments, root galling was reduced and top fresh weight increased in nematode-infected tomatoes following soil pretreatment with Trichoderma peat-bran preparations. The use of a proteinase Prb1-transformed line (P-2) that contains multiple copies of this gene improved biocontrol activity in the greenhouse experiments compared with the nontransformed wild-type strain (WT). All the Trichoderma strains showed the ability to colonize M. javanica-separated eggs and second-stage juveniles (J2) in sterile in vitro assays, whereas P-2 also penetrated the egg masses. This protease-transformed line presented the same nematicidal and overall proteolytic activity as the WT in in vitro tests in which concentrated soil extracts from Trichoderma-treated soils immobilized the infective J2. However, the J2 immobilization and proteolytic activities of both P-2 and the WT were higher than those obtained with strain T-203. Characterization of the activity of all Trichoderma strains soil extracts on J2 showed that it was heat resistant and restricted to the low-molecular-weight fraction (less than 3 kDa). It is suggested that improved proteolytic activity of the antagonist may be important for the biological control of the nematodes.     

哈茨木霉Th-33全基因组序列测定与分析

利用Hiseq2500高通量测序平台对哈茨木霉Th-33全基因组进行序列测定,获得196个scaffolds,共预测了10849个基因,平均长度为1776 bp(GenBank登录号:PRJNA272949)。以GO(gene ontology)数据库对预测出的基因做基因注释,共注释基因6238个;以KEGG(kyoto encyclopedia of genes and genomespathway database)数据库对预测出的基因做基因注释,有6789个基因注释到279条KEGG代谢途径。KEGG富集分析显示,对氨基苯甲酸甲酯降解代谢通路涉及基因最多,有232个基因;其次是双酚降解代谢通路,有206个基因。利用Rfam数据库对基因组序列进行RNA分类预测,共分为25个类别,包含7123个基因,其中涉及基因最多的为转录后修饰、蛋白质翻转和分子伴侣一类。比较了哈茨木霉、深绿木霉、绿木霉以及里氏木霉基因组中重寄生相关的碳水化合物活性酶、蛋白酶及次生代谢相关基因。研究结果有助于深入了解木霉菌的生防机制、推动木霉菌功能基因的挖掘和利用。     

哈茨木霉S30胞外几丁质酶的纯化及特性

哈茨木霉Ｓ３０在ＳＭＣＳ液体培养基中恒温摇床（２００ｒ／ｍｉｎ,２７．５℃）上培养１５天,培养滤液经硫酸铵分级沉淀、ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ Ｆａｓｔ Ｆｌｏｗ阴离子交换柱层析、Ｐｈｅｎｙ。－Ｓｅｐｈａｒｏｓｅ Ｆａｓｔ Ｆｌｏｗ疏水层析、ＣＭ－Ｓｅｐｈａｒｏｓｅ Ｆａｓｔ Ｆｌｏｗ阳离离子柱层析、Ｐｈｅｎｙｌ－Ｓｅｐｈａｒｏｓｅ ＨＰ疏水柱层析获得了凝胶电泳谱带单一的几丁质酶。几丁质酶反应的     

Biological control by Trichoderma harzianum of damping-off of lettuce caused by Rhizoctonia solani1

Ten isolates of Trichoderma harzianum were tested for their ability to control lettuce seedling damping-off caused by introduced Rhizoctonia solani. T. harzianum isolates TRC 9 and 28 both reduced damping-off. Dual culture experiments were used to select isolates for the study of antibiotic production and mycoparasitism. T. harzianum isolate TRC 12 produced volatile and non-volatile antibiotics, whilst TRC 33 produced only non-volatile antibiotics. T. harzianum isolates 018-2/Y and TRC 9,15 and 28 mycoparasitized R. solani by coiling around and lysing the host hyphae. It appeared that mycoparasitism was more important than antibiosis in the biological control of damping-off.     

哈茨木霉对水稻恶苗病菌的拮抗作用

PDA平板拮抗试验表明 ,哈茨木霉对水稻恶苗病菌有强烈的拮抗作用 ,其孢子悬浮液的含孢量为 10⁶～10⁷个 /mL时 ,对恶苗病菌的抑制力达 92.33%。通过哈茨木霉菌液和 3种药剂对水稻恶苗病菌抑制效果的比较 ,哈茨木霉孢子悬浮液含孢量为 10⁷个 /mL与施保克质量浓度 1μg/mL的抑菌效果接近 ,分别为 76.7%、75.4%。显微摄影结果显示 ,哈茨木霉以附着胞附着在恶苗病菌菌丝上 ,然后穿透菌丝在其内生长 ,或与恶苗病菌的菌丝平行生长 ,然后再侵入病菌内寄生。