木霉几丁质酶及其基因的研究进展

现已报道8种木霉的39个几丁质酶基因,其中22个为42kD内切酶基因.这些42kD内切酶基因的核苷酸序列及其编码的前体蛋白的氨基酸序列极为相似.42kD几丁质酶基因开放阅读框一般有3个内含子,前体蛋白一般有约22AA的信号序列,引导输出.成熟蛋白具有催化域、磷酸化域和耱基化域.基因的表达是诱导型的,受几丁质及真菌几丁质细胞壁的强诱导,受代谢物阻遏.转入植物的木霉几丁质酶基因可正确表达.本文就木霉生防菌株几丁质酶及其基因克隆、几丁质酶基因结构和功能、几丁质酶基因的表达和调控,以及木霉几亍质酶的生防应用前景等几方面进行了综述.     

拟康宁木霉T 51几丁质酶活性及内切几丁质酶基因克隆与分析

木霉是一类重要的生防真菌,木霉产生的几丁质酶在其生物防治的重寄生过程中起着重要作用。拟康宁木霉Trichoderma koningiopsis T-51是一株对番茄灰霉病有生防潜力的木霉菌株,本文测定了T-51菌株产生几丁质酶的活性,结果表明T-51在PDB中液体培养以及与灰霉病菌在PDA上对峙培养时产生的几丁质酶活性显著受到灰霉病菌的诱导。采用RACE技术首次克隆了拟康宁木霉T-51中一个几丁质酶基因Tkchit42,全长为1 817bp,包含4个外显子和3个内含子。预测该基因有一个1 275bp的开放阅读框,编码424个氨基酸,预测的蛋白总分子量为46.378kDa,预测的等电点(pI)为5.16,与T.koningii中42kDa内切几丁质酶的氨基酸序列相似性达到99%。     

哈茨木霉几丁质酶诱导及其对水稻纹枯病菌的拮抗作用

 在实验室条件下分别以几丁质和水稻纹枯病菌(Rhizoctonia solani)细胞壁作唯一碳源诱导哈茨木霉(Trichoderma harzianum)菌株NF9、TC3和P1产生几丁质酶,用硫酸铵沉淀法制备几丁质酶粗提液。上述木霉菌株内切几丁质酶活性(对胶体几丁质浑浊度的减少率)分别为79.8%、74.4%和76.0%,均显著高于非诱导的阳性对照。培养第5 d几丁质诱导的木霉菌株NF9和TC3内切几丁质酶活性显著高于由水稻纹枯病菌细胞壁诱导的酶活性。体外测定表明,通过诱导的木霉菌株TC3、NF9和P1几丁质酶粗提液对水稻纹枯病病菌的拮抗圈直径可达38、21和23 mm,与非诱导的阳性对照比较有显著性差异。对木霉几丁质酶拮抗作用的特点及生防应用进行了讨论。     

寄生于南方根结线虫卵的长梗木霉几丁质酶基因TlChi46的克隆

为进一步筛选高效寄生线虫真菌和阐明其寄生线虫卵的机理,本研究从湖北省烟草南方根结线虫雌虫分离到1株具高效生防潜力的菌株HBF1。形态学、rDNA-ITS和翻译延长因子tef1-α序列分析鉴定该菌为长梗木霉菌Trichoderma longibrachiatum,其对南方根结线虫卵第10 d寄生率为80.45%。通过简并引物设计和RACE技术克隆其几丁质酶基因,分析该基因序列及与其他几丁质酶的同源性。该菌是1株可以产生几丁质酶的南方根结线虫卵寄生真菌,第10 d几丁质酶的活性达到高峰,为24.88μmol/h/mL。本研究首次从长梗木霉HBF1菌株中克隆到1个几丁质酶基因TlChi46,该基因DNA全长1 793 bp,含3个内含子和1个1 272 bp的开放阅读框,编码423个氨基酸,理论分子量45.9 kDa,等电点5.23。同源性比对表明和昆虫寄生菌几丁质酶的亲缘关系较远。从长梗木霉HBF1中克隆得到的几丁质酶基因编码的几丁质酶的功能域可供进一步研究,高效产几丁质酶并有效寄生南方根结线虫卵的长梗木霉对南方根结线虫具有良好的生防潜力。     

木霉菌重寄生过程中的酶学研究进展

木霉是一种重要的生防真菌,重寄生作用是木霉的主要生防机制之一。本文对国内外在木霉重寄生过程中产生的主要酶类及其作用的研究进展进行综述。     

蝇蛆甲壳素对深绿木霉T2产生几丁质酶的诱导作用

分别采用EDTA除钙和氢氧化钠-戊醇反应体系,并结合微波加热处理2次的优化工艺,从蝇蛆中提取甲壳素、壳聚糖,探讨二者对深绿木霉T2菌株产生几丁质酶的影响,并对深绿木霉T2几丁质酶活性及其对4种病原真菌的拮抗作用进行了研究。结果表明,提取的蝇蛆甲壳素的产出率为75.0%,灰分含量为17.3%;蝇蛆壳聚糖制备获得脱乙酰度为95.5%、含水量为8.36%的高质量壳聚糖产品,且碱用量明显下降,反应时间由4 h缩短为6 min;蝇蛆甲壳素培养基诱导培养的深绿木霉T2几丁质酶活性为0.9721 U/mL,高于PDB液体培养基、SCMS营养液和蝇蛆壳聚糖培养基;不同营养条件下,蝇蛆甲壳素培养基均能够诱导深绿木霉T2菌株产生几丁质酶,且最佳产酶培养时间为120 h,酶活性峰值为1.8340 U/mL;经蝇蛆甲壳素诱导后的深绿木霉T2菌株对4种病原菌的抑菌效果明显高于对照菌株T2。     

重寄生真菌盾壳霉产生几丁质酶的条件优化

本试验采用摇瓶培养的方法研究了盾壳霉产生胞外几丁质酶的条件。结果表明：培养液组分、通气状况、表面活性剂和草酸等因子对盾壳霉产生几丁质酶均有影响。改良的马铃薯蔗糖培养液（mPSB）较合成培养基SMCS更适宜作为几丁质酶产生的基础培养基,其几丁质酶产量达到616.8U/L;以mPSB为基础培养基,供试的9种碳源和7种氮源中5g/L葡萄糖和1g/L硝酸钾可使几丁质酶的产量分别达到756U/L和672U/L,较适宜几丁质酶的产生;生长曲线试验显示20℃培养15d几丁质酶产量达到高峰。另外,两种供试表面活性剂对几丁质酶的产生均有抑制作用;一定浓度的草酸溶液（0.1-3g/L）有助于盾壳霉几丁质酶的产生。     

通过染色体整合几丁质酶基因提高伯克氏菌生防能力

为了提高伯克氏菌Burkholderia vietnamiensis的生防能力,通过Tn5转座子介导,将其携带的来自枯草芽孢杆菌Bacillus subtilis的几丁质酶基因整合到野生伯克氏菌B418染色体DNA上,获得工程菌株B418-37。对获得的转化子进行Southern杂交和几丁质酶活性检测,结果证实几丁质酶编码基因已整合到伯克氏菌B418-37染色体DNA上并能表达几丁质酶。生物学特性研究发现几丁质酶基因整合到伯克氏菌染色体中,没有影响野生菌的解磷、解钾、固氮及其在植物根际的定殖能力。平板抑菌试验和温室盆栽试验显示,与野生型相比,该工程菌株对多种病原真菌的抑菌效果增强,说明几丁质酶在植物病害的防治中具有重要作用。上述结果说明通过染色体整合几丁质酶基因是获得多功能生防工程菌的有效途径之一。     

产生几丁质酶的食线虫真菌绿粘帚霉Gliocladium virens CFCC80915对南方根结线虫卵孵化的影响

 根结线虫卵壳主要由几丁质和蛋白质构成。寄生根结线虫卵或产毒真菌产生几丁质酶特性是评价食线虫真菌生物防治潜力的重要生化指标之一。利用还原糖法和pNP法分别测定绿粘帚霉(Gliocladium virens)的几丁质酶的内切酶和外切酶活性。第14d内切酶活性达到最高值,其酶活为53.1μmol/h mL;外切酶活性第26d达到高峰,其酶活为0.432μmol/h mL。利用活性染色电泳测其几丁质酶的分子量分别为75.8kDa、42.8kDa和39.6kDa。表明筛选到的绿粘帚霉CFCC80915菌株具有较高产生几丁质酶的活性。绿粘帚霉12d的培养滤液对根结线虫卵孵化7d后的抑制率达到92.9%。显微观察线虫卵壳变形和破坏情况的结果表明,绿粘帚霉CFCC80915产生的几丁质酶可以引起根结线虫卵壳的裂解,抑制根结线虫卵的孵化。     

哈茨木霉LTR-2双价基因重组菌株的构建及其防治效果

本文利用限制性内切酶介导法转化哈茨木霉LTR-2,获得具有双价基因(glu14、chi42)的重组生防菌株.试验得到的木霉转化子具有遗传稳定性,于无药PDA平板上连续转接6次后,在Hyg 200 μg/mL的PDA平板上仍可继续生长.PCR扩增及Southern杂交证实目的基因已随机插入木霉基因组DNA上,本试验转化子均为单拷贝插入.转化子的β-1,4-葡聚糖酶和几丁质酶水解活性较出发菌株LTR-2显著提高(P＜0.01),其中转化子L-15的两种水解酶活性均最高.转化子在温室条件下对番茄晚疫病、茄子猝倒病和黄瓜灰霉病均有较好的防治作用,但不同处理间存在显著性差异(P＜0.01),其中转化子L-15最高,对3种病害的防治效果分别达到了91.5％、94.9％和83.8％,较出发菌株LTR-2处理分别提高了53.5％、55.9％和33.5％.结果表明,利用REMI技术,将β-1,4-葡聚糖酶基因glu14和几丁质酶基因chi42重组到木霉染色体DNA上,是获得高效重组菌株的有效手段.     

洋葱伯克氏菌群不同基因型菌株对几种重要植物病原真菌的抑制作用及其潜在致病性

从玉米和水稻根围分离到70株不同基因型的洋葱伯克氏菌,对这些菌株进行了拮抗植物病原菌的筛选,并对高拮抗菌株进行了潜在致病性和安全性分析。结果表明,有46株洋葱伯克氏菌对一种或多种病菌有较高的拮抗活性。在不同的洋葱伯克氏菌基因型内,以基因型Ⅴ内拮抗菌株占的比例最高,对所测5种病原真菌的平均拮抗菌株比率为80．0％,其中部分菌株表现出很强的拮抗活性。筛选出的4株高拮抗菌株对洋葱不具有致病性,同时也未检测到与人体致病相关的BCESM毒力基因。     

Chitinase modifying proteins from phylogenetically distinct lineages of Brassica pathogens

Chitinase modifying proteins, cmps, are secreted fungal proteases that truncate specific plant class IV chitinases by cleaving peptide bonds in their amino termini. We recently identified a cmp from the Zea mays (maize) pathogen Fusarium verticillioides and found that it is a member of the fungalysin class of proteases. We also found that Alternaria brassicae, a pathogen of the mustard plant family Brassicaceae, secretes a protease with the same activity. To determine which pathogens of Brassicaceae plants secrete fungalysin cmps, we tested protein extracts from twenty fungi that had been isolated from diseased plants. Each fungal isolate was grown saprotrophically on maize and canola seeds. Secreted fungal proteins were extracted from cultures and incubated with three purified plant chitinases: ChitA and ChitB from maize, and AtchitIV3 from Arabidopsis thaliana. We found that fungalysin cmps were secreted by fungal pathogens distributed among five families in three major Ascomycota classes (Dothideomycetes, Leotiomycetes and Sordariomycetes). Of four fungal species that did not secrete fungalysin cmp activity, three secreted other cmps that truncated maize ChitA and ChitB by cleaving other peptide bonds. AtchitIV3 was only susceptible to truncation by fungalysin cmps. These results show that cmps are commonly secreted by fungal pathogens of Brassicaceae and suggest that interfering with fungalysin cmp activity may improve plant resistance to multiple fungal diseases.     

木霉菌对黄瓜生理特性及立枯病防治效果的影响

黄瓜立枯病是由立枯丝核菌（Rizoctonia solani,简称Rs）侵染引起的苗期土传病害。本研究采用前期筛选获得的对黄瓜立枯病菌Rs具有较强拮抗作用的木霉菌,分别为棘孢木霉Trichoderma asperellum 437、哈茨木霉T.harzianum 670和752,通过盆栽试验和田间试验,测定对黄瓜幼苗的生长和生理特性、产量、品质以及立枯病防效的影响。结果显示：3株木霉菌均能显著促进黄瓜幼苗的生长,提高黄瓜幼苗的生理活性和保护性酶活性,对黄瓜立枯病的防效均在75%以上;木霉菌处理的黄瓜幼苗株高、茎粗、叶面积、根体积、全株鲜重、叶绿素含量、硝酸还原酶活性、根系活力、根系总吸收面积,以及抗逆性酶活性包括超氧化物歧化酶、过氧化物酶、过氧化氢酶、多酚氧化酶均显著高于对照。木霉与病原菌Rs共同处理的黄瓜幼苗,上述生长和生理生化指标均显著高于病原菌Rs单独处理。其中木霉670处理的黄瓜幼苗各项检测指标高于木霉437和752。此外,木霉菌处理的黄瓜单瓜重、产量、可溶性固形物含量、可溶性糖含量、Vc含量、可溶性蛋白含量均不同程度的增加。上述结果说明,本研究的3株木霉菌通过提高黄瓜幼苗生理特性和保护性酶活性,促进了幼苗生长,提高了对黄瓜立枯病的抗性,改善了黄瓜产量和品质。     

An antibiotic complex from Lysobacter enzymogenes strain C3: antimicrobial activity and role in plant disease control

Lysobacter enzymogenes C3 is a bacterial biological control agent that exhibits antagonism against multiple fungal pathogens. Its antifungal activity was attributed in part to lytic enzymes. In this study, a heat-stable antifungal factor (HSAF), an antibiotic complex consisting of dihydromaltophilin and structurally related macrocyclic lactams, was found to be responsible for antagonism by C3 against fungi and oomycetes in culture. HSAF in purified form exhibited inhibitory activity against a wide range of fungal and oomycetes species in vitro, inhibiting spore germination, and disrupting hyphal polarity in sensitive fungi. When applied to tall fescue leaves as a partially-purified extract, HSAF at 25 mug/ml and higher inhibited germination of conidia of Bipolaris sorokiniana compared with the control. Although application of HSAF at 12.5 mug/ml did not reduce the incidence of conidial germination, it inhibited appressorium formation and suppressed Bipolaris leaf spot development. Two mutant strains of C3 (K19 and DeltaNRPS) that were disrupted in different domains in the hybrid polyketide synthase-nonribosomal peptide synthetase gene for HSAF biosynthesis and had lost the ability to produce HSAF were compared with the wild-type strain for biological control efficacy against Bipolaris leaf spot on tall fescue and Fusarium head blight, caused by Fusarium graminearum, on wheat. Both mutant strains exhibited decreased capacity to reduce the incidence and severity of Bipolaris leaf spot compared with C3. In contrast, the mutant strains were as efficacious as the wild-type strain in reducing the severity of Fusarium head blight. Thus, HSAF appears to be a mechanism for biological control by strain C3 against some, but not all, plant pathogenic fungi.     

The Molecular Biology of the Interactions Between Trichoderma spp., Phytopathogenic Fungi, and Plants

ABSTRACT Trichoderma-based biofungicides are a reality in agriculture, with more than 50 formulations today available as registered products worldwide. Several strategies have been applied to identify the main genes and compounds involved in this complex, three-way cross-talk between the fungal antagonist, the plant, and microbial pathogens. Proteome and genome analysis have greatly enhanced our ability to conduct holistic and genome-based functional studies. We have identified and determined the role of a variety of novel genes and gene-products, including ABC transporters, enzymes and other proteins that produce or act as novel elicitors of induced resistance, proteins responsible for a gene-for-gene avirulent interaction between Trichoderma spp. and plants, mycoparasitism-related inducers, plant proteins specifically induced by Trichoderma, etc. We have transgenically demonstrated the ability of Trichoderma spp. to transfer heterologous proteins into plant during root colonization, and have used green fluorescent protein and other markers to study the interaction in vivo and in situ between Trichoderma spp. and the fungal pathogen or the plant.     

茶树内生菌株TL2对茶轮斑病防治效果的研究

从茶树上分离筛选到14株能在茶树体内内生、对茶叶斑病菌拮抗作用较强的芽孢杆菌菌株。14株目标菌株都具有在茶树体内内生定殖的能力,对茶叶斑病菌及其它植物病原菌有一定的拮抗能力。其中,菌株TL2具有较强的内生定殖能力、拮抗能力强且拮抗谱广,对茶轮斑病防病效果强。     

木霉对植物的促生及诱导抗性研究进展

木霉不仅能够直接抑制植物病原菌的生长,还可通过定殖于植物的根部引起植物新陈代谢的改变。本文综述了木霉在植物根部定殖后,通过产生植物生长调节剂、抑制或降解根际有害物质、增加养分利用率以促进植物生长以及通过产生激发子诱导植物形成胞壁沉积物、合成抗菌物质以诱导植物局部或系统抗性的研究进展,以期为进一步扩大木霉在农业生产中的应用提供参考。     

Individual-based approach to modeling hyphal growth of a biocontrol fungus in soil

ABSTRACT Fungi in soil perform beneficial roles that include biological control of soilborne plant pathogens. However, relatively little predictive information is available about the growth and activity of fungal hyphae in soil habitats. A stochastic computer simulation model ("Fungmod") was developed to predict hyphal growth of the biocontrol fungus Trichoderma harzianum ThzID1 in soil. The model simulates a fungal colony as a population of spatially referenced hyphal segments, and is individual-based, in that records of spatial location and branching hierarchy are maintained for individual hyphal nodes. In this way, the entire spatial structure of the fungal colony (hyphal network) can be explicitly reconstructed at any point in time. Also, the soil habitat is modeled as a population of spatially referenced 1-mm(3) soil cells, allowing for the simulation of a spatially heterogeneous environment. Initial hyphal growth parameters were derived from previously published results, and the model was tested against new data derived from image analysis of hyphal biomass accumulation in soil. The ability to predict fungal growth in natural habitats will help to improve the predictability of successful myco-parasitic events in biological control systems.