玉米大斑病菌（Exserohilum turcicum）原生质体的分离及转化

玉米大斑病是严重危害玉米生产的一个世界性真菌病害。由于玉米大斑病菌（Exserohilum turcicum）在无性生长过程中迅速产生黑色素,致使原生质体难以分离。测试了包括Fungase、Funcelase、Novozyme、Glucanex、Driselase、Uskizyme、Kitalase在内的7种细胞壁降解酶及其组合、病原菌菌株和培养基对原生质体分离效果的影响。结果表明菌株的培养形态和菌丝的生长状态显著影响原生质体的分离效率;酶组合Kitalase+Glucanex+Driselase, Kitalase+Glucanex和Kitalase+Uskizyme能够有效地分离玉米大斑病菌的原生质体。初步的转化试验表明,质粒pAN71可以用于该病原菌的转化。这些结果将为E.turcicum和Exserohium属其它真菌的基因克隆提供一些有用的信息。     

西藏地区土壤放线菌种群多样性及拮抗活性研究

从西藏不同海拔、不同气候条件的5个地区采集的10份土样中,使用选择分离培养基、ISP 2（Yeast extract_malt extract agar）和高氏一号培养基,通过分散差速离心法（Dispersion and differential centrifugation,DDC）分离得到放线菌156株。根据形态和培养特征将其归入32个类群,并从中选出65个代表菌株进行全细胞壁氨基酸组分分析,结果表明：９株菌为非链霉菌胞壁类型,其余为链霉菌胞壁类型。16S rDNA扩增片段长度多态性（ARDRA）分析得到不同带型,对其序列分析结果表明：分离菌株分属链霉菌属（Streptomyces）和5个稀有放线菌属。同时测试了代表菌株抗耐药细菌和真菌的活性,其中38.5%的菌株具有拮抗活性。     

六六六（HCH）降解菌Sphingomonas sp. BHC_A的分离与降解特性的研究

从长期受六六六污染的土壤中分离得到一株能以HCH为唯一碳源的高效降解菌株 BHC_A。通过对其主要生理生化特征分析,以及16S rDNA序列的测定和同源性比较分析,将BHC_A鉴定为鞘氨醇单胞菌属（Sphingomonas sp.）。BHC_A菌株在12h以内能够完全矿化浓度分别为5mg/L的α_、β_、γ_、δ_HCH 4种异构体,特别是对β_HCH的降解在国际上也属少例。而前人所报道的γ_HCH降解菌Sphingomonas paucimobilis UT26菌株对β_HCH和δ_HCH不产生降解作用,即使经过24h的培养,对5mg/L的α_HCH的降解率也只有12.6%。在黄瓜的盆钵试验中发现,15d后BHC_A在土壤中对α、β_、γ_、δ_HCH 4种异构体的降解率为84.3%,能够有效地消除土壤中六六六的污染,缓解植株受药害症状。     

新疆青海中度嗜盐放线菌生物多样性初步研究

从我国新疆、青海等地采集数份盐碱土样或泥样,采用淀粉-酪素琼脂培养基、甘油天门冬酰胺琼脂培养基、土壤浸汁琼脂培养基分别从中分离到8株、32株中度嗜盐放线菌菌株。经形态、生理学特性与全细胞壁氨基酸组分分析结果比较,选取其中的14株进行16S rDNA序列分析。就物种多样性而言,新疆、青海分离到中度嗜盐放线菌分布至少有3个科,5个属。其中有拟诺卡氏菌科（Nocardiopsaceae）的拟诺卡氏菌属(Nocardiopsis)和链单孢菌属（Streptomonospora）;假诺卡氏菌科(Pseudonocardiaceae)的普氏菌属(Prauserella)和糖单孢菌属(Saccharomonospora);链霉菌科(Streptomycetaceae)的链霉菌属(Streptomyces)。就地区分布来讲,新疆分离到的中度嗜盐放线菌种类要远高于青海。     

链霉菌S01菌株几丁质酶对植物病原真菌的拮抗作用

纯化后的链霉菌S01菌株几丁质酶用环柱法在PDA平板上对杨树腐烂病菌（Valsasordida）、葡萄孢菌（Botrytissp．AS3．266）、黄瓜黑腥病菌（Cladosporiumcucumerinrm）、辣椒疫病菌（Phytophlhoracopsici）、棉花黄萎病菌（Verliilliumalbo-atrum)、立枯丝核菌（Rhizoctonasolani）等植物病原真菌及产黄青霉（Penlcillumc     

带cry3Aa启动子的aiiA基因在苏云金芽胞杆菌中的表达

N-乙酰高丝氨酸内酯（N-cyl-homoserine lactones,AHLs）,是一类数量感知（Quorum-sensing）系统中的信号分子,它参与诱导调控许多植物病原菌致病基因的表达。苏云金芽胞杆菌的AiiA蛋白能降解这类AHLs分子,进而可减弱病原菌致病基因表达产生的病害。苏云金芽胞杆菌杀虫晶体蛋白基因cry3Aa的启动子是一种不依赖芽胞形成的启动子,它相对于其它cry类基因的启动子有启动基因转录时间早,转录时间长的优点。通过重叠延伸PCR,用杀虫晶体蛋白基因cry3Aa启动子替换编码AiiA蛋白的基因aiiA自身的启动子,构建了融合基因pro3A-aiiA。将融合基因装入穿梭载体pHT304的BamHI/SphI位点,得到重组质粒pBMB686并转化苏云金芽胞杆菌无晶体突变株BMB171,重组菌株BMB686的AiiA蛋白表达量在各个生长时期均高于对照菌株,对AHLs分子的降解活性和对胡萝卜软腐欧文氏菌感染马铃薯产生病害的抑制能力也明显优于对照菌株。     

编码1,3-丙二醇氧化还原酶基因的克隆和表达

采用PCR法克隆了巴氏梭菌（Clostridium pasteurianum）CpN86菌株编码1,3丙二醇氧化还原酶基因（dhaT基因）;完成了dhaT基因测序、表达载体构建和在大肠杆菌中表达;分离和纯化了dhaT基因表达的重组蛋白。实验结果：（1）PCR法克隆的dhaT基因和肺炎克雷伯氏菌Klebsiella pneumoniae菌株dhaT基因的序列同源性为829%;（2）dhaT基因表达蛋白的酶活为108U/mg;（3）dhaT基因表达的蛋白分子量为43kD;（4）Western blot确定了dhaT基因表达的蛋白和 CpN86菌株天然蛋白有相同的抗原反应。     

几种药用植物内生真菌抗真菌活性的初步研究*

从三尖杉 ,南方红豆杉及香榧中分离出 1 72株内生真菌 ,对其进行抗菌活性检测 ,结果表明共 90株内生真菌对一种或多种植物病原真菌 ,如红色面孢霉 (Neurosporasp .) ,木霉 (Trichodermasp .) ,镰刀菌 (Fusariumsp .)等有抑制作用 ,来自三尖杉、南方红豆杉和香榧的抗菌活性菌株比例分别为 40 %,54.2 %及 57.1 %。其中平板抑菌圈直径大于1 5mm的高抗菌株有 3 5株。按Ainsworth等鉴定系统和方     

木霉菌防治植物真菌病害研究进展

木霉菌是一种重要的植物病害生防因子,尤其在防治植物病原真菌病害中一直受到极大的关注。木霉菌依靠其菌株在包括趋向生长、识别、接触、缠绕与穿透等步骤的真菌寄生过程中分泌产生的几丁质酶、葡聚糖酶、纤维素酶、蛋白酶等一系列细胞壁降解酶,进行重寄生作用,拮抗其他植物病原菌,行使其生防功能。我们简要概述了木霉菌的种类、拮抗对象、抑菌机制、诱导抗性、促生作用、基于分子生物学的转基因工程研究,以及木霉菌在植物病原真菌生物防治中的应用。     

芒果炭疽病菌β-微管蛋白基因的克隆及其与多菌灵抗药性发生的关系

参照豆科合萌属（Aeschynomene）作物炭疽病菌的tub1和tub2基因序列设计了2对引物,分别从芒果（Mangifera）炭疽病菌对多菌灵(MBC)田间抗药性（MBCR）和敏感（MBCS）的菌株中扩增β_微管蛋白基因。结果只有以tub2为参照设计的引物扩增到了特异片段。进一步对全基因进行了克隆和测序。该基因序列全长1344bp,编码447aa,其核苷酸和氨基酸序列与豆科合萌属炭疽病菌的tub2基因高度同源。对芒果炭疽病菌抗、感菌株β_微管蛋白氨基酸序列进行比较分析,发现第181、237和363位氨基酸发生了突变,而其它位置（如第198位或200位）均不变。     

Purification of a chitinase from Trichoderma sp. and its action on Sclerotium rolfsii and Rhizoctonia solani cell walls

Trichoderma harzianum is an effective biocontrol agent of several important plant pathogenic fungi. This Trichoderma species attacks other fungi by secreting lytic enzymes, including beta-1,3-glucanase and chitinolytic enzymes. Superior biocontrol potential may then be found in strains having a high capacity to produce these enzymes. We have therefore evaluated the capacity of six unidentified Trichoderma spp. isolates to produce chitinolytic enzymes and beta-1,3-glucanases in comparison with T. harzianum 39.1. All six isolates demonstrated substantial enzyme activity. However, while the isolates hereafter called T2, T3, T5, and T7 produced lower amounts of enzymes, the activity of isolates T4 and T6 were 2-3 fold higher than that produced by T. harzianum 39.1. A chitinase produced by the T6 isolate was purified by a single ion-exchange chromatography step and had a molecular mass of 46 kDa. The N-terminal amino-acid sequence showed very high homology with other fungal chitinases. Its true chitinase activity was demonstrated by its action on chitin and the failure to hydrolyze laminarin and p-nitrophenyl-beta-N-acetylglucosaminide. The hydrolytic action of the purified chitinase on the cell wall of Sclerotium rolfsii was convincingly shown by electron microscopy studies. However, the purified enzyme had no effect on the cell wall of Rhizoctonia solani.     

Mycolytic effect of extracellular enzymes of antagonistic microbes to Colletotrichum falcatum,red rot pathogen of sugarcane

Strains of selected bacteria and Trichoderma harzianum isolated from sugarcane rhizosphere and endosphere regions were tested for the production of chitinolytic enzymes and their involvement in the suppression of Colletotrichum falcatum, red rot pathogen of sugarcane. Among several strains tested for chitinolytic activity, 12 strains showed a clearing zone on chitin-amended agar medium. Among these, bacterial strains AFG2, AFG 4, AFG 10, FP7 and VPT4 and all the tested T. harzianum strains produced clearing zones of a size larger than 10 mm. The antifungal activity of these strains increased when chitin was incorporated into the medium. Trichoderma harzianum strain T5 showed increased levels of activity of N-acetylglucosaminidase and -1,3-glucanase when grown on minimal medium containing chitin or cell wall of the pathogen. Lytic enzymes of bacterial strains AFG2, AFG4, VPT4 and FP7 and T. harzianum T5 inhibited conidial germination and mycelial growth of the pathogen. Enzymes from T. harzianum T5 were found to be the most effective in inhibiting the fungus. When mycelial discs of the pathogen were treated with the enzymes, electrolytes were released from fungal mycelia. The results indicated that antagonistic T. harzianum T5 caused a higher level of lysis of the pathogen mycelium, and the inhibitory effect was more pronounced when the lytic enzymes were produced using chitin or cell wall of the pathogen as carbon source.     

Improvement of Trichoderma strains for biocontrol

The use of the fungal genus Trichoderma to control fungal plant diseases is a promising alternative to the use of chemical compounds. The aim of this work has been to obtain Trichoderma strains with improved capacity as biological control agents. To do so, the hydrolytic capacity on fungal cell walls of strains of the fungus Trichoderma harzianum has been increased. On one hand, transformation experiments with genes which coded for chitinases and glucanases have been carried out in T. harzianumstra ins. On the other hand, the medium composition has also been modified in order to eliminate proteolytic degradation of some of the overproduced enzymes. Finally, hybrid chitinolytic enzymes with substrate-binding domains have been produced as an alternative to obtain improved biocontrol strains. The transformant strains, when compared with the wild type, showed improved antifungal capacity against the phytopathogenic fungus Rhizoctonia solani, in in vitro experiments.     

Enzyme activity of extracellular protein induced in Trichoderma asperellum and T. longibrachiatum by substrates based on Agaricus bisporus and Phymatotrichopsis omnivora

Antagonistic Trichoderma spp. are used throughout the world for the biological control of soil-borne plant diseases. This approach has stimulated an on-going search for more efficient mycoparasitic strains with a high potential for producing extracellular lytic enzymes. This study compares the production of lytic enzymes by native strains of Trichoderma asperellum and Trichoderma longibrachiatum on substrates of differing complexity. The quantity of protein induced by Agaricus bisporus-based medium was higher than that induced by Phymatotrichopsis omnivora-based medium. In P. omnivora medium, T. asperellum exhibited higher chitinolytic and β-1,3-glucanolytic activities than T. longibrachiatum. The enzyme profile was related to the previously reported ability of these strains to inhibit the growth of several soil-borne plant pathogens. NAGase production was similar among the tested indigenous strains of T. longibrachiatum; T479 and T359 produced more endochitinase, T479 produced more glucanase, and T341 and T359 produced more β-1,3-glucanase. The detected variations in glucanase and β-1,3-glucanase activities suggest that the production of these enzymes is strongly influenced by the substrate. Strains T397 and T359 exhibited xylanase activity, which triggers defence mechanisms in plants. Thus, these strains may utilise an additional mechanism of biocontrol.     

Potential of genes and gene products fromTrichoderma sp. andGliocladium sp. for the development of biological pesticides

Fungal cell wall degrading enzymes produced by the biocontrol fungiTrichoderma harzianum andGliocladium virens are strong inhibitors of spore germination and hyphal elongation of a number of phytopathogenic fungi. The purified enzymes include chitinolytic enzymes with different modes of action or different substrate specificity and glucanolytic enzymes with exo-activity. A variety of synergistic interactions were found when different enzymes were combined or associated with biotic or abiotic antifungal agents. The levels of inhibition obtained by using enzyme combinations were, in some cases, comparable with commercial fungicides. Moreover, the antifungal interaction between enzymes and common fungicides allowed the reduction of the chemical doses up to 200-fold. Chitinolytic and glucanolytic enzymes fromT. harzianum were able to improve substantially the antifungal ability of a biocontrol strain ofEnterobacter cloacae. DNA fragments containing genes encoding for different chitinolytic enzymes were isolated from a cDNA library ofT. harzianum and cloned for mechanistic studies and biocontrol purposes. Our results provide additional information on the role of lytic enzymes in processes of biocontrol and strongly suggest the use of lytic enzymes and their genes for biological control of plant diseases.     

Effect of biocontrol strains of Trichoderma on plant growth, Pythium ultimum polulations, soil microbial communities and soil enzyme activities

Five strains of Trichoderma with known biocontrol activities were assessed for their effect upon pea growth and their antagonistic activity against large Pythium ultimum inocula. The effect of Trichoderma inocula upon the indigenous soil microflora and soil enzyme activities in the presence and absence of Pythium is assessed. In the absence of Pythium, Trichoderma strain N47 significantly increased the wet shoot weight by 15% but did not significantly affect the dry weight, whilst strains T4 and N47 significantly increased the root weights by 22% and 80%) respectively. Strains TH1 and N47 resulted in significantly greater root lengths. Pythium inoculation significantly reduced the root length and the number of lateral roots and nodules, and significantly increased the root and rhizosphere soil fungal populations. Pythium inoculation significantly reduced the plant wet and dry shoot weights and significantly increased the wet and the dry shoot/root ratio. All the Trichoderma strains reduced the number of lesions caused by Pythium and increased the number of lateral roots. The effect of the Pythium on emergence and shoot growth was significantly reduced by all the Trichoderma strains except strain To10. Inoculation with Trichoderma strains TH1 and T4 resulted in significantly greater wet root weights (62% and 57%, respectively) in the presence of Pythium compared to the Pythium control. Strain N47 significantly increased the shoot/root ratio compared to the Pythium control. Inoculation with Trichoderma strains T4, T12 and N47 significantly reduced Pythium populations. Pythium increased the activity of C, N and P cycle enzymes, whilst four Trichoderma strains reduced this effect, indicating reduced plant damage and C leakage. Overall, strains T4 and N47 had the greatest beneficial characteristics, as both these strains improved plant growth in the absence of Pythium and reduced plant damage in the presence of Pythium. The dual properties of these strains improve the commercial application, giving them an advantage over single action inocula, especially in the absence of plant pathogens.     

Chronological Events Associated with the Antagonistic Properties of Trichoderma harzianum against Botrytis cinerea: Indirect Evidence for Sequential Role of Antibiosis and Parasitism

Chronological events associated with the degradation of Botrytis cinerea by a strain of Trichoderma harzianum selected for superior biocontrol ability were studied using ultrastructure and cytochemical investigations in an attempt to define the relative roles of antibiosis and parasitism in the antagonistic process. The first ultrastructural changes were observed 12 h before contact between the organisms, and were characterized by punctuated invaginations of the Botrytis plasmalemma. These reactions were followed by a gradual retraction of the plasmalemma, disorganization of the cytoplasm, loss of turgor pressure and cell death within 48 h of contact between hyphae of the interacting fungi. The first evidence of penetration of B. cinerea by T. harzianum was recorded 72 h after contact. This penetration was apparently mediated by either mechanical pressure or localized wall digestion at points of entry, as there was no clear evidence of chitinolytic degradation of the B. cinerea cell wall, as determined by cytochemical labelling of chitin with a lectin-gold conjugate. However, after 10 days there was clear indication of chitin degradation, based on the random and reduced presence of gold particles over the cell wall of B. cinerea. These results suggest that the strain of T. harzianum antagonized first and foremost by antibiosis, leading to cell death, followed by degradation of the cell by means of chitinolytic enzymes. The production of antibiotics may, therefore, be more important than that of chitinolytic enzymes in conferring superior biocontrol properties to T. harzianum.     

Larvicidal potential of cell wall degrading enzymes from Trichoderma asperellum against Aedes aegypti (Diptera: Culicidae)

Aedes aegypti is a mosquito vector of arboviruses such as dengue, chikungunya, zika and yellow fever that cause important public health diseases. The incidence and gravity of these diseases justifies the search for effective measures to reduce the presence of this vector in the environment. Bioinsecticides are an effective alternative method for insect control, with added ecological benefits such as biodegradability. The current study demonstrates that a chitinolytic enzyme complex produced by the fungus Trichoderma asperellum can disrupt cuticle formation in the L3 larvae phase of A. aegypti, suggesting such biolarvicidal action could be used for mosquito control. T. asperellum was exposed to chitin from different sources. This induction of cell wall degrading enzymes, including chitinase, N-acetylglucosaminidase and β-1,3-glucanase. Groups of 20 L3 larvae of A. aegypti were exposed to varying concentrations of chitinolytic enzymes induced with commercial chitin (CWDE) and larvae cell wall degrading enzymes (L-CWDE). After 72 h of exposure to the CWDE, 100% of larvae were killed. The same percent mortality was observed after 48 h of exposure to L-CWDE at half the CWDE enzyme mixture concentration. Exoskeleton deterioration was further observed by scanning and electron microscopy. Our findings indicate that L-CWDE produced by T. asperellum reflect chitinolytic enzymes with greater specificity for L3 larval biomolecules. This specificity is characterized by the high percentage of mortality compared with CWDE treatments and also by abrupt changes in patterns of the cellular structures visualized by scanning and transmission electron microscopy. These mixtures of chitinolytic enzymes could be candidates, as adjuvant or synergistic molecules, to replace conventional chemical insecticides currently in use.     

Extracellular proteases of Trichoderma species. A review

Cellulolytic, xylanolytic, chitinolytic and beta-1,3-glucanolytic enzyme systems of species belonging to the filamentous fungal genus Trichoderma have been investigated in details and are well characterised. The ability of Trichoderma strains to produce extracellular proteases has also been known for a long time, however, the proteolytic enzyme system is relatively unknown in this genus. Fortunately, in the recent years more and more attention is focused on the research in this field. The role of Trichoderma proteases in the biological control of plant pathogenic fungi and nematodes has been demonstrated, and it is also suspected that they may be important for the competitive saprophytic ability of green mould isolates and may represent potential virulence factors of Trichoderma strains as emerging fungal pathogens of clinical importance. The aim of this review is to summarize the information available about the extracellular proteases of Trichoderma. Numerous studies are available about the extracellular proteolytic enzyme profiles of Trichoderma strains and about the effect of abiotic environmental factors on protease activities. A number of protease enzymes have been purified to homogeneity and some protease encoding genes have been cloned and characterized. These results will be reviewed and the role of Trichoderma proteases in biological control as well as their advantages and disadvantages in biotechnology will be discussed.