苜蓿轮枝菌的生物学特性分析及ATMT遗传转化体系的建立

 为了揭示苜蓿轮枝菌Verticillium alfalfae的生物学特性及其致病机理,在室内条件下测定不同温度、pH值、碳源和氮源对菌株Ms198的生长速率和产孢量的影响,同时利用农杆菌转化法(Agrobactirium tumfacience-mediated transformant,ATMT)将带有潮霉素(Hygromycin,Hyg)抗性标记和绿色荧光蛋白(green fluorescent protein,GFP)报告基因的双元载体转入苜蓿轮枝菌的分生孢子,获得147株阳性转化子,以野生型菌株为对照,对挑取的15株阳性转化子的菌落形态、生长速率、产孢量、孢子萌发率、粗毒素分泌量和致病力进行研究。结果表明,苜蓿轮枝菌具有较宽的温度和酸碱度适应范围,最适生长和产孢温度分别为25 ℃和20 ℃,最适生长pH值为6 ~ 9,最适产孢pH值为9。可以利用多种碳、氮源,最适生长的碳、氮源分别为可溶性淀粉和牛肉膏,最适产孢的碳、氮源分别为D-牛乳糖和胰蛋白胨。与野生型菌株相比,15株供试转化子中有66.67%的转化子在菌落形态方面与野生型菌株无明显差别,而其生长速率、产孢量和孢子萌发率均有不同程度的降低。在产毒能力和致病力方面,1株转化子的粗毒素分泌量显著高于野生型菌株,9株显著低于野生型菌株,其余5株与野生型菌株无显著差异;4株转化子的致病力显著低于野生型菌株,其余11株转化子的致病力与野生型菌株均无显著差异。研究结果表明,转化子的生物学特性以及产毒能力和致病力,随外源基因插入位点的随机性而有所变化。     

棉花黄萎病菌低致病力突变体的表型分析及致病相关基因克隆

 本研究通过对菌核型强致病力菌株Vd080产生的800个T-DNA插入突变体进行致病力测定,筛选得到31株致病力极显著降低的突变体,并对其进行了生物学性状分析。分子验证结果表明,这些低致病力突变体均为阳性,且有25株的T-DNA为单拷贝插入。与野生型菌株Vd080相比,这25株低致病力突变体菌落形态变异丰富,除了8株与野生型菌株形态一致的菌核型突变体外,还有3株黄色菌丝型,2株白色菌丝型和12株中间型突变体。此外,多数突变体的生长速率、产孢量和粗毒素产量都发生了显著变化,且各生理指标之间并无明显相关性。借助TAIL-PCR技术和VdLs.17的基因组数据库,进一步获得了这25株单拷贝插入低致病力突变体T-DNA的侧翼序列,并从Vd080中成功克隆得到了影响黄萎病菌致病力的相关基因。     

转DsRed荧光蛋白的新月弯孢Curvularia-lunata菌株构建

本研究通过农杆菌介导转化方法(ATMT),利用DsRed荧光蛋白基因对玉米弯孢叶斑病致病菌新月弯孢进行遗传转化。通过转化子的荧光蛋白基因和潮霉素B抗性基因的PCR检测,菌丝体和分生孢子的荧光观察,hyg基因的Southern杂交验证,以及荧光蛋白基因插入位点的TAIL-PCR分析,确定了DsRed荧光蛋白基因插入与表达对新月弯孢转化子的影响。结果表明:测定的4株转化子基因组中均成功整合了DsRed荧光蛋白目的基因片段;转化子在生长发育和致病性方面与野生型菌株存在一定差异,分别有2株在产孢量方面略高于野生型菌株,4株转化子在纤维素酶活性和粗毒素致病力方面均低于野生型菌株,有3株转化子在果胶酶活性上较野生型菌株有提高,1株转化子的致病力显著低于野生型菌株;获得其中3个转化子插入位点的侧翼序列。     

高温胁迫对禾谷镰孢生长和致病力的影响

为探究高温胁迫对禾谷镰孢生长和致病力的影响, 本研究测定了禾谷镰孢5株耐高温菌株和4株温度敏感型菌株在25℃和30℃下的菌丝生长速率、产孢量、孢子萌发率以及不同胁迫压力下的生长速率、致病力和DON毒素含量等。结果表明, 不论耐高温菌株还是温度敏感型菌株, 30℃高温对其菌丝生长均有抑制作用, 但对产孢量和孢子萌发有促进作用;30℃高温能减轻NaCl和CaCl2胁迫对禾谷镰孢生长的抑制, 但是不影响KCl、刚果红, SDS和H2O2对病原菌的抑制作用;在30℃下, 大部分耐高温菌株的致病力不变或降低, 而大部分温度敏感型菌株的致病力反而增加, 30℃对大部分菌株的DON毒素产量有一定促进作用。研究结果可为研究气候变化下小麦赤霉病的流行和预测提供理论基础。     

烟草赤星病菌致病力分化与弱毒株抗性诱导作用的研究

对我国不同地区烟草赤星病菌(Alternaria alternata)20个菌株致病力的测定结果表明,病菌不同菌株致病力有明显的差异。菌株致病力的强弱与其生长量和产孢量有一定的关系。致病力强的菌株菌落生长慢、气生菌丝生长好、产孢量低,致病力弱的菌株则相反。据此选出11个弱毒株对烟草幼苗作诱导接种,其中菌株 TBA16可明显诱导烟苗对赤星病的抗性,当用菌株 TBA28和 TBA19挑战接种时,抗性诱导效应分别为50%—61%和60%—71.4%。     

感染星天牛幼虫高致病力金龟子绿僵菌菌株的筛选

室内测定了6株金龟子绿僵菌菌株的菌落生长速率、产孢量及其对星天牛幼虫的致病力。结果表明：不同菌株生长速率和产孢量存在显著差异,在PPDA培养基上,菌株MaYTTR04和MaZPTR01的菌落生长较快,培养15 d后,菌落直径分别为6.3 cm和5.9 cm;产孢量以菌株MaYTTR04最大,MaZPTR01其次,分别为2.0×10^8孢子·cm^-2和1.1×10^8孢子·cm^-2,显著高于其他菌株。生物测定结果表明,不同绿僵菌菌株对星天牛幼虫的致病力差异显著,接种20 d后,星天牛幼虫累积死亡率在40%～96.7%之间;菌株MaZPTR01和MaYTTR04对星天牛幼虫致死率分别达到96.7%和93.3%;僵虫率也高,分别达到86.7%和80%;致死中时（LT50）短,分别为5.71 d和5.80 d。研究结果表明,菌株MaZPTR01和MaYTTR04对星天牛幼虫致病力较强,同时具备良好的生产性能,故可作为优良菌株用于星天牛的防治。     

三株病原真菌对稗草生防潜力的研究

从稻田中采集自然感病的稗草标样,分离获得10余种病原真菌,经生测,链格孢菌、尖角突脐孢菌和内脐孢菌是3株很有希望的稗草生防潜力菌。对它们的生长及产孢特性、寄主范围及孢子悬浮液喷雾接种对水稻和稗草的致病性等进行了研究。结果表明,3个菌各有优缺点。链格孢菌对稗草致病力稍差,对水稻有轻度感染,但其产孢速度快,产孢量大;尖角突脐孢菌对稗致病力强,对稻无影响,但产生孢子慢,产生菌丝多;内脐孢菌对稗致病力强,对稻安全,菌落生长较慢,但产孢速度快,产孢量多,气生菌丝少。黑暗、空气充足有利于3菌株的生长及产孢。     

突脐蠕孢与弯孢原生质的双亲灭活融合

采用灭活原生质体方法,对致病力强、产孢量低的突脐蠕孢EM菌株和致病力较弱、但产孢量高的弯孢CL菌株两株稗草致病菌进行原生质体融合,通过观察形态、生物学及抑草活性等特性筛选融合子。结果表明,2%溶壁酶+2%蜗牛酶处理获得两亲株的原生质体释放数量最高,CL和EM分别达1.25×106个/ml和1.21×106个/ml;再生培养基○c处理的CL和EM原生质体再生率分别达14.7%和13.2%。EM利用热灭活55℃5min;CL利用紫外灭活30s,距离20cm,照射时间4min;聚乙二醇促融,获得3个融合子。融合子在孢子形态上与亲株存在明显差异,其菌丝生长速度、孢子产量、对稗草根生长的抑制作用等也与双亲菌株有差异。     

高毒力白僵菌的筛选及对花生甜菜夜蛾的致病力

为了筛选高毒力的生防白僵菌菌株,本研究对分离获得的4株白僵菌F1、F5、F6、F8的生物学特性进行分析,并在室内检测对甜菜夜蛾的致病力,最后通过大田试验验证了高毒力菌株对花生甜菜夜蛾的防治效果。结果表明,上述4株白僵菌的菌落形态、生长速率、产孢量等生物学性状及致病力存在显著差异。生长最快以及产孢量最高的是菌株F1,菌落呈粉状。菌株F6的分生孢子较其他菌株的分生孢子大。致病力检测结果表明,菌株F1对甜菜夜蛾的致病力最强,LC₅₀和LT₅₀分别是2.9383×10⁶ cfu/mL和5.79 d;其次是菌株F8,菌株F5和F6的毒力相对较弱。大田防效试验表明,菌株F1对花生甜菜夜蛾的防治效果可达75%以上,并在花生叶片具有较强的定殖能力。     

抗氰烯菌酯假禾谷镰孢突变体的诱导及其生物学特性

为评估引起小麦茎基腐病的病原菌假禾谷镰孢Fusarium pseudograminearum对氰烯菌酯的抗性风险,对5株敏感菌株进行了室内药剂驯化,获得33株抗性突变体,突变频率为16.5%,其对氰烯菌酯的抗性水平范围为7.39～1 665.76倍,3株表现低抗,4株表现中抗,26株表现高抗;发现在myosin-5基因上存在11种抗性突变类型,其中217位的丝氨酸突变为亮氨酸(S217L)、420位的谷氨酸突变为赖氨酸(E420K)和135位的丙氨酸突变为苏氨酸(A135T)为主要突变类型,其比例分别为45.5%、15.2%和9.1%。S217L型抗性突变体的产孢量显著下降,菌丝生长速率和致病力与亲本菌株无显著差异。E420K型抗性突变体的菌丝生长速率和致病力显著下降,产孢量与亲本菌株无显著差异。A135T型抗性突变体的菌丝生长速率和产孢量与亲本菌株无显著差异。研究结果表明假禾谷镰孢在药剂选择压力下易形成氰烯菌酯的抗性群体,对氰烯菌酯存在中到高等的潜在抗性风险,其myosin-5的点突变与其对氰烯菌酯的抗性相关。     

Isolation of pathogenicity- and gregatin-deficient mutants of <Emphasis Type="Italic">Phialophora gregata</Emphasis> f. sp. <Emphasis Type="Italic">adzukicola</Emphasis> through <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation

Phialophora gregata f. sp. adzukicola, a causal agent of brown stem rot in adzuki beans, produces phytotoxic compounds: gregatins A, B, C, D, and E. Gregatins A, C, and D cause wilting and vascular browning in adzuki beans, which resemble the disease symptoms. Thus, gregatins are considered to be involved in pathogenicity. However, molecular analyses have not been conducted, and little is known about other pathogenic factors. We sought to isolate nonpathogenic and gregatin-deficient mutants through Agrobacterium tumefaciens-mediated transformation (ATMT) for cloning of pathogenicity-related genes. The co-cultivation of P. gregata and A. tumefaciens for 48 h at 20°C with 200 μM acetosyringone resulted in approximately 80 transformants per 10⁶ conidia. The presence of acetosyringone in the A. tumefaciens pre-cultivation period led to an increase in T-DNA copy number per genome. Of 420 and 110 transformants tested for their pathogenicity and productivity of gregatins, one nonpathogenic and three gregatin-deficient mutants were obtained, respectively. The nonpathogenic mutant produced gregatins, whereas the gregatin-deficient mutants had pathogenicity comparable to the wild-type strain. This is the first report of ATMT of P. gregata. Further analysis of these mutants will help reveal the nature of the pathogenicity of this fungus including the role of gregatin in pathogenesis.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation as a useful tool for the molecular genetic study of the phytopathogen <Emphasis Type="Italic">Curvularia lunata</Emphasis>

In order to explore the molecular mechanisms of virulence and genetic variance of Curvularia lunata in maize, an ATMT (Agrobacterium tumefaciens-mediated transformation) system was established in order to create a wide range of insertional transformants of C. lunata. Our results showed that the germinating conidia were the ideal starting material for transformation. Based on our optimised transformation condition, the transformation efficiency of C. lunata with T-DNA was improved greatly, and the average transformation frequency was as high as 84 ± 5 transformants per 1 × 10⁶ germlings. Southern blotting results of 39 randomly-selected transformants showed a unique hybridisation pattern and predominant single-copy insertions. An ATMT library containing approximate 3000 transformants was generated, and four types of transformants were screened in terms of growth rate, sporulation, mycelial pigmentation, and toxin production in vitro. This library will be used to identify genes involved in the virulence of the pathogen.     

Disruption of Cerevisin via Agrobacterium tumefaciens‐mediated transformation affects microsclerotia formation and virulence of Verticillium dahliae

Agrobacterium tumefaciens‐mediated transformation (ATMT) was used to obtain a large number of Verticillium dahliae (Vd991) T‐DNA insertion mutants that were randomly integrated. Insertion mutants that produced significantly fewer microsclerotia were chosen for further analysis. Mutant T0065 was identified as having the Cerevisin gene interrupted by T‐DNA, and it was named cerevisin. The cerevisin protein showed a high amino acid sequence similarity with the vacuolar protease B. The mutant strain cerevisin displayed decreased production of microsclerotia and conidia, significantly reduced growth rate and reduction in virulence compared to the wild type. Moreover, the composition of secreted proteins differed between the cerevisin mutant and the wild type. Loss of function of Cerevisin decreased secretion of proteins of low molecular weight (14–25 kDa). Upon treatment with the secreted proteins of the mutant, the degree of leaf wilting decreased, indicating that Cerevisin is implicated in production of these proteins, which are putative pathogenicity factors of V. dahliae. The results suggest that Cerevisin is involved in controlling multiple processes of development and metabolism, plays an important role in vegetative growth and microsclerotia formation and affects virulence of V. dahliae.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation for random insertional mutagenesis in <Emphasis Type="Italic">Colletotrichum lagenarium</Emphasis>

Random insertional mutagenesis using a marker DNA fragment is an effective method for identifying fungal genes relevant to morphogenesis, metabolism, and so on. Agrobacterium tumefaciens-mediated transformation (AtMT) has long been used as a tool for the genetic modification of a wide range of plant species. Recent study has indicated that A. tumefaciens could transfer T-DNA not only to plant cells but also to fungal cells. In this study, AtMT was applied to Colletotrichum lagenarium for random insertional mutagenesis. We constructed a binary vector pBIG2RHPH2 carrying a hygromycin-resistant gene cassette between the right and left borders of T-DNA. Optimal co-cultivation of C. lagenarium wild-type 104-T with pBIG2RHPH2-introduced A. tumefaciens C58C1 led to the production of 150–300 hygromycin-resistant transformants per 10⁶ conidia. Southern blot analysis revealed that T-DNA was mainly integrated at a single site in the genome and at different sites in transformants. The T-DNA inserts showed small truncations of either end, but the hygromycin-resistant gene cassette inside the T-DNA was generally intact. The mode of T-DNA insertion described above resulted in highly efficient gene recovery from the transformants by thermal asymmetrical interlaced-polymerase chain reaction. The fungal genomic DNA segments flanking T-DNA were identified from five of eight mutants that had defective melanin biosynthesis. The sequence from one of the segments was identical to that of the melanin biosynthesis gene PKS1 of C. lagenarium, which we previously characterized. These results strongly support our notion that AtMT is a possible tool for tagging genes relevant to pathogenicity in the plant pathogenic fungus C. lagenarium.     

番茄灰霉病菌对咯菌腈的抗性诱变及抗药突变体的生物学特性

为评估番茄灰霉病菌Botrytis cinerea对咯菌腈的抗性风险,就室内经紫外照射获得抗药突变体的方法及抗性突变体的生物学性状进行了研究。结果表明:番茄灰霉病菌分生孢子的紫外照射亚致死时间为90~120 s;经亚致死时间紫外照射后,4个亲本菌株中有2个菌株共产生了6个抗咯菌腈的突变体,其EC50值是亲本菌株的310倍以上,抗性突变频率为3.13×10-7;经紫外照射诱变获得的所有抗性突变体在菌丝生长速率、产孢量、产菌核能力及其在番茄果实上的致病性方面均比其亲本菌株明显降低。相关分析显示,所得抗咯菌腈突变体对氟啶胺、啶菌唑、啶酰菌胺和嘧霉胺无交互抗性。表明番茄灰霉病菌对咯菌腈的抗药性风险较低。     

西瓜噬酸菌hflX基因功能分析

为明确GTP结合蛋白基因hflX在西瓜噬酸菌Acidovorax citrulli中的功能尤其是对该菌致病力的影响,以西瓜噬酸菌Aac5菌株为材料获得hflX基因的缺失突变菌株及互补菌株,通过对致病力等表型以及相关基因表达量的测定初步探究西瓜噬酸菌hflX基因的功能。结果显示,同野生型菌株Aac5相比,缺失突变菌株的致病力显著下降,III型分泌系统基因hrpG和hrpE的表达量显著下调,仅为野生型菌株表达量的54.99%和27.39%;hflX基因的缺失并未影响其引起烟草过敏性坏死反应的能力;但缺失突变菌株的运动能力显著下降,鞭毛基因fliR和fliC的表达量显著下调,仅分别为野生型菌株的37.04%和29.68%。此外,缺失突变菌株的生物膜形成能力相较于野生型菌株显著增强了26.32%,同时,其体外生长能力也有所增强。表明hflX基因可能通过调控III型分泌系统基因hrpG和hrpE、鞭毛基因fliR和fliC的表达以及运动能力来影响西瓜噬酸菌Aac5菌株的致病力。     

胶孢炭疽菌G蛋白信号调控因子CgRGS3的生物学功能

G蛋白信号调控因子(regulators of G-protein signaling,RGS)是G蛋白的一类负调控因子,在植物病原真菌生长发育及致病过程中起着重要作用。为探究胶孢炭疽病菌1个RGS基因CgRGS3的生物学功能,利用PCR技术扩增CgRGS3基因并进行生物信息学分析,通过同源重组的方法获得该基因的敲除突变体,并在突变体的基础上获得互补株,通过表型分析确定CgRGS3的生物学功能。结果表明:CgRGS3编码1个含有367个氨基酸的蛋白,包含1个RGS功能域和3个跨膜区域,表型分析发现与野生型菌株相比,敲除突变体营养生长缓慢,分生孢子产量附着胞形成率显著降低,对Cu~(2+)、Fe~(2+)、Zn~(2+)离子更加敏感,细胞壁完整性发生改变,黑色素产量降低及致病力减弱等。由此可见,CgRGS3参与调控胶孢炭疽菌的营养生长,分生孢子产生及附着胞的分化、细胞壁完整性、黑色素产生及致病性等。     

Nitric oxide-overproducing transformants of Pseudomonas fluorescens with enhanced biocontrol of tomato bacterial wilt

The relation between nitric oxide (NO) production and the protective ability of Pseudomonas fluorescens T5 against bacterial wilt disease in tomato was examined. The endogenous nitric oxide reductase gene of T5 was disrupted by homologous recombination using a suicide plasmid. Three disruptants were obtained, and all had higher levels of NO production. Infection with Ralstonia solanacearum was reduced in tomato plants treated with the NO-overproducing transformants compared with the wild type. These results suggest that the modification of pseudomonads to increase their level of NO production is a new approach to enhancing their biocontrol efficacy.     

木霉耐盐突变菌株的紫外诱变选育

为了获得高效耐盐木霉突变菌株,以盐碱土中分离的一株深绿木霉T-YM作为原始菌株进行紫外诱变处理,并筛选了突变菌株的最佳诱变条件,利用NaCl溶液模拟盐胁迫条件测定了突变菌株耐盐指标生长速率、产孢量和菌丝生长量。结果表明,与对照(野生型菌株)相比,当诱变时间为3 min时能够显著提高深绿木霉T-YM突变菌株菌落生长速度和产孢量,分别增加17.69%和28.82%;诱变时间为0.5 min和5 min时能够显著提高突变菌株菌丝生长量,分别增加42.22%和46.67%。利用10 mg·mL^-1 NaCl溶液模拟盐胁迫条件时,与对照相比突变菌株菌落生长速度、产孢量和菌丝干重整体高于野生型菌株,尤其当诱变时间为3 min时,不同浓度盐胁迫下其产孢量平均增加51.18%,诱变时间为0.5 min时其菌丝干重平均增加23.65%;NaCl胁迫(10 mg·mL^-1)下,诱变处理0.5 min获得的正突变菌株经传代接种培养后其耐盐性较为稳定。因此,紫外诱变可作为一种选育高效耐盐木霉突变菌株的有效方法。