弯孢菌引起的变色米初步研究

 本文报道了弯孢菌引起的变色米的症状,弯孢苗的种类及其对水稻的致病性。变色米的症状,因水稻品种、弯孢菌种类和接种方法而有差异,但一般在糙米上为浅至深色的褐色米;在稻谷上产生全褐型、半褐型、褐斑型、褐点型和退色型五种类型。后各地采集的变色谷(米)上分离到弯孢属9个种,即新月弯孢、膝曲弯孢、近缘弯孢、不正弯孢、中隔弯孢、棒状弯孢、苍白弯孢、画眉弯孢、假弯孢,其中6个种为国内首次报道。据在水稻孕穗至开花期人工接种结果,上述9个种对水稻均有致病性,并产生大量的变色谷(米)。     

水稻叶尖枯病病原种类研究

 从江苏13个县市采集病叶标本,经分离、纯化后获280株菌。据鉴定结果和接种试验,稻生叶点霉(Phyllosticta oryzicola Hara)为主要致病菌,占74.6%。此外,还分离到稻盘多毛孢(Pestalotia oryzae Hara)、链格孢(Alternaria alternata(Fr.) Keissl.)、稻喙孢(Rhynchosporium oryzae Hash.et York.)、苍白弯孢(Curvularia pallescens Boed.)、新月弯孢(C.lunata(Walk.) Boed.)、膝曲弯孢(C.geniculata(Tracy et Earle) Boed.)、镰刀菌(Fusarium spp.)、平脐蠕孢(Bipolaris spp.)、稻黑孢(Nigrospora oryzae(Berk.et Br.) Perch)、球黑孢(N.sphaerica(Sacc.) Mason)等真菌。首次以透射电镜观察表明,稻生叶点霉产孢方式为全壁芽生单体式(hb-sol)。     

瓜拟多隔孢引起的砂田西瓜叶疫病

 近年来,甘肃省白银市靖远县的一些砂田西瓜中出现了严重的叶疫病,严重田块病株率超过70%。2018年7月,从靖远县高湾乡罹病西瓜叶片上分离得到拟多隔孢属Stagonosporopsis真菌,病叶检出率达100%。采用离体叶片和植株接种法评价单孢分离菌株XG-3对西瓜和甜瓜的致病性,所有接种处理在24 h内均发病显症而对照未发病,其中发病叶片原接种菌的检出率为100%。菌株XG-3在PDA和OA平板培养基上20和25 ℃培养20 d,未见产孢。在自然感病的西瓜叶片上观察到少量分生孢子器,在人工接种发病的西瓜和甜瓜茎、叶上产生大量分生孢子器和分生孢子:分生孢子器球形至亚球形,大小为(82.4~243.3) μm×(82.4~188.4)μm,分生孢子器具1~2个孔口,孔口直径15.7~27.5 μm;分生孢子无色,0~3个隔膜,杆状、柱状、长椭圆形、花生形及不规则形,直或稍弯曲,分隔处不缢缩或缢缩,大小为(5.2~28.3)μm×(2.2~6.0)μm,分生孢子形态和大小依基质和环境条件的不同而有较大差异。BLASTn分析结果显示,菌株XG-3(GenBank登录号:MW282128)的rDNA-ITS序列与瓜拟多隔孢Sta. cucurbitacearum分离物287ITS(GenBank登录号:AY293804.1)的序列相似性达99.80%。在基于rDNA-ITS构建的系统发育树中,菌株XG-3与Sta. cucurbitacearum聚为一组,与西瓜拟多隔孢Sta. citrulli和木瓜拟多隔孢Sta. caricae区分开来。依据病菌形态学和分子生物学特征,将菌株XG-3鉴定为瓜拟多隔孢Sta. cucurbitacearum [Basionym: Sphaeria cucurbitacearum]。     

马唐病原真菌的分离筛选及其致病力测定

从马唐罹病植株上分离到 6种病原真菌 :中隔弯孢、新月弯孢、多节长蠕孢、灰梨孢、链格孢和粉红镰孢。其中新月弯孢和多节长蠕孢对玉米有致病作用 ,灰梨孢对水稻有轻微的致病作用 ;另3种病原真菌中 ,链格孢和粉红镰孢对马唐致病性弱 ,而中隔弯孢菌株对 4叶期以下的马唐有极强的致病作用 ,室内控制效果可达 1 0 0 % ,田间控制可达 75%以上。对中隔弯孢菌株QZ 2 0 0寄主范围测定表明 :该菌对水稻、玉米、大豆、棉花、小麦、向日葵、花生等作物及黑麦草、高羊茅和狗牙根等草坪草十分安全 ,具有开发为作物田和草坪真菌除草剂的潜力     

稻曲菌交配型初探

 稻曲菌（有性态：Villosiclava virens;无性态：Ustilaginoidea virens）有性繁殖产生的子囊孢子是水稻稻曲病可能的初侵染源之一,交配型基因座对真菌有性繁殖中的性别控制起着决定性作用。为进一步揭示稻曲菌的有性繁殖方式,本研究首次克隆了稻曲菌交配型基因mat1-1-1的α-结构域（α-domain）相应核苷酸序列,发现其与麦角菌科真菌Cordyceps militaris、Cor. bassiana和Claviceps purpurea的mat1-1-1基因中α-结构域相应核苷酸序列同源性分别为61%、63%和68%;根据mat1-1-1和mat1-2-1部分基因序列设计特异性引物,并使用PCR方法检测了来源于3个异源子座的240个子囊孢子单孢菌株和50个田间菌株的交配型基因,结果显示稻曲菌mat1-1-1和mat1-2-1基因分别存在于不同菌株中;将具有相同或不同交配型基因的菌株配对接种,发现菌核通常产生在mat1-1-1和mat1-2-1基因型菌株配对接种产生的稻曲球上,其中大部分菌核可萌发产生子座,而菌株单独接种或具有相同交配型基因的菌株配对接种水稻后多数不能形成菌核;根据以上结果初步推断稻曲菌为异宗配合真菌。     

重寄生菌葡酒锈生座孢(Tuberculina vinosa)和白蜡锈生座孢(T.fraxinis)的寄生专化性

 用葡酒锈生座孢(Tuberculina vinosa)的孢子悬浮液,在贴梗海棠、木瓜、苹果和垂丝海棠锈病发生期,喷雾接种于叶面锈菌性子器处,7-12 d出现重寄生现象。表明葡酒锈生座孢除能在自然条件下寄生梨锈病菌外,在人为接种条件下,其寄主范围包括贴梗海棠锈病菌、木瓜锈病菌(Gymnosporangium asiaticum)、苹果锈病菌和垂丝海棠锈病菌(G.yamadai)。用葡酒锈生座孢和白蜡锈生座孢(T.fraxinis)在梨锈病菌和大叶白蜡锈病菌(Aecidium fraxinibungeanae)上交互接种,结果表明,两者不能交互寄生。研究T.vinosa和T.fraxinis的寄生专化性,对2种重寄生菌的应用及锈生座孢属(Tuberculina)种的鉴定具有意义。     

玉米新月弯孢菌(Curvularia lunata)的RAPD分析

 对分离自玉米弯孢菌叶斑病标样中的77株新月弯孢菌和1株来自水稻的新月弯孢菌进行RAPD分析表明,菌株间具有丰富的遗传多样性,在相似系数约0.60处,所有菌株被聚为3个组,但88.0%的菌株聚入第Ⅰ组内,其余菌株被聚入另外2个组内。第Ⅰ组内共有69个菌株,包含来自不同区域的致病性较强的菌株,是玉米弯孢菌叶斑病的优势类群,其余2个类群主要是一些致病能力较弱或不致病的菌株。结果表明,新月弯孢菌种内菌株的遗传多样性与致病性相关,但与菌株地理来源无明显的直接关系。     

香梨果萼黑斑病菌Alternaria alternata遗传转化体系的建立及GFP标记菌株的获得

 为建立香梨果萼黑斑病菌链格孢(Alternaria alternata)的原生质体遗传转化体系,本实验以香梨果萼黑斑病菌强致病性菌株LI1为供试材料,研究菌龄、酶系统、酶解时间等对链格孢菌原生质体制备的影响。链格孢菌菌丝在CM液体培养基中培养20 h,以0.7 mol·L^-1 NaCl为稳渗剂,1%裂解酶+1%崩溃酶+1%蜗牛酶的酶液组合下,28 ℃酶解4 h,原生质体制备效率最高。通过PEG/CaCl₂介导法将含有潮霉素B抗性基因和绿色荧光蛋白基因的质粒转入链格孢菌LI1,转化子生长表型及外源基因的PCR鉴定结果表明抗性基因已成功整合到香梨果萼黑斑病菌中。成功建立了香梨果萼黑斑病菌链格孢菌的原生质体遗传转化体系,并成功获得GFP标记菌株,为病原菌侵染定殖过程及致病机制研究奠定了基础。     

京津冀设施大棚根际土壤中筛选出的防治根腐病的生防镰孢菌

 由致病性尖孢镰孢菌(Fusarium oxysporum)引起的根腐病严重危害果蔬生产,但非致病性镰孢菌可作为潜在的生防菌。为筛选防治根腐病的非致病生防镰孢菌,从京津冀设施大棚采集茄科、葫芦科果蔬78份根际土样中分离2 402株真菌,筛选出对致病性尖孢镰孢菌(F. oxysporum)具有拮抗效果的真菌173株。利用镰孢菌通用引物进行PCR扩增,从中筛选出28株候选镰孢菌;通过镰孢菌发酵液泡根进行安全性测试,筛选出对寄主黄瓜幼苗安全无害的镰孢菌菌株4株(1418、1441、1436和1473)。进一步通过镰孢菌测序通用引物TEF1αF/TEF1αR结合菌落和分生孢子的形态学特征,1418菌株和1441菌株被鉴定为尖孢镰孢菌(F. oxysporum)、1436菌株被鉴定为茄病镰孢菌(F. solani)。盆栽测试发现,除1441菌株外,1418菌株、1436菌株和1473菌株对黄瓜根腐病的防效均在50%以上,其中1418菌株的防效为70%,与杀菌剂咪酰胺的防效相当,具有很好的应用潜力。本研究筛选获得的具有生防潜力的镰孢菌不仅为镰孢菌致病力分化的研究提供了实验材料,也为新型生防产品的研制奠定了基础。     

胶孢炭疽菌及两种疫霉菌对苯酰菌胺的敏感性与β-微管蛋白氨基酸突变的相关性分析

选择对多菌灵、乙霉威和苯酰菌胺具有不同敏感性的胶孢炭疽菌 Colletotrichum gloeosporioides、辣椒疫霉菌 Phytophthora capsici 及恶疫霉菌 P.cactorum,采用菌丝生长速率抑制法及氨基酸序列比对法分析了其 β-微管蛋白氨基酸突变与敏感性的关系。结果表明,胶孢炭疽菌对苯酰菌胺、多菌灵和乙霉威的敏感性与 β-微管蛋白198位或200位氨基酸突变有关:对多菌灵敏感、对苯酰菌胺和乙霉威不敏感的胶孢炭疽菌 β-微管蛋白氨基酸198位为谷氨酸(E),200位为苯丙氨酸(F);对多菌灵已产生抗性而对苯酰菌胺和乙霉威不敏感的菌株,其 β-微管蛋白氨基酸200位由苯丙氨酸(F)突变为了酪氨酸(Y);对多菌灵高抗、对苯酰菌胺和乙霉威敏感的菌株其 β-微管蛋白氨基酸198位由谷氨酸(E)突变为了丙氨酸(A)。辣椒疫霉菌和恶疫霉菌对苯酰菌胺敏感,对多菌灵和乙霉威均不敏感。检测疫霉菌菌株 β-微管蛋白未发现氨基酸突变,但发现其 β-微管蛋白氨基酸在196~200位与胶孢炭疽菌差异较大,这可能是导致苯酰菌胺仅对疫霉菌有抑制效果的原因。     

疏水表面诱导橡胶树炭疽菌侵染结构的发育分化过程

为了解橡胶树2种炭疽病菌的侵染结构发育分化过程,采用平板菌落生长速率法测定了3株胶孢炭疽菌Colletotrichum gloeosporioides和3株尖孢炭疽菌C.acutatum的菌丝生长速率,测量其分生孢子大小,显微观察2种炭疽菌在疏水表面诱导下侵染结构的发育分化过程。结果表明,胶孢炭疽菌菌丝生长速率为0.96~1.36 cm/d,显著高于尖孢炭疽菌的菌丝生长速率0.72~0.89 cm/d,但二者分生孢子大小无显著差异。在疏水表面诱导下,2种炭疽菌分生孢子在接种2~6 h后开始萌发,12 h孢子萌发率为71.70%~88.05%,13~16 h开始分化附着胞,24 h附着胞形成率为48.99%~70.74%,36 h菌丝诱发形成大量附着枝,48 h后分生孢子产生的次生菌丝也可诱发形成附着枝,附着枝呈圆形、姜瓣形、梨形或不规则形。分生孢子极易产生,可在菌丝顶端成簇或菌丝侧面排列产生,也可由分生孢子形成的芽管产生,或在芽管分化附着胞过程分枝形成分生孢子;附着胞多着生于芽管顶端,少数附着胞顶端可继续萌发类似短芽管结构,再次分化形成可黑色化的次级附着胞。表明橡胶树2种炭疽菌不同菌株间分生孢子萌发时间、孢子萌发率、附着胞形成时间和形成率有一定差异,但种间无明显差异;橡胶树炭疽菌分生孢子极易形成,在疏水表面容易分化形成附着胞和附着枝,说明具有极强的适生性。     

大丽轮枝菌侵染抗感棉种的组织学过程观察

 棉花黄萎病是一种极难防治的土传性真菌病害,研究病原菌侵染棉花的组织学过程对致病机理解析和抗病资源利用具有重要意义。本研究利用绿色荧光蛋白标记的大丽轮枝菌系统研究了其对抗病棉种海岛棉7124和三裂棉、感病棉种军棉1号和戴维逊棉的侵染过程。结果表明,大丽轮枝菌对抗/感棉种的初始侵染没有明显差异,接菌5 h后,分生孢子均能吸附在感病和抗病棉种的根表面。但侵染过程存在显著差异,侵染感病棉种中病原菌3~5 d到达皮层,5~7 d达到维管束,随后迅速扩展繁殖,侵染14 d后即完成系统侵染,并开始产生黄萎病症状;而病原菌侵染抗病棉种,5~7 d才侵入皮层,7~10 d到达维管束,14 d后仍无法扩展,病原菌的定殖与发展受到限制,无法形成系统侵染,较少形成黄萎病症状。本研究通过绿色荧光蛋白标记大丽轮枝菌对抗/感棉种的侵染过程研究,为大丽轮枝菌致病机理研究和抗性资源利用提供了强有力的理论依据。     

非亲和性稻瘟病菌对水稻的诱导抗性

为研究稻瘟病菌Magnaporthe oryzae不同菌株间的相互作用,选择与单抗性基因系水稻IRBL5-M （携带抗性基因Pi5）表现为亲和性的菌株HN52与非亲和性的菌株HN119为研究对象,将其单独或混合接种到单抗性基因系水稻IRBL5-M中,并通过荧光显微镜观察接种后水稻叶鞘的发病情况及病斑面积,测定接种后水稻内相关抗性基因OsWRKY45、OsNPR1、OsPR10、OsMAPK2的表达量以及活性氧的变化。结果显示,相较于单独接种亲和性菌株,混合接种后单抗性基因系水稻IRBL5-M病斑发病面积减少;混合接种中亲和性菌株HN52菌丝侵染能力降低,侵染菌丝细胞间扩展率显著降低73.13%;同时单抗性基因系水稻IRBL5-M中OsWRKY45、OsNPR1、OsPR10和OsMAPK2抗性基因表达量显著增加,水稻叶片中活性氧含量增加,表明在菌株混合侵染过程中,非亲和性菌株可通过激发水稻的抗性反应来降低亲和性菌株对水稻的侵染程度。     

Influence of bacteria isolated from rice plants and rhizospheres on antibiotic production by the antagonistic bacterium <Emphasis Type="Italic">Serratia marcescens</Emphasis> strain B2

Serratia marcescens strain B2 is an antagonistic bacterium that produces the red-pigmented antibiotic prodigiosin and suppresses rice sheath blight caused by Rhizoctonia solani AG-1 IA. Rice sheath blight disease was suppressed when plants were inoculated with this bacterium an hour before pathogen inoculation but not when plants were treated 4 weeks before pathogen inoculation. In both cases the bacteria were detected in the rice rhizosphere 4 weeks after inoculation. Bacteria isolated from the rice plant and rhizosphere inhibited biosynthesis of prodigiosin in S. marcescens strain B2. We suggest that bacteria isolated from rice plants and rhizospheres mediate the suppression of antibiotic production of biological control agents and that such suppression is common under field conditions.     

利用环介导等温扩增(LAMP)技术快速检测辣椒疫霉菌

<正>辣椒疫霉(Phytophthora capsici)是一种重要植物病原菌,能造成植株坏死、果实腐烂,严重影响产量[1]。辣椒疫霉侵染植物的早期病症并不明显,易被忽视。因此,对辣椒疫霉早期快速、准确检测显得尤为重要。聚合酶链式反应(PCR)为动植物病原物检测的重要方法,但需要较昂贵的仪器、试剂与耗材,后期的电泳检测也费时费力,致使这一技术很难在生产一线普及推广。Notomi等2000年研发了环介     

Control of rice sheath blight by phyllosphere epiphytic microbial antagonists

Epiphytic microorganisms on the phyllosphere of traditional and high-yielding rice varieties were isolated from different agroecological zones of Sri Lanka and screened for theirin vitro andin vivo antagonism againstRhizoctonia solani Kühn AG-1 1A, the sheath blight pathogen of rice. Among a total of 196 bacterial and 91 fungal isolates, 12 bacterial and two fungal isolates which showed more than 50% growth inhibition ofR. solani were tested for theirin vivo antagonism. Among the 14 antagonists tested, six bacterial and one fungal isolate substantially reduced the incidence of sheath blight (by more than 82%) and severity (by more than 92%) of the rice varieties BG94-1 and IR8 grown in a pot experiment under open field conditions. Using five antagonists that showed the bestin vitro antagonism, a pot experiment was conducted to determine whether the presence of indigenous microflora on the rice sheath had any effect on the effectiveness of antagonism. Three isolates (B4, GbB5 and HMWB4) controlled sheath blight incidence and severity equally well in the presence and absence of indigenous microflora. Two isolates (BG352B1 and BG300B1) were more effective when they were introduced into the rice sheath without indigenous microflora. Among the effective antagonists determined by the pot experiment, isolates B4, B16, BG94-1B5, GbB5, HMWB4 and BG379-F2 were tested under field conditions for two consecutive growing seasons. Under field conditions, severity of rice sheath blight was significantly reduced by the application of all the tested antagonists as a spray on rice sheath at a concentration of 10⁸ cfu ml⁻¹, starting 3 days after the development of symptoms and continuing for three applications at 10-day intervals. Antagonistic performances were consistent in the two seasons under field conditions andB. megatarium A (isolate B16) andAspergillus niger (isolate BG379-F2) performed as the most effective antagonists in both seasons. When disease severity was quantified as percentage sheath area covered by the disease lesions, the respective reductions in disease severity were greater than 50% and 61% byB. megaterium A (isolate B16) andAspergillus niger (isolate BG379-F2), respectively, in both seasons. http://www.phytoparasitica.org posting Jan. 10, 2008.     

Resistance in leaf blades assessed by counting conidia correlates with whole-plant-specific resistance in leaf sheaths in a compatible rice–Magnaporthe oryzae interaction

Resistance in the leaf blades of rice plants against a virulent race of the rice blast fungus Magnaporthe oryzae was quantitatively examined using a modified spot inoculation method. Numbers of conidia produced in the lesions were affected by plant age and paralleled the frequency of resistance infection types, which is indicative of whole-plant-specific resistance (WPSR), in the inoculated leaf sheaths of the corresponding plants. Exogenous abscisic acid treatment, which suppresses WPSR, also increased the susceptibility of the leaf blades. These results indicate a correlation between the resistance of the leaf blades and the WPSR in the leaf sheaths.     

Recovery and characterization of asexual recombinants ofMagnaporthe grisea

Eleven nitrate non-utilizing (nit) mutants were recovered from six field isolates ofMagnaporthe grisea that had different degrees of sensitivity to the blasticide kitazin P (iprobenfos, IBP). Allnit mutants were resistant to chlorate and there were no significant differences in hyphal growth rate, conidial production, sensitivity to IBP, and pathogenicity betweennit mutants and their parent isolates.nit phenotypes and IBP-resistance were used as two independent genetic markers to study asexual recombination inM. grisea. Asexual recombinants were recovered from the heterokaryotic mycelium of three inter-strain pairings, namely, DY2-3(nitl-LR) + A7-3 (nitM-S); A7-3 (nitM-S) + F4-2 (nitl-MR); and F4-2 (nitl-MR) + DY2-4 (nitA-LR), at a frequency of 7.31%, 14.00% and 8.63%, respectively. The growth rate, conidial production and pathogenicity of asexual recombinants were similar to those of parental strains. We concluded that asexual recombination resulting from hyphal fusion might contribute to variability inM. grisea. http://www.phytoparasitica.org posting Nov. 14, 2005.     

鞭毛素蛋白FliCxcv对水稻免疫反应的诱导功能鉴定

 为揭示来源于番茄细菌性斑点病菌(Xanthomonas campestris pv. vesicatoria, Xcv)菌株XV18鞭毛素(FliCxcv)作为一种病原相关分子模式(PAMP)诱导水稻免疫反应的功能,本研究对FliCxcv编码基因flicxcv进行了基因克隆、序列分析、原核表达、蛋白纯化和诱导活性测定。结果表明,通过PCR特异性扩增,从Xcv菌株XV18中克隆了1 200 bp的fliCxcv基因片段,其序列与GenBank中己测序菌株的完全一致。在大肠杆菌中对该基因全长、N端和C端截短序列进行了原核表达,并获得了纯化的FliCxcv全长及其截短蛋白。将纯化蛋白浸润接种到水稻品种日本晴叶片组织,发现FliCxcv全长及其截短蛋白均能诱导水稻叶片细胞死亡、H₂O₂产生以及防卫基因(OsPAL和OsPR1b)表达等免疫反应,但诱导活性存在差异。因此,本研究验证了FliCxcv具有激发水稻细胞免疫反应的PAMP功能,为水稻免疫诱导制剂的研发提供了材料。     

Bacterial black spot caused by <Emphasis Type="Italic">Burkholderia andropogonis</Emphasis> on <Emphasis Type="Italic">Odontoglossum</Emphasis> and intergeneric hybrid orchids

A new bacterial black spot disease was observed on Odontoglossum, Odontioda, Odontocidium, and Vuylstekeara orchids in Japan. Typical symptoms on the leaves were dark or black spots (or both) with a yellow halo. The causal agent was identified as Burkholderia andropogonis (Smith 1911) Gillis, Van Van, Bardin, Goor, Hebbar, Willems, Segers, Kersters, Heulin and Fernandez 1995. The isolates were pathogenic on four original host orchids, Phalaenopsis orchid, and tulip; they were not pathogenic on white clover or corn after needle stab inoculation. An antibiotic bactericide (oxytetracycline/streptomycin mixture WP) was most effective for controlling the disease.