西北地区马铃薯疮痂病病原菌鉴定及其生物学特性

为明确西北地区马铃薯疮痂病病原菌的种类和生物学特性,分别采用常规组织分离法和土壤混悬液分离法从宁夏、陕西和甘肃3个省区采集的29份疮痂病发病薯块和8份发病地块土壤中进行病原菌分离,并利用形态特征、生理生化特性和16S rDNA序列分析对病原菌进行鉴定。结果表明,从发病薯块和发病土壤中共分离到50株链霉菌Streptomyces spp.,通过回接法验证获得6株马铃薯疮痂病致病菌株。6株致病菌株的培养特性和形态特征差别较大;其中菌株G4-1、G9和SYN13不能以果糖和木糖为单一碳源,菌株SYNT3不能以棉子糖为单一碳源;除菌株NLG4-1外,其余5株菌株均能在络氨酸琼脂培养基上产生黑色素。经16S rDNA序列分析,菌株G4-1、G9与疮痂病链霉菌S. scabiei的相似率分别达99.47%和99.34%,菌株NLG4-1、SYNT3与S. enissocaesilis的相似率分别达97.90%和98.18%,菌株GBH2与加利利链霉菌S. galilaeus的相似率达99.93%,菌株SYN13与S. turgidiscabies的相似率达97.56%,表明西北地区马铃薯疮痂病病原菌至少存在4个种。     

云南省马铃薯疮痂病致病链霉菌种类组成研究

 为明确云南省马铃薯疮痂病病原菌(Streptomyces spp.)的种类及其生物学特征,自2013年从云南省13个马铃薯主产区采集疮痂病病样,共分离到200株链霉菌,通过温室盆栽致病性试验筛选出67株致病菌。通过形态学、生理生化指标、致病性测定及16S rDNA序列分析对获得的菌株进行鉴定。结果显示,引起云南地区马铃薯疮痂病的病原为10种链霉菌,分别为S. caviscabies、S. anulatus、S. scabies、S. turgidiscabies、S. acidiscabies、S. europaeiscabiei、S. luridiscabiei、S. enissocaesilis、S. griseus和S. aureofaciens。其中S. enissocaesilis和 S. anulatus为优势种群,S. caviscabies、S. anulatus和S. luridiscabiei为国内首次报道的病原菌。因此,认为云南省马铃薯疮痂病菌种类复杂多样。     

马铃薯疮痂病菌在植株和田间的分布与动态分析

为探明马铃薯疮痂病菌在植株和土壤中的分布情况及种群动态变化特点,利用常规PCR和定量PCR(qPCR)技术对不同环境的马铃薯疮痂病株和田间植株不同生育期的土壤样品进行病原菌的定性定量检测.结果 表明,病田、温室盆栽和微型薯苗床中马铃薯疮痂病重度发病植株的根、匍匐茎、块茎、地上茎、叶片等组织样品均可检测到184 bp的疮...     

一株枯草芽孢杆菌挥发性物质的抑菌作用初步研究

初步研究了分离自石榴果实中的一株枯草芽孢杆菌Bacillus subtilis所产生的挥发性物质对几种常见林果病原菌的抑菌作用。发现该挥发性物质对不同病原菌菌丝生长具有一定的抑制作用,其中对杨树溃疡病菌Botryosphaeria dothidea的抑制率达60%。将不同病原菌株与该挥发性物质互作后的菌丝形态与正常菌丝进行比较,结果表明:处理后的杨树溃疡病菌、石榴疮痂病菌B.dothidea、苹果轮纹病菌B.dothidea及葡萄白腐病菌Coniothyrium diplodiella的菌丝分支增多,扭曲,菌丝顶端和中部细胞膨大;杨树腐烂病菌Valsa sordida及桑紫纹羽病菌Helicobasidium purpureum的菌丝变粗;合欢枯萎病菌Fusarium oxysporum的菌丝变细;苹果霉心病菌Trichothecium roseum菌丝形态无明显变化。同时发现,不同病原菌菌丝色素产生的速度均受到了抑制。表明该枯草芽孢杆菌所产生的挥发性物质对不同病原菌的抑菌效果和作用方式不同。     

玉米茎腐病菌毒素致病力初报

通过提取玉米茎腐病菌毒素粗提液,证实玉米茎腐病原菌(镰刀菌和腐霉菌为主)在镰刀菌毒素培养液、查氏培养液和普通培养液中,能产生和玉米小斑病菌毒素(简称HM毒素,下同)类似的致病毒素。该物质能抑制种子根的生长。接种在玉米叶片上能产生典型的萎蔫枯死斑;用上述3种培养液培养病原菌,产生的毒素致病力差异不显著;不同类型病原菌产生的毒素致病力和同一类型病原菌不同菌株产生的毒素致病力差异都极显著。玉米品系对茎腐病原菌产生的毒素抗病性差异也极显著。     

侧孢短芽胞杆菌JYC 688鉴定及抑菌促生特性研究

马铃薯疮痂病是由多种致病链霉菌侵染引起薯块表面结痂导致薯块外观品质下降的病害,防控困难,生物防治是其主要防治措施之一。筛选具有良好拮抗作用的生防菌,可为马铃薯病害的生物防治提供优良的微生物资源。本研究从健康马铃薯植株根际土壤中分离获得JYC 688菌株,采用平板对峙和纸碟法筛选验证菌株拮抗性,结合形态学观察、生理生化特性测定及分子生物学手段确定其种属地位,并检测其产生水解酶和嗜铁载体的能力。结果表明,菌株JYC 688是侧孢短芽胞杆菌Brevibacillus laterosporus,其菌株培养液能够显著抑制引起马铃薯疮痂病的酸性疮痂链霉菌Streptomyces acidiscabies和肿胀疮痂链霉菌S.turgidiscabies的生长,抑菌圈直径分别为27.86 mm和31.90 mm;其对马铃薯晚疫病菌Phytophthora infestans、早疫病菌Alternaria alternata、黑痣病菌Rhizoctonia solani也具有良好的抑制活性;菌株JYC 688能够分泌β-1,3葡聚糖酶、纤维素酶、蛋白酶、嗜铁素等多种抗菌相关物质,可溶解大豆卵磷脂、碳酸钙、碳酸镁3种难溶性化合物,有利于磷、钙、镁3种元素的释放,是一株具有开发潜力的生防菌。     

黑痣病菌毒素对马铃薯幼苗生理生化抗性相关物质的诱导

为了探讨马铃薯抗黑痣病机制,采用毒素接种脱毒组培苗的方法,研究了黑痣病菌毒素对马铃薯不同抗性品种幼苗体内病程相关蛋白、木质素及游离脯氨酸含量的影响及其与抗黑痣病的关系。毒素处理后,感病品种大西洋和抗病品种底西芮幼苗几丁质酶和β-1,3-葡聚糖酶活性均明显升高,36 h升高幅度均达到最大,感病品种升幅大于抗病品种。底西芮和大西洋木质素及游离脯氨酸含量也大幅增加,但抗病品种的增加幅度大于感病品种。研究表明,黑痣病菌毒素诱导几丁质酶和β-1,3-葡聚糖酶活性升高以及木质素和游离脯氨酸含量增加是马铃薯幼苗抵抗毒素胁迫的内在机制;木质素、游离脯氨酸含量与品种抗病性呈正相关,而几丁质酶、β-1,3-葡聚糖酶活性变化则不是引起品种抗性差异的主要原因。     

我国马铃薯疮痂病及其防治研究进展

马铃薯疮痂病(potato common scab)是由放线菌目链霉菌属的链霉菌Streptomyces spp.引起的土传兼种传病害,广泛分布于世界各马铃薯种植区,不仅影响马铃薯的外观品质和销售价格,严重时还会导致马铃薯出苗延迟甚至引起幼苗死亡,造成产量下降,给马铃薯产业带来巨大的经济损失,已经成为全球危害马铃薯生产的第四大病害。2015年我国确立马铃薯主粮化战略,推动了马铃薯产业的发展。近年来,随着种植区域和规模不断扩大,马铃薯疮痂病在我国很多省(自治区)有不同程度的发生,并有逐年扩大和加重的趋势,严重影响商品薯、加工原料薯和种薯的生产,成为制约我国马铃薯生产的主要病害。本文对马铃薯疮痂病症状、发病因素、传播规律、致病机理、分类方法以及我国马铃薯疮痂病发生情况、种类及分布进行归纳,并对马铃薯疮痂病防治措施进行总结,以期为我国马铃薯疮痂病的研究和防治奠定理论基础。     

薄壳山核桃疮痂病菌TaqMan荧光定量PCR检测方法的建立及应用

疮痂病是薄壳山核桃上最具毁灭性的病害,带菌植物材料是传播疮痂病的重要来源。准确、灵敏、快速的检测方法可为该病害流行规律调查和防控提供有力的依据。本文通过比较薄壳山核桃疮痂病菌Venturia effusa及其近似种之间的ITS序列差异,设计了特异性引物和TaqMan探针,建立了薄壳山核桃疮痂病菌的荧光定量PCR检测方法。特异性检测结果表明,该方法可以检测不同地区的薄壳山核桃疮痂病菌菌株,而对其近似种以及薄壳山核桃上的其他真菌均没有信号。本研究建立的检测方法对薄壳山核桃疮痂病菌DNA的最低检测限可达0.5 pg/μL。该方法用于田间样品检测时,检测时间仅需1 h,远快于常规的分离培养法。本研究建立的基于TaqMan探针的荧光定量PCR检测方法为薄壳山核桃疮痂病菌的快速检测和监测提供了有力工具。     

香蕉假茎细胞对枯萎病菌不同小种及其粗毒素的病理反应

 以香蕉枯萎病菌(Fusarium oxysporum f.sp.cubense)1号小种和4号小种及其粗毒素分别接种香牙蕉和粉蕉的组培苗及离体假茎后,用组织切片法观察香蕉假茎细胞的病理反应,以探明香蕉枯萎病菌不同小种及其粗毒素的致病作用。结果表明,枯萎病菌不同小种人工接种仅能感染相应的香蕉种类,但不同香蕉种类的离体假茎细胞用不同小种接种及其粗毒素处理,均产生褐变等病理反应,且病变程度不存在小种间的差异。表明枯萎病菌不同小种对香蕉不同种类的致病力差异可能与存在其它致病因子或专化性识别的因子有关。同时证实了病菌不同小种的毒素对蕉类不存在着选择毒性     

甘薯青枯病菌毒素产生条件的研究

 用致病力不同的甘薯青枯菌株(Pseudomonas solanacearum)进行毒素产生条件研究。试验结果表明,不同pH值、培养天数、菌株浓度及不同致病力的菌株所制备的毒素毒力差异达显著和极显著,但不同培养温度所制备的毒素毒力差异不显著。用Lotus1-2-3电脑软件综合分析以上产毒条件对所制备的甘薯青枯菌毒素的毒力影响程度,计算出强、弱和混合菌株产生毒素的3个最优回归方程。分别用Basic和Fortran语言编写程序,求出强、弱和强弱混合的甘薯青枯病菌产生毒素的3个最优模式为:①强菌株:pH=7,菌株浓度10⁸菌细胞/m l,培养天数3 d。②弱菌株:pH=7,菌株浓度10¹²菌细胞/ml,培养天数2 d。③混合菌株:pH=7,菌株浓度10¹⁴菌细胞/ml,培养天数1 d。培养温度均为25~35℃。     

Using the TxtAB operon to quantify pathogenic Streptomyces in potato tubers and soil

The phytotoxin thaxtomin, produced by plant pathogenic Streptomyces species, is the only known pathogenicity determinant for common scab diseases of potato and other root and tuber crops. Genes encoding thaxtomin synthetase (txtAB) are found on a pathogenicity island characteristic of genetically diverse plant pathogenic Streptomyces species. In this study, an SYBR Green quantitative real-time polymerase chain reaction (PCR) assay using primers designed to anneal to the txtAB operon of Streptomyces was developed to quantify pathogenic bacterial populations in potatoes and soil. The real-time PCR assay was specific for pathogenic Streptomyces strains. The detection limit of the assay was 10 fg of the target DNA, or one genome equivalent. Cycle threshold (Ct) values were linearly correlated with the concentration of the target DNA (correlation coefficient R(2) = 0.99) and were not affected by the presence of plant DNA extracts, indicating the usefulness of the assay for quantitative analyses of the pathogenic bacteria in plant tissues. The amount of pathogenic Streptomyces DNA in total DNA extracts from 1 g asymptomatic and symptomatic tubers was quantified using the assay and ranged from 10(1) to 10(6) pg. A standard curve was established to quantify pathogenic Streptomyces in soil. Using the standard curve, numbers of pathogenic Streptomyces colony forming units were extrapolated to range from 10(3) to 10(6) per gram of soil from potato fields where common scab was found. This real-time PCR assay using primers designed from the txtAB operon allows rapid, accurate, and cost effective quantification of pathogenic Streptomyces strains in potato tubers and in soil.     

梨果生刺盘孢子囊孢子单孢菌系的生物学特性及致病性研究

 梨果生刺盘孢的2种致病型菌株FJ-85(产生黑点症状)和菌株FJ-11-2(产生坏死斑症状)的子囊孢子单孢培养均可产生正、负2种类型的单孢菌系。正型单孢菌系菌落浅白色,其上形成的子囊孢子再培养,可形成正、负2种类型的单孢菌系;负型单孢菌系菌落深灰色,其上形成的子囊孢子再培养,只形成负型一种单孢菌系。2个菌株的单孢菌系中,负型的比例明显多于正型。2种类型的单孢菌系对峙培养,在菌落内及菌落交界处均可形成子囊壳,而同种类型的单孢菌系对峙培养仅在菌落内产生子囊壳。结果表明同种类型的单孢菌系可同宗配合,而正、负2种类型的单孢菌系还可异宗配合。正型单孢菌系的致病力与原始菌株相近,而负型单孢菌系较原始菌株弱。2个菌株的单孢菌系在翠冠梨上引起的症状差异与其原始菌株相同。研究结果为解析梨果生刺盘孢的有性生殖与致病的关系提供了新的有用信息。     

Toxin Profile, Fertility and AFLP Analysis of Fusarium verticillioides from Banana Fruits

Gibberella fujikuroi is composed of at least nine mating populations (MPs), corresponding to biological species and assigned letters (from A to I). Each MP possesses a specific toxicological profile and a preferential host. Members of Fusarium verticillioides and F. thapsinum, anamorphs respectively of MPs A (G. moniliformis) and F (G. thapsina), share identical morphological traits, but they have a different preferential hosts (maize and sorghum, respectively) and toxin profiles, beingable the only member of MP A to produce fumonisins and the only member of MP F to produce moniliformin. Isolates from banana fruits were identified morphologically as F. verticillioides. The isolates were analyzed for fumonisin and moniliformin production. While none of the isolates produced fumonisin, all the isolates produced moniliformin. The isolates were crossed with tester strains of MPs A and F, showing ability to produce fertile perithecia only when crossed by MP A tester strains isolated from maize. However, the time required for the formation of fertile perithecia and their size differed significantly from the usual fertile crosses of strains belonging to MP A. Pathogenicity tests using such isolates of F. verticillioides isolated from banana and a set of F. verticillioides isolates isolated from maize were also performed on banana fruits. The data showed that the isolates from banana were more pathogenic. Finally, isolates from banana and maize were compared using AFLP. The results revealed that isolates from banana and maize produced two distinctly different clusters. In conclusion, isolates of F. verticillioides from banana showed specific traits (toxin production, in vitro fertility, pathogenicity and molecular profiles), that were different to those of the same species from maize. This could reflect important differences in the ecology and natural history of the population from banana and should encourage further investigations into the mechanisms of toxin production and pathogenicity within the same MP.     

Phylogenetic Analysis of 16S rRNA Genes and PCR Analysis of the nec1 Gene from Streptomyces spp. Causing Common Scab, Pitted Scab, and Netted Scab in Finland

ABSTRACT The sequences of the 16S rRNA genes (nucleotides 29 to 1,521) from various Streptomyces strains pathogenic to potato were compared. These included 10 pathogenic Streptomyces strains isolated from potato scab lesions in Finland, the type strains of S. aureofaciens NRRL 2209(T) and S. lydicus ATCC 25470(T), 'S. griseus subsp. scabies' ATCC 10246, and two S. griseus strains that were originally deposited to the collection as pathogens. The nucleotide sequence (>94.5% sequence identity [SI]) and length (1,469 to 1,481 nucleotides) of the analyzed region varied. Phylogenetic analysis of 16S rRNA genes placed Finnish strains into three species, supported by previously characterized morphological and physiological traits. Six Finnish strains, including two strains that deviated from the others in one trait (no spiral sporophores or D-xylose utilization), had identical 16S rRNA genes and were identified as S. scabies (99.9% SI to S. scabies ATCC 49173). Three Finnish strains were identified as S. turgidiscabies, a species previously described only in Japan (99.9% SI to S. turgidiscabies ATCC 700248). Finnish strain 317 and S. aureofaciens NRRL 2209 (99.8% SI) were placed in a distinct phylogenetic cluster together with Kitosatospora spp., which suggests that S. aureofaciens may belong to the recently revived genus Kitosatospora. In pathogenicity tests, S. scabies caused characteristic symptoms of common scab, S. turgidiscabies caused mainly pitted scab, and S. aureofaciens caused netted scab and necrotic lesions on stolons of potato cultivars Bintje and Matilda in the greenhouse. The nec1 gene and the intergenic region between nec1 and the 5' transposase pseudogene ORFtnp were successfully amplified by polymerase chain reaction from S. scabies ATCC 49173 and the pathogenic Finnish strains of S. scabies, but not from a nonpathogenic strain of S. scabies, three pathogenic and two nonpathogenic strains of S. turgidiscabies, and S. aureofaciens.     

利迪链霉菌A02的抑菌谱及其抑菌活性的稳定性

离体抑菌试验表明,利迪链霉菌A02对植物病原真菌具有广谱的抑制活性,不同病原菌种类对其敏感性有一定的差异,果蔬灰霉病菌、番茄早疫病菌、小麦赤霉病菌和小麦纹枯病菌的敏感性较高;无菌发酵滤液稳定性测定显示,A02抑菌活性物质具有很好的热稳定性和耐酸碱、抗日光照射的特性,在70℃以下、pH5～8的条件下能较好地保持其抑菌活性。研究结果表明,拮抗菌A02在植物真菌性病害,尤其是半知菌引起的病害的生物防治中具有较高的应用价值。     

毒素在稻纹枯病菌致病中的作用分析

用水稻胚根生长抑制法测定了10个稻纹枯病菌菌株在寄主体外产生毒素的能力,发现不同菌株的产毒能力各不相同,且产毒量差异极显著。用水稻离体叶接种法测定了这10个菌株的致病力,结果表明：不同菌株的致病力也各不相同,且差异极显著。相关分析发现,菌株的体外产毒能力与致病力高度正相关（R =0.915 2）。这些结果表明：稻纹枯病菌毒素的分泌可能在其致病过程中起重要作用。     

芸苔链格孢菌种内和链格孢菌种间的酯酶同工酶比较

 对链格孢菌(Alternaria)不同种和芸苔链格孢菌(Alternaria brassicae)不同菌系的10个菌株的酯酶同工酶进行聚丙烯酰胺凝胶电泳(IF-PAGE)和TL紫外扫描分析。试验发现,链格孢菌种间酯酶同工酶的扫描结果差异比种内菌株间要大;对来自不同地区,不同品种的芸苔链格孢菌的酯酶同工酶扫描结果表明,白帮菌系和青帮菌系间的差异大于各菌系内菌株的差异,说明白菜青帮菌系内和白帮菌系内各菌株间各自的同源性较近。可见,IF-PAGE技术可用来鉴定病原菌的致病小种和菌系,也可用来研究病原菌的致病遗传机理。     

产纳他霉素生防菌A01和A02与已知产生菌的RAPD比较

为了明确自主分离的天然抗真菌活性产物——纳他霉素产生菌A01和A02与已知纳他霉素产生菌的遗传相似性,采用RAPD技术对这2株菌和3个产纳他霉素的标准菌株进行了比较分析。利用筛选出的7个随机引物对5个菌株的PCR扩增共得到154条清晰稳定的DNA条带,其中同源性条带86条,多态性条带68条,分别占总条带数的55.8%和44.2%,平均每个引物扩增出9.7个多态性条带,得到了丰富的DNA指纹图谱。所得数据经NTSYS pc 2.10e软件聚类分析,表明5个菌株间的遗传距离为0.0991~0.4738,其中A02与其它菌株间的遗传距离均较远。结合前期的分类研究结果,确证了菌株A02为新的纳他霉素产生菌。