福建省大豆疫病病原鉴定及其核糖体DNA-ITS序列分析

 从福建省龙海大豆根腐病株上分离的疫霉菌株中,选取6个代表菌株,对病原菌进行了形态特征、致病性、寄主范围鉴定及核糖体DNA-ITS序列分析,结果表明,该菌为疫霉属真菌,在黑麦琼脂培养基上生长缓慢,菌丝致密、无隔,形成菌丝膨大体,近直角分支,分支处稍缢缩。水培后产生大量椭圆形孢子囊,不形成乳突,通过内层出方式产生新孢子囊,游动孢子在孢子囊内形成,同宗配合,藏卵器球形,雄器侧生;接种后可出现典型的大豆疫病症状;人工接种只侵染大豆、豇豆和菜豆等少数豆科植物。其核糖体DNA-ITS序列分析表明,分离菌株与GenBank中大豆疫霉的ITS序列的同源性均为99.8%,仅有2个碱基的差异,结合形态特征和致病性测定,将这些病原菌鉴定为Phytophthora sojae. 这是首次报道大豆疫霉菌在福建省存在。     

海南橡胶树棒孢霉落叶病菌毒素蛋白基因检测与致病性分析

Cassiicolin毒素蛋白是橡胶树棒孢霉落叶病菌的主要致病因子。本研究对分离自海南省5个市县的橡胶树棒孢霉落叶病菌进行了鉴定,形态学鉴定结合分子生物学鉴定结果表明,病原菌为多主棒孢。利用Cassiicolin毒素不同亚型编码基因特异性引物和菌饼活体接种法分别鉴定了分离菌株的毒素蛋白类型和致病性,结果显示,分离的35个菌株都含有Cas5基因,未发现含其他类型的毒素基因,表明海南橡胶树棒孢霉落叶病菌为含Cas5毒素蛋白优势群体;所有菌株对橡胶树品种‘RRIM600’都能够致病,但是在不同抗性品种上致病性存在一定差异。     

蒙城大豆疫霉菌的鉴定及其生理小种

 在安徽省蒙城县对大豆疫霉根腐病的发生情况进行调查。应用选择性培养基对类似大豆疫霉根腐病症状的病株进行病原菌分离,在春大豆蒙城早熟青豆病株上分离到2株疫霉菌PMC1、PMC2和一些Fusarium spp.,在夏大豆上分离到的主要病原菌为Pythium spp.和Fusarium spp.,未分离到疫霉菌。根据疫霉菌分离物PMC1和PMC2形态和生理学特征以及对大豆的专化致病性,2个分离物被鉴定为大豆疫霉菌(Phytophthora sojae Kaufmann&Gerdemann)。应用国际通用鉴别寄主进行生理小种鉴定,PMC1和PMC2的毒力公式分别为1b,1d,3a,3c,5,7和1b,1d,4,5,为新的小种类型,定名为中国6号小种、中国7号小种(CNR-6和CNR-7)。这是首次报道大豆疫霉菌在我国淮北地区存在。     

柑桔脚腐病研究

 1986-89年.通过病原菌分离、培养.纯化和接种试验,结合形态、生物学特性,菌体蛋白质的聚丙烯酰胺凝胶盘状电泳和扫描等,鉴定出了四川柑桔脚腐病的病原是疫霉属寄生疫霉、柑桔褐腐疫霉、棕榈疫霉、恶疫霉、甜瓜疫霉等.其中寄生疫霉所占比例最大.     

雪松疫霉(Phytophthora lateralis)的快速分子检测

由雪松疫霉(Phytophthora lateralis)引起的疫病是一类植物检疫性病害。为建立该病原菌的快速检测技术,本文比较分析了雪松疫霉和其他疫霉的tRNA序列,在此基础上设计了一对检测雪松疫霉的特异性引物T1/T2,该对引物从雪松疫霉中扩增得到1条192 bp的条带,而其他15种疫霉和其他真菌菌株均无扩增条带,表明该对引物对雪松疫霉具有特异性。在25μL PCR反应体系中,引物T1/T2检测灵敏度为10 pg基因组DNA;而以引物T3/T4和T1/T2进行巢式PCR扩增,能够检测到1 fg基因组DNA,使检测灵敏度提高了10 000倍。该检测体系对灭菌水中游动孢子的检测灵敏度可达0.5个游动孢子,对人工接种发病的植物组织能够特异性地检测到该病原菌。此外,进一步建立了该病原菌的实时荧光定量PCR检测体系。     

大雄疫霉与大豆疫病

前言大雄疫霉为藻菌纲、霜霉目、腐霉科、疫霉属真菌的一个种。在近代分类史上,真菌学家们虽根据其孢子形态和致病性等对此菌进行了多次分类与修订,在种内又区分出几种形态和致病性不同的类型,而大豆疫病菌仅是其中的一种。伴随着大雄疫霉菌的分类进展,致使大豆疫病菌的命名也发生了相应的变化:由Phvtophthora megasperma 发展为 P.     

重庆地区辣椒疫霉菌的分离培养及生理小种鉴定

本试验分离鉴定了来源于重庆不同辣椒产区的14个病原菌分离物,经形态特征鉴定及回接发病特征观察,确定这些菌株均为辣椒疫霉(Phytophthora capsici)。14个菌株在OMA培养基上诱导产生的孢子囊形态类似,形状多为卵圆形或长椭圆形,乳突明显。对PDA、OMA、CA及V8汁4种培养基上的培养性状观察表明,辣椒疫霉菌株在OMA培养基上生长速度最快,但在V8汁培养基上产孢最多。采用灌根法对14个菌株进行生理小种鉴别,其中10个为race 3,2个为race 2,2个为race 1,初步推定race 3为重庆地区辣椒疫病病原菌的优势小种。     

安徽省大豆疫霉根腐病菌的鉴定及rDNA-ITS序列分析

为明确安徽省夏大豆疫霉根腐病的病原菌种类,对采集自涡阳、怀远、固镇3个县的夏大豆病株及土样分离纯化后获得28株菌株,选取6株代表性菌株,通过形态学观察及核糖体DNA-ITS序列分析对其进行鉴定,并测定了其致病型。结果表明,6株菌株在利马豆培养基上菌落白色,质地均匀;菌丝无隔,致密,具近直角分枝;在10%V8C培养液中,游动孢子囊顶生,不脱落,卵形至椭圆形,无明显乳突,有内层出现象,长宽比大于1.6∶1;同宗配合,在利马豆培养基上单株培养产生大量卵孢子,藏卵器球形,雄器大多侧生;接种合丰35大豆品种后出现典型的大豆疫霉根腐病症状。r DNA-ITS序列分析表明,6株菌株与Gen Bank中大豆疫霉Phytophthora sojae的ITS序列同源性高达100%;菌株GY4、GY8、HY11、HY16、GZ10、GZ21的毒力公式分别为1b,2,3a,3b,4,5,6,7;1b,1d,3a,3b;1d,3a,3b,3c,4,5,6,7;2,3c,4,5,6,7;1b,3a,3c,5,8;3a,3b,5,6,7,8;属于6个不同的致病型。研究表明,这6株菌株均为大豆疫霉。     

冬生疫霉(Phytophthora hibernalis)的快速分子检测

 由冬生疫霉(Phytophthora hibernalis)引起的疫病是一类植物检疫性病害。为建立该病原菌的快速检测技术,本文比较分析了冬生疫霉和其它疫霉的ITS序列,在此基础上设计了一对检测冬生疫霉的特异性引物751F/752R,该对引物从冬生疫霉中扩增得到一条616bp的条带,而其它19种疫霉和其它真菌菌株均无扩增条带,表明该对引物对冬生疫霉具有特异性。在25μL PCR反应体系中,引物751F/752R检测灵敏度为10龟基因组DNA;而以卵菌ITS区通用引物ITS1/ITS4和751F/752R进行套式PCR扩增,能够检测到10ag的基因组DNA,使检测灵敏度提高了1000倍。该检测体系对灭菌水中游动孢子的检测灵敏度可达0.5个游动孢子。结合快速碱裂解法提取发病组织的DNA,采用该PCR检测技术,在1个工作日内即可从人工接种发病的植物组织中特异性的检测到该病原菌。表明本研究建立的检测方法可用于冬生疫霉的快速分子检测。     

中国樟疫霉A1交配型的研究

 1985—1986年自福建、浙江、江苏等省的鳄梨（Persea americana）、山茶花（Camellia sp.）、雪松（Cedrus deodara）、上分离到疫霉属（Phytophthora de Bary）真菌17个菌株,全部鉴定为樟疫霉（Phytophthora cinnamomi Rands）。其中来自福建漳州鳄梨、浙江杭州山茶花上的5个菌株全部为樟疫霉A₁交配型.自江苏南京雪松上共分得樟疫霉12个菌株,11个菌株属A₂交配型,只有一个菌株为A₁交配型.雪松是樟疫霉A₁交配型的寄主新记录.不同寄主植物上的樟疫霉A₁交配型菌株的形态有一定的差异,樟疫霉的A₁、A₂交配型的培养性状和形态特征差别不明显.     

新疆主要农作物疫霉菌种类鉴定

 1993~1995年,对侵染新疆主要农作物的疫霉菌进行了较全面的调查研究,共从9个地区的28种作物上采集表现根腐、根颈腐、果腐症状(包括病土)的病样1531个,结果从其中17种作物上分离到452个疫霉菌株。根据形态特征、生理生化特性、致病性和菌体可溶性蛋白电泳测定,鉴定为7个种:辣椒疫霉(Phytophthora capsici Leon.),恶疫霉[P.cactorum(Leb.et Cohn) Schroeter],掘氏疫霉(P.drechsleri Tucker),菸草疫霉(P.nicotianae van Breda de Haan),苎麻疫霉(P.boehm eriaeSaw.),柑桔褐腐疫霉[P.citrophthora (Sm.et Sm.) Leonian],隐地疫霉(P.cryptogea Pethyb.etLaff.)。这些疫菌是造成新疆茄果类、瓜类、棉花、苹果、梨、枸杞、草霉、红花、白术等幼苗及成株大量死亡及烂果的主要病原,在农业生产中具有十分重要的意义。     

甘蓝枯萎病病原菌的鉴定

 采用温室接种致病性测定、形态学观察和分子鉴定方法对甘蓝枯萎病病原进行了研究。从北京市延庆县9个甘蓝生产基地采集甘蓝枯萎病病样96份,分离获得来自甘蓝病株根、短缩茎、叶片等器官的30个真菌分离物。经过致病性试验证实,分离物GLW3和GLW8为甘蓝枯萎病病原菌。经形态学鉴定,GLW3为尖孢镰刀菌(Fusarium oxysporum),GLW8为轮枝样镰刀菌(Fusarium verticillioides)。利用镰刀菌的核糖体DNA(rDNA)内转录间隔区(internal transcribed spacer,ITS)的保守性和变异性,分别采用真菌通用引物和镰刀菌属及轮枝样镰刀菌特异性引物对GLW3和GLW8进行PCR扩增,并将测序结果在GenBank中进行同源性比对分析,分子鉴定与形态学鉴定结果一致。轮枝样镰刀菌作为甘蓝枯萎病的病原菌系首次报道。     

黑龙江省籽用南瓜疫病病原菌致病力分析与抗性种质资源评价

由辣椒疫霉菌(Phytophthora capsici Leonian)引起的南瓜疫病是黑龙江省籽用南瓜生产中的首要病害,造成严重的死瓜烂秧现象,籽用南瓜在黑龙江省的种植面积逐年减小.本研究在对2017～2019年黑龙江省籽用南瓜种植以及疫病发生危害情况进行分析的基础上,采用组织分离法对疫病高发区采集的病样进行分离和纯...     

玉米穗腐病样品中层出镰刀菌的分离与鉴定

为了解豫西南玉米穗腐病的病原菌,于2009年9月在河南省南阳市6个县区采集玉米穗腐病样品,进行病原菌的分离与单孢纯化.依据致病性、形态及分子检测结果进行病原菌鉴定.研究结果表明,分离得到的105株镰刀菌中,层出镰刀菌Fusarium proliferatum为70株,占分离镰刀菌的66.7％,属优势种群.在基于核糖体基因内转录间隔区(rDNA-ITS)与翻译延长因子1α基因(tef)序列构建的系统发育树中,3株代表菌株Fp1、Fp2和Fp3分别与GenBank登记的层出镰刀菌在自举值89％和91％水平相聚同一群.层出镰刀菌的生长温度为13～34℃,最适温度为28℃;在pH5.0和6.0各有1个生长高峰;碳源对该菌营养生长的影响较为稳定,而氮源对其营养生长影响的变幅较大.     

Occurrence and Identification of Phytophthora spp. Pathogenic to Pear Fruit in Irrigation Water in the Wenatchee River Valley of Washington State

ABSTRACT Seven hundred forty-nine isolates of Phytophthora spp. were obtained from irrigation canals in eastern Washington State during the 1992 to 1995 and 1999 growing seasons. Isolates were retrieved using pear baiting techniques. All isolates were pathogenic to pear and were present in irrigation water beginning early in fruit development. Over the course of the 5 year study, 10 and 5% of isolates were identified as P. cactorum and P. citricola, respectively, using morphological criteria. The remaining isolates could not be identified using morphological criteria. Colony morphology of these isolates was characterized during all years of the study. In 1999, more detailed studies utilizing polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis of entire internal transcribed spacer (ITS) regions (ITS1, 5.8S, and ITS2) of ribosomal DNA for 180 isolates, and sequence analysis of ITS2 for 50 isolates, were used to investigate genetic variation and phylogenetic relationships among isolates. Isolates were divided into 12 groups based on their growth type on corn meal agar. Restriction digestion of the entire ITS region with three enzymes revealed 11 restriction digestion patterns among 180 isolates. PCR-RFLP and sequence data were obtained for 12 reference Phytophthora spp. (two species in each of Waterhouse's six morphological groups). Phylogenetic analysis of ITS2 regions revealed nine clades, each with strong bootstrap support. Molecular analyses revealed 23 isolates that were in the P. gonapodyides clade, 9 in the P. parasitica clade, 1 in the P. cactorum clade, 7 in the P. citricola/capsici clade, and 4 in the P. cambivora/pseudotsugae clade. The three isolates comprising clade 5 were significantly distinct from all other Phytophthora spp. in the databases and may represent a new Phytophthora sp. Colony morphology was not consistently correlated to PCR-RFLP pattern or ITS2 phylogeny, suggesting that the former criterion is insufficient for species identification. The results of this study indicate that at least nine phylogenetically distinct taxa of Phytophthora pathogenic to pear are present in irrigation water in North Central Washington.     

Inter- and intraspecific morphometric variation and characterization of Phytophthora isolates from cocoa

Difficulties in the accurate identification of the Phytophthora species responsible for black pod disease of cocoa continue to hamper effective disease control. A re-evaluation of morphological characters ( Brasier & Griffin, 1979 ) and a detailed morphometric analysis of 161 Phytophthora isolates largely associated with black pod disease of cocoa from 17 countries worldwide have shown considerable inter- and intraspecific variation. Stable and more reliable parameters for the identification of the species responsible for the disease have been determined. Colony characteristics such as pattern and growth rate on V8 agar are reasonably characteristic for the cocoa Phytophthora species, and can be used to make preliminary identification to species level. Significant sporangial character variation was found within isolates of species from the same and different sources, highlighting the difficulties in making accurate identification on the basis of raw morphological data. Pedicel length was found to be the most consistent species-linked sporangial characteristic. Cluster plots of length/breadth ratios of sporangia versus reciprocals of sporangial pedicel length clearly separated all isolates into distinct species groups ( P. capsici , P. citrophthora , P. palmivora and P. megakarya ) and can be used reliably to identify accurately those pathogens involved in black pod disease outbreaks.     

海南省胡椒瘟病病原鉴定及发生规律

采用形态特征观察、致病性测定及rDNA-ITS序列分析等方法,对海南省主产区胡椒瘟病病原进行鉴定,通过田间实地调查对胡椒瘟病的发生规律进行了研究。结果表明,该病病原为辣椒疫霉(Phytophthora capsici),月平均降雨量对该病的发生有着显著的影响。降雨量越大,持续降雨天数越多,发病率越高。     

Genetic variation and host specificity of <Emphasis Type="Italic">Phytophthora citrophthora</Emphasis> isolates causing branch cankers in Clementine trees in Spain

Considerable tree losses have been observed during the past few years in Spain due to Phytophthora branch canker of clementines caused by Phytophthora citrophthora. The emergence of this disease led to the speculation that either the pathogen has evolved increasing its aggressiveness or specificity to clementines. A total of 134 isolates of P. citrophthora collected from 2003 to 2005 in 135 citrus orchards in Spain and 22 reference isolates were analyzed genotypically and phenotypically to determine the structure of the population. Genotypic diversity was evaluated by means of Inter-Simple Sequence Repeat (ISSR) markers. Among the phenotypic characteristics examined, sporangial characters, sexual behavior, growth rates and colony morphology of the isolates at different temperatures were studied. The aggressiveness and host-specificity of selected isolates were evaluated by pathogenicity tests on sweet oranges and clementines under field conditions. Phytophthora branch canker of clementines was associated mainly with one genotype (P-1), which included 88% of the isolates obtained from branches. Strains isolated years before the first disease outbreak clustered also with this major genotype, thus it may be considered as a predominant population. Thirteen other minor genotypes were determined, but most contained only one isolate. Although there was wide variation in the morphological and physiological characters, all Phytophthora isolates obtained from branch cankers were sexually sterile and showed a characteristic petalloid colony pattern. As in previous greenhouse studies, pathogenicity tests under field conditions demonstrated that clementines and their hybrids were more susceptible to P. citrophthora than sweet oranges. However, no evidence was found to support the hypothesis that the emergence of the disease was associated with more aggressive or host-specific forms of P. citrophthora.