基于Dickeya dadantii特有标志基因检测甘薯茎腐病菌

 甘薯细菌性茎腐病是由达旦提狄克氏菌(Dickeya dadantii)引起的一种检疫性病害,近年来在我国多地发生,严重威胁我国甘薯产业的发展。建立特异灵敏的检测D. dadantii的方法对于鉴定检疫病原菌、田间监测病原菌和防控病害有重要意义。本研究对Dickeya属菌株的全基因组序列进行比较基因组学分析,筛选到D. dadantii特有的标志基因,针对标志基因设计引物,其中针对编码登录号为WP_077245517未知蛋白基因的1对引物Dad1-F(5′-CATATCAACCAGACCAGCCGTT-3′)和Dad1-R(5′-CGGCCTGCTTTTAAACAACGTATTA-3′)能只从D. dadantii扩增到167 bp目的片段。由此建立了特异灵敏的常规PCR和实时荧光定量PCR检测D. dadantii的方法,为鉴定检疫甘薯茎腐病病原菌和田间监测病害提供了有效方法。     

基于Dickeya dadantii特有标志基因检测甘薯茎腐病菌

 甘薯细菌性茎腐病是由达旦提狄克氏菌(Dickeya dadantii)引起的一种检疫性病害,近年来在我国多地发生,严重威胁我国甘薯产业的发展。建立特异灵敏的检测D. dadantii的方法对于鉴定检疫病原菌、田间监测病原菌和防控病害有重要意义。本研究对Dickeya属菌株的全基因组序列进行比较基因组学分析,筛选到D. dadantii特有的标志基因,针对标志基因设计引物,其中针对编码登录号为WP_077245517未知蛋白基因的1对引物Dad1-F(5′-CATATCAACCAGACCAGCCGTT-3′)和Dad1-R(5′-CGGCCTGCTTTTAAACAACGTATTA-3′)能只从D. dadantii扩增到167 bp目的片段。由此建立了特异灵敏的常规PCR和实时荧光定量PCR检测D. dadantii的方法,为鉴定检疫甘薯茎腐病病原菌和田间监测病害提供了有效方法。     

一株地黄茎部致病菌的鉴定及防治药剂筛选

 在河南省温县地黄(Rehmannia glutinosa)种植园采集茎部感病的地黄植株,从感病部位分离、纯化出4株病原菌。通过对分离菌的形态学观察、致病性检测及ITS、EF1-α、TUB、CAL、HIS多基因核酸序列分析,鉴定其中1株分离菌为扁桃腐皮壳菌(Diaporthe amygdali),为地黄茎部病害的新型致病菌。生物学特性研究表明,最适宜该致病菌生长的为PDA培养基;菌丝生长的最佳温度为25 ℃、最适pH值为6、对光照不敏感;最适碳氮源分别是麦芽糖和蛋白胨。药剂抑菌活性筛选结果表明,选用的10种杀菌剂对D. amygdali生长都有一定的抑制作用,其中500 g·L^-1氟啶胺悬浮剂活性最高,EC₅₀为0.092 5 mg·L^-1。上述结果为D. amygdali引起的地黄茎部病害防治提供了科学依据。     

甘薯茎腐病症状及其病原鉴定

 甘薯茎腐病是危害甘薯的一种严重病害,甘薯茎、叶柄、叶片和块根均可被害。该病典型症状是茎基部发黑和变软腐烂,叶发黄,茎和块根维管束黑褐色,引起块根腐烂、有臭味。通过对甘薯茎腐病典型症状样本的采集、病原菌的分离和纯化以及致病性测定,明确该病害是一种细菌病害。通过对甘薯茎腐病菌的菌体形态和培养特性观察发现,病原菌是革兰氏阴性细菌,菌体短杆状,大小约为2.36 μm×0.4 μm, 周生鞭毛,可在烟草上激发过敏性反应(HR)。Biolog测定、脂肪酸分析、16S rDNA序列分析、MALDI-TOF质谱鉴定和8个看家基因(dnaX、rplB、fusA、gapA、gyrA、purA、recA和rpoS)的序列系统发育分析,发现该病原菌与达旦提狄克氏菌Dickeya dadantii高度一致。这些结果说明,浙江省发生的小番薯病害是甘薯茎腐病,病原为D. dadantii。     

浙麦冬黑斑病病原菌的分离鉴定及淀粉酶产色链霉菌对其生物防治效果

黑斑病是浙江省慈溪市浙麦冬种植基地常发病害,传染速度快且严重影响麦冬的产量与品质。为了明确引起黑斑病的病原菌以便后续有效防治该病害,本文通过对染病组织培养、病原菌分离纯化和柯赫法则验证、形态学观察与基因序列分析比对（ITS、EF-1α、RPB2、Alt a 1、His 3、ATP）,最终鉴定出引起浙麦冬黑斑病的病原菌是链格孢菌Alternaria alternata。研究淀粉酶产色链霉菌Streptomycesdiastatochromogenes 1628代谢产物对黑斑病的防效结果表明,1628代谢产物对链格孢菌菌丝生长具有较强的抑制作用,其EC₅₀=0.764%（体积浓度）。田间防效结果表明7和14 d校正防效分别可达74.08%和65.28%,均高于阳性对照多菌灵。本研究明确了浙麦冬黑斑病病原菌的分类地位,并筛选到能有效抑制该病原菌的生防菌株淀粉酶产色链霉菌,为后续浙麦冬黑斑病的防治提供依据。     

新疆榆树黄萎病病原菌鉴定

2013年以来,在石河子大学农学院试验站榆树苗上,出现了一种病害,可导致叶片褪绿,变黄,萎蔫,甚至枯死,剖茎检查可见维管束变成褐色。采用常规组织分离法对病茎组织进行分离、纯化获得单孢纯培养菌株;通过常规纸钵撕底蘸根法对其进行致病性测定;用形态学和rDNA-ITS序列分析方法对病原菌进行鉴定。结果表明,分离菌株的菌落形态、分生孢子梗和分生孢子的形态都与大丽轮枝菌(Verticillium dahliae)一致;经分子生物学鉴定,该菌的rDNA-ITS序列与中国棉花黄萎病菌(V.dahliae)的ITS序列(登录号EU 835817.1)相似性达99.0%,故将引起新疆榆树黄萎病的病原菌鉴定为大丽轮枝菌(V.dahliae)。     

红掌细菌性疫病的病原菌初步鉴定

 在云南西双版纳的红掌上发现一种由细菌侵染引起的病害,从叶片的病组织上分离到具有致病性的杆状细菌,将分离的病原菌接种于健康的红掌上,表现出与田间一致的症状。感病初期在叶缘或叶脉间出现水渍状小点,后期病斑多呈棕褐色至黑色坏死,病健交界处多呈黄色。从接种发病的病斑与田间标样上分离的病原菌菌落形态完全相同。通过菌株的培养性状、常规生理生化特性及透射电镜下菌体形态观察结果,将病原菌初步鉴定为黄单胞菌属(Xanthomonas)。BIOLOG鉴定和致病性测定结果进一步鉴定病原菌为地毯草黄单胞菌花叶万年青致病变种Xanthomonas axonopodis pv. dieffenbachiae(Xad)(McCulloch & Pirone) Vauterin et al.     

洛阳牡丹新病害—柱枝孢叶斑病

为了明确河南省洛阳地区牡丹叶部一种新病害的病原种类,对采自田间的典型症状叶片进行了常规组织分离,对单孢菌株进行了形态鉴定、rDNA ITS序列分析及致病性测定.结果表明,病原菌在PDA培养基上菌落初期白色绒毛状,后期红褐色.分生孢子梗通常呈扫帚状,有2～3个分支,末端膨大呈泡囊状.分生孢子直棒状,两端钝圆,有0～1个隔膜,大小为(38.4～86.4)μm× (4.6～5.9)μm,平均60.1 μm×5.2 μm.离体叶片与活体植株接种结果表明,该菌均能侵染牡丹叶片并引起与田间相同的症状.据此将分离物鉴定为Cylindrocladium canadense,将该病害命名为牡丹柱枝孢叶斑病.     

高粱刺盘孢Colletotrichum sublineola是四川地区高粱炭疽病的主要致病菌

 高粱是四川省主要经济作物,在高粱生产上普遍发生炭疽病。关于高粱炭疽病菌多数文献描述为禾生刺盘孢Colletotrichum graminicola。已有文献通过分子生物学和基因组学分析证明高粱炭疽病的病原菌是高粱刺盘孢C. sublineola,并非C. graminicola。为明确四川省和国内高粱主产区炭疽菌的种类,本研究从四川省泸州泸县等11个地区以及重庆等其他6个省市,总共采集了198份病样,经分离纯化共获得225个炭疽菌株。通过形态学观察,结合ITS和GAPDH基因序列及进化树分析,发现其中215个来源于17个地区的菌株为C. sublineola,且分生孢子形态均为月牙型。其余10个分别来源于两个地区的菌株为兰花刺盘孢C. cliviicola,其分生孢子形态均为圆柱型。通过另外3个基因(CHS、ACT和TUB2)的系统发育分析,进一步证明来自不同地区的17个代表性的炭疽菌菌株分类相同,且均属于C. sublineola。研究结果表明,分生孢子形态及ITS和GAPDH等基因序列可以作为高粱炭疽菌的主要分类依据。在常规高粱品种Hui1等3个材料上接种19个代表性菌株,发现17个C. sublineola菌株的致病能力存在差异,2个C. cliviicola菌株均不能致病。本文研究结果表明,C. sublineola是四川高粱主产区炭疽病的致病菌。另外,通过人工接种试验,证明高粱品种402B对强致病力菌株ZG-FS-1具有较强抗性,402B具有抗病育种应用潜力。     

白术枯斑病原鉴定、生物学特性及其对不同药剂的敏感性研究

 白术(Atractylodes macrocephala Koidz.)是浙江省传统中药材“浙八味”之一。近年来在磐安县白术主产区发生一种新的枯斑病。为明确致病菌及其生物学特性和对杀菌剂的敏感性,本研究通过病原菌分离培养、柯赫氏法则验证、形态学特征观察,及ITS、LSU、rpb2和tub2多基因序列分析,发现引起该枯斑病的病原菌为Phoma exigua。生物学特性测定表明:该菌菌丝生长的最适温度为28℃,最适pH为8,最适碳源与氮源分别为可溶性淀粉和蛋白胨;分生孢子萌发最适温度24℃、相对湿度100%;菌丝和分生孢子的致死温度分别为47℃和54℃。不同药剂敏感性测定结果表明:咯菌腈对该菌菌丝生长的抑制作用最强,EC₅₀为0.24 μg·mL^-1;吡唑醚菌酯和嘧菌酯对其分生孢子萌发具有较强的抑制作用,EC₅₀分别为0.10和0.16 μg·mL^-1。由P. exigua 引起的白术枯斑病为国内外首次报道。     

越南槐圆斑病病原鉴定

The pathogen causing round leaf spot of Sophora tonkinensis (Lour.) was identified by Koch's postulates, morphological characteristics and combined gene sequences analysis on ITS, TUB, and ACT. The results showed that morphological characteristics of a pathogenic representative isolate SDG-1 was similar to that of Didymella glomerata. The isolate SDG-1 was clustered into one clade in phylogenetic tree with D. glomerata. Taken together, these results indicated that the round leaf spot of S. tonkinensis was caused by D. glomerata. This is the first report of a round leaf spot on S. tonkinensis was identified as D. glomerata.     

南天竹一种新的叶斑病病原菌的分离鉴定

<正>南天竹(Nandina domestica)为小檗科南天竹属,常绿灌木,形态优雅,果实球形鲜红且经冬不落,是赏果观叶的园林绿化植物。南天竹具有重要的药用价值,有“镇咳止喘、兴奋神经、强壮身体”之效[1]。在生长过程中,南天竹叶片易受炭疽病(Colletotrichum)危害[2],也可受叶斑病(Pestalotia microspore)、褐斑病(Cercospora nandinae)、     

青苹果竹芋平脐蠕孢叶斑病病原菌的鉴定

A new leaf spot disease on Calathea rotundifolia cv. fasciata was observed in Guangdong, China. The pathogen of this disease was identified by pathogenicity, morphological characteristics and multi-gene sequences analysis of ITS, TEF and GAPDH. The result showed that leaf spot of C. rotundifolia cv. fasciata was caused by Bipolaris sivanesaniana. This is the first report of leaf spot disease on C. rotundifolia cv. fasciata caused by B. sivanesaniana.The results of host range determination showed that B. sivanesaniana could also infect Calathea veitchiana and cause leaf spot. This study will facilitate the timely detection and management of Bipolaris leaf spot.     

浙江甘薯蔓枯病病原菌鉴定

Recent years, gummy stem blight caused by Diaporthe spp. is an important disease on Ipomoea batatas. In this study, infected plant tissues, collected from Ningxi, Taizhou, were used for isolating pathogens from the gummy stem. Three pure strains with similar cultural characteristics were isolated, tested for their pathogenicity against stems of I. batatas, and re-isolated from the tissue inoculated, and Koch's postulate was fulfilled. The rRNA intergenic spacer (ITS) region of these isolates was amplified and the sequences were used for the phylogenetic analysis. The aliment result showed that the sequences (MT107556, MT107557 and MT107558) of three strains were 100% similarity with that of D. batatas CBS 122.21. These isolates were identified as D. batatas based on the morphological and molecular characteristics. Therefore, gummy stem blight on Ipomoea batatas was caused by D. batatas in Zhejiang province. Meanwhile, this study firstly showed that the stroma of D. batatas was coralloid.     

引起甜樱桃叶斑病的葡萄座腔菌的鉴定

Cherry leaf spot disease is one of the most common diseases of sweet cherry. In 2018 and 2019, a leaf spot symptom was observed on sweet cherry in Beijing city, Shandong province and Liaoning province, and the pathogen fungi were isolated from diseased samples. Seven fungal isolates (JZB310197-JZB310203) were identified as Botryosphaeria dothidea according to morphological characteristics and multi-gene (ITS, EF 1-α and TUB2) phylogenetic analysis. To fulfill the Koch′s postulation, pathogenicity assay was carried out on detached leaves and tissue cultured seedlings. The inoculation results showed that all isolates were pathogenic to cherry leaves, proving that B. dothidea was a pathogen of cherry leaf spot disease. This is the first report of B. dothidea causing leaf spot of sweet cherry in China.     

北京市月季丽赤壳褐斑病病原菌鉴定

In September 2018, a new leaf spot on Rosa chinensis with incidence of up to 60% was observed in a park in Miyun district of Beijing. Disease samples were collected and three strains (JZB3210001, JZB3210002 and JZB3210003) were obtained by isolation and single spore purification. According to the morphological characteristics, they were identified as Calonectria sp.. For molecular phylogenetic analysis, β-tub, Cal, H3, EF1-α genes were amplified with genomic DNA of these three isolates. Phylogenetic trees constructed by the Maximum likelihood method showed that three strains clustered with Calonectria blephiliae CBS136425 as a distinct clade with 91% bootstrap support. Based on both morphological characteristics and phylogenetic analysis, the three strains were identified as C. blephiliae. The pathogenicity assay confirmed that C. blephiliae was the pathogen causing this disease. This is the first report of leaf spot caused by C. blephiliae on Rosa chinensis in China.     

中华常春藤细菌性叶斑病病原菌的鉴定

 2015年春季在昆明市区绿化带种植的中华常春藤上发现一种由细菌侵染而引起的病害,称为中华常春藤细菌性叶斑病。通过发病症状、菌落形态观察、致病性测定、Biolog分析,16S rDNA序列和核糖体DNA内转录间隔区(internal trans-cribed spacer, ITS )序列分析比较,对昆明地区的常春藤叶斑病病原菌及其系统进化关系进行研究。分离病原菌接种中华常春藤叶片完成科赫法则验证,发病初期在叶片表面形成带有黄色叶晕的不规则褐色斑点,后期叶片边缘形成倒V字型坏死并起皱。将菌株CCT1和CCT6测序结果与现有的黄单胞菌菌株的16S rDNA序列和核糖体DNA的ITS序列构建进化树,结果均显示病原菌与野油菜黄单胞菌的序列相似度最大,属于同一支。研究确定该病原菌为野油菜黄单胞菌(Xanthomonas campestris)。这是中国首次报道由X. campestris 引起的中华常春藤叶斑病。     

草莓根腐病的Dactylonectria病原菌鉴定

<正>0引言草莓(Fragaria×ananassa Duch．)为蔷薇科草莓属多年生草本植物^[1],是北京市特色优势果品之一,为北京农民增收,区县经济发展,发挥了重要作用。引起草莓根腐病的病原菌种类众多,是较难防治的草莓病害。据报道,引起草莓根腐病常见的病原菌有镰刀菌属(Fusarium)、刺盘孢属(Colletotrichum)、疫霉属(Phytophthora)和丝核菌属(Rhizoctonia)^[1]。     

抗生素溶杆菌13-6对玉米小斑病的生防作用机制初步研究

 玉米小斑病是玉米生产中的重要病害之一,本研究利用传统植物病理学和荧光定量PCR方法,研究抗生素溶杆菌对玉米小斑病菌(Bipolaris maydis)的生防效果和作用机制,为玉米小斑病的生物防控提供理论依据。结果显示,抗生素溶杆菌13-6对玉米小斑病菌丝生长和孢子萌发具有显著抑制作用,造成菌丝顶端生长受阻、表面粗糙,部分菌丝破裂,代谢粗提物抑制孢子萌发率高达92.19%,PI荧光染色显示病原菌孢子破裂并死亡。温室叶面喷施13-6发酵液、发酵液粗提物后显著降低玉米小斑病病情指数,相对防效分别达到42.43%和68.06%。诱导抗性实验表明:13-6对玉米小斑病的诱导防效达49.57%。玉米植株体内相关抗性基因表达结果显示,在灌根处理3 d后,抗病相关基因PR1和PR5、脂氧合酶LOX、乙烯受体ETR1均被激活表达,9 d达到最大值。研究证实抗生素溶杆菌13-6对玉米小斑病具有较好的生防效果,直接喷施可减缓玉米小斑病的发生,灌根施用使植株产生诱导抗性。