宁夏葡萄霜霉病菌致病型鉴定及葡萄品种抗性评价

为合理利用葡萄抗性品种以及田间病害综合治理,采用离体叶盘接种法分别对2018年和2019年自宁夏回族自治区银川市、石嘴山市和吴忠市采集的43株葡萄霜霉病菌Plasmopara viticola菌株进行致病型鉴定及聚类分析,并对17个主栽葡萄品种进行抗性评价及聚类分析。结果表明：2018、2019年供试菌株致病力和供试葡萄品种抗性之间均存在显著差异,菌株平均病情指数与发病率呈显著正相关,皮尔逊相关系数r和回归系数R²分别介于0.45~0.96和0.20~0.92之间;2018、2019年供试菌株的强致病力菌株和弱致病力菌株分别为CS-YL和VI-BYDD、RG-JSJG和CS-YM;2018年和2019年供试菌株致病型被划分为强、中、弱3种类群,2年所占比例分别为10.00%、15.00%、75.00%和34.78%、39.13%、26.09%;供试17个葡萄品种的抗性类型可被划分为感病、抗病、高抗3种类群,2年所占比例均为17.65%、29.41%和52.94%。不同葡萄品种对不同菌株的抗性类型多样化。表明宁夏回族自治区葡萄霜霉病菌菌株致病力分化明显,不同菌株的致病型与其地域来源无关,不同葡萄品种的抗感程度不同。     

上海地区草莓灰霉病菌对啶酰菌胺的抗性现状研究

为研究上海地区草莓灰霉病菌对啶酰菌胺的抗性现状,本文在上海采集了草莓灰霉病样,共分离纯化得到115株草莓灰霉病菌(Botrytis cinerea)。采用菌丝生长速率法测定了草莓灰霉病菌对啶酰菌胺的敏感性数据。结果表明,所测定的115株灰霉菌株中抗性菌株的比例占13. 9%,以中抗水平为主,共检测到16株抗性菌株,其中12株为中抗菌株,低抗和高抗菌株各检测到2株。田间试验数据显示,啶酰菌胺对草莓灰霉病3次药后防效在80%左右。综合两方面的数据,目前上海地区草莓灰霉病菌对啶酰菌胺已经产生了一定的抗性,需要引起重视,但啶酰菌胺在实际生产中用以防治草莓灰霉病仍是可行的。     

北京地区草莓灰霉病菌对异菌脲的抗性及抗性分子机制

由灰葡萄孢(Botrytis cinerea Pers.)引起的草莓灰霉病是草莓上危害严重的病害之一。为了解北京地区草莓灰霉病菌对二甲酰亚胺类常用杀菌剂异菌脲的抗性,本研究采用最低抑制浓度法分别检测了2013年和2014年从北京地区15个草莓园采集的共计121株草莓灰霉病菌对异菌脲的抗性。结果表明,北京地区草莓灰霉病菌对异菌脲存在较高的抗性频率,2014年较2013年的抗性频率略有上升,由40.4%上升为45.3%。不同草莓园菌株的抗性频率差异很大,可能与用药水平有关。2014年的低抗、中抗和高抗菌株数分别占检测菌株总数的9.4%、28.1%和7.8%。利用PCR技术扩增编码组氨酸激酶基因BcOS1中与二甲酰亚胺抗性相关的区段,对抗性菌株的分子机制进行了初步研究,结果表明BcOS1基因第1 214位核苷酸发生了2类突变:以第Ⅰ类为主,菌株的抗性水平为中抗和高抗,由ATC突变为AGC,导致第365位氨基酸由异亮氨酸变为丝氨酸;第Ⅱ类菌株为低抗,由ATC突变AAC,导致第365位氨基酸由异亮氨酸变为天冬酰胺。     

马铃薯黄萎病病原菌营养亲和群、生理小种、交配型鉴定以及致病力差异分析

为明确引起马铃薯黄萎病的大丽轮枝菌Verticillium dahliae的遗传变异和致病力的差异,对从内蒙古及其周边地区马铃薯发病株上分离得到的29株大丽轮枝菌的营养亲和群、生理小种和交配型进行了测定,利用伤根接种法进行致病力分化研究。结果表明,供试的29株马铃薯大丽轮枝菌菌株被划分为VCG2B、VCG4B和VCG4A三个营养亲和群,其菌株数分别为11、2和16株;供试的29株菌株均鉴定为单一的2号生理小种和MAT1-2-1交配型。供试的29株大丽轮枝菌菌株间致病力存在一定的差异,其中NCP-1菌株的致病力最强,病情指数为83.33;而NWS-5菌株的致病力最弱,病情指数仅为10.85;不同营养亲和群菌株的平均致病力也存在显著差异,VCG4B型菌株的平均致病力最高,平均病情指数为67.18;其次为VCB2B型菌株,平均病情指数为42.50,而VCG4A型菌株致病力最弱,平均病情指数仅为20.54。     

不同年代大丽轮枝菌菌株的生物学特性和ISSR分析

 为了探究不同年代的大丽轮枝菌(Verticillium dahliae Kleb.)菌株生物学特性和遗传多样性方面的差异,本文以我国黄河流域和长江流域主产棉区6个省的不同年代大丽轮枝菌菌株为研究对象,观察培养性状,测定致病类型(致病力、落叶型),同时采用ISSR指纹图谱分析其遗传多样性。结果显示,不同年代大丽轮枝菌之间菌丝生长速率无显著差异,但菌核型菌株所占比例有减少趋势;2007~2009年和2017年的菌株全部为落叶型菌株,而1983~2000年的菌株中落叶型菌株仅占28.6%,表明随着年代的推移,黄河流域和长江流域的落叶型菌株所占比例呈上升趋势;不同年代菌株之间致病力存在显著差异,1983~2000年、2007~2009年和2017年的菌株中强致病力类型菌株分别占21.4%、25.0%和38.9%,仅有的5株弱致病力类型菌株均为1983~2000年的菌株;与2000年后的菌株相比,1983~2000年的菌株,Nei′s基因多样性指数为0.205 1,Shannon信息指数为0.299 0,表现出更丰富的多样性,利用NTSYS软件和Structure软件对ISSR指纹图谱进行聚类分析,两种方法均将所有供试菌株分为4个类群,且聚类结果与致病力和不同年代之间均具有一定的相关性,与地理来源无明显相关性。本研究结果表明,过去30年间我国黄河流域和长江流域棉田黄萎病菌落叶型和强致病力类型菌株所占比例逐渐升高,且不同年代和不同致病力的菌株在遗传上有差异,为进一步探究大丽轮枝菌的遗传与进化奠定了基础。     

湖南省草莓灰霉病菌对4种杀菌剂的抗药性检测

为明确湖南省草莓灰霉病菌菌株的抗药性状况,2013-2015年从湖南省不同地区草莓灰霉病病果或病叶上经单孢分离获得草莓灰霉病菌454株,采用最小抑制浓度法(MIC)检测不同地区草莓灰霉病菌株对多菌灵、腐霉利、异菌脲、嘧霉胺的抗药性。结果表明:草莓灰霉病菌对多菌灵、腐霉利、异菌脲、嘧霉胺的抗性频率分别为55.73%,77.31%、3.52%和77.31%;所测菌株对4种杀菌剂的敏感性类型有Cbz~RPcm~RIpd~RPmt~R、CbzSPcm~RIpd~RPmt~R、Cbz~RPcm~RIpdSPmt~R、Cbz~SPcm~RIpdSPmt~S、Cbz~RPcm~SIpd~SPmt~R、Cbz~SPcm~RIpd~SPmt ~R等6种,其所占比例分别为33.26%、5.07%、41.41%、4.63%、3.96%、11.67%,未发现对4种杀菌剂均敏感的类型,表明湖南省草莓灰霉病菌已对多菌灵、腐霉利和嘧霉胺产生抗药性,对异菌脲的抗药性较低。     

江苏省大丽轮枝菌的培养、遗传及致病特性分析

由大丽轮枝菌引起的黄萎病是我国棉花和茄子的主要病害之一,对棉花和茄子的生产造成巨大损失。为了研究大丽轮枝菌的群体遗传变异以及对棉花和茄子的交互致病性,本文对分离自江苏省的63个棉花黄萎菌和10个茄子黄萎菌进行了培养、遗传和致病特性分析。根据菌株在PDA培养基上生长时形成微菌核的多少来划分培养类型,结果菌核型占83.6%,成为主要的培养类型。用PCR技术检测菌株的致病类型、交配型以及是否具有无毒基因Ave1,结果落叶型菌株占86.3%,为优势种群,但是10株茄子黄萎菌100%都是非落叶型菌株;供试的所有江苏菌株交配型都是MAT1-2型,并且都没有无毒基因Ave1。选择江苏省的6个棉花黄萎菌和4个茄子黄萎菌在室内苗期接种棉花和茄子,进行交互致病性测定,结果这10个菌株都可以侵染棉花和茄子,而且来源于不同寄主作物的菌系之间致病力分化明显,表现在不同菌株对同一寄主的致病力不同,同一菌株对不同寄主的致病力也不同。研究结果为深入研究大丽轮枝菌群体遗传结构和制定黄萎病防治措施提供了理论支持。     

不同年代大丽轮枝菌菌株的生物学特性和ISSR分析

 为了探究不同年代的大丽轮枝菌(Verticillium dahliae Kleb.)菌株生物学特性和遗传多样性方面的差异,本文以我国黄河流域和长江流域主产棉区6个省的不同年代大丽轮枝菌菌株为研究对象,观察培养性状,测定致病类型(致病力、落叶型),同时采用ISSR指纹图谱分析其遗传多样性。结果显示,不同年代大丽轮枝菌之间菌丝生长速率无显著差异,但菌核型菌株所占比例有减少趋势;2007~2009年和2017年的菌株全部为落叶型菌株,而1983~2000年的菌株中落叶型菌株仅占28.6%,表明随着年代的推移,黄河流域和长江流域的落叶型菌株所占比例呈上升趋势;不同年代菌株之间致病力存在显著差异,1983~2000年、2007~2009年和2017年的菌株中强致病力类型菌株分别占21.4%、25.0%和38.9%,仅有的5株弱致病力类型菌株均为1983~2000年的菌株;与2000年后的菌株相比,1983~2000年的菌株,Nei′s基因多样性指数为0.205 1,Shannon信息指数为0.299 0,表现出更丰富的多样性,利用NTSYS软件和Structure软件对ISSR指纹图谱进行聚类分析,两种方法均将所有供试菌株分为4个类群,且聚类结果与致病力和不同年代之间均具有一定的相关性,与地理来源无明显相关性。本研究结果表明,过去30年间我国黄河流域和长江流域棉田黄萎病菌落叶型和强致病力类型菌株所占比例逐渐升高,且不同年代和不同致病力的菌株在遗传上有差异,为进一步探究大丽轮枝菌的遗传与进化奠定了基础。     

草莓灰霉病菌对嘧菌酯的抗性检测及抗性菌株的生物学特性研究

由灰葡萄孢(Botrytis cinerea Pers.)引起的草莓灰霉病是严重危害草莓的病害之一。为了解草莓灰霉病菌对杀菌剂嘧菌酯的抗性情况,本研究针对2012年从国内10省市采集和分离的134株草莓灰霉病菌菌株,利用特异性引物BcAR-F/BcAR-R扩增其cytb基因的部分序列,进行了嘧菌酯抗性的分子检测;对检测出的抗性菌株在嘧菌酯浓度分别为22.4、112和224μg·mL~(-1)的含药培养基上进行抗性验证;此外,还比较了抗性菌株与敏感菌株主要生物学性状的差异。结果表明,有54个菌株对嘧菌酯表现抗性,均属于中等抗性水平,占检测总数的40.3%,分布于北京市、湖北、江苏、河北、辽宁和四川各省;对15个抗性菌株cytb基因的序列分析表明,其第143位发生突变,由甘氨酸变为丙氨酸;对随机选取的4株抗性菌株和2株敏感菌株的适合度测定结果发现,抗性菌株和敏感菌株在最适生长温度、菌落生长速度、孢子萌发率方面没有表现出规律性差异,但抗性菌株的产孢量和致病力均显著低于敏感菌株。     

不同来源番茄溃疡病菌致病力差异研究

采用打顶法接种、半选择性培养基再分离发病植株中的病原菌,以及特异性PCR验证方法,对来自3个国家9个不同地区的46株番茄溃疡病菌进行了致病性测定,以病情指数评价不同菌株的致病力。结果显示,分离自我国河北滦平县、内蒙古包头市等地的24株菌株的病情指数达到75以上,属于强致病力水平;11株菌株的病情指数为50~75,属于中等致病力;而9株菌株的病情指数为50以下,属于弱致病力;检测同时证实,有2株属于无致病力菌株。强致病力、中等致病力、弱致病力和无致病力菌株占供试菌株总数的比例分别为52.2%、23.9%、19.6%和4.3%,表明供试的46株番茄溃疡病菌存在不同程度的致病力差异。     

Two Sibling Species of the Botrytis cinerea Complex, transposa and vacuma, Are Found in Sympatry on Numerous Host Plants

ABSTRACT Strains of Botrytis cinerea (the anamorph of Botryotinia fuckeliana) were collected from 21 different plant species around vineyards in the Champagne region (France). Strains were analyzed using three new polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) markers that were found by SWAPP (sequencing with arbitrary primer pairs), in addition to 15 other markers (PCR-RFLP, transposable elements, and resistance to fungicides). The markers revealed a high degree of genetic diversity and were used to investigate population structure. The two sympatric species transposa and vacuma, previously identified on grapes in these vineyards, were also detected on many of the plant species sampled. A new type of strain was also detected, having only the transposable element Boty. We did not detect any differentiation between strains from different organs or locations, but the prevalences of transposa and vacuma were significantly different on the different host plants. Fungicide resistance frequencies were significantly different in transposa and vacuma species. This study confirms that B. cinerea is a complex of sibling species and shows that the sibling species occur sympatrically on many host plants. However, the two species seemed to have different pathogenic behaviors. These findings contradict the traditional view of B. cinerea as a clonal population without specialization.     

Genetic diversity of Botrytis in New Zealand vineyards and the significance of its seasonal and regional variation

Species‐ and population‐specific differences in fungicide resistance and aggressiveness within Botrytis makes basic data on genetic diversity important for understanding disease caused by this fungus. Genetic diversity of Botrytis was surveyed between 2008 and 2012 from grapes from five New Zealand wine‐growing regions. A total of 1226 isolates were gathered from symptomless flower buds at the start of the growing season and 1331 isolates from diseased fruit at harvest. Two species were found, B. cinerea and B. pseudocinerea. Botrytis pseudocinerea was common in both Auckland vineyards sampled, and infrequent elsewhere. However, even in Auckland, it was rarely isolated from diseased fruit. The presence of the Boty and Flipper transposons was assessed. Isolates with all four transposon states (Boty only, Flipper only, both Boty and Flipper, no transposons) were found for both species. Both vineyards in the Auckland region had high numbers of Flipper‐only isolates at flowering; both vineyards from the Waipara region had high numbers of Boty‐only isolates at flowering. Most isolates from diseased fruit at harvest contained both transposons. These observations suggest that B. pseudocinerea, and isolates with one or both of the transposons missing, may be less aggressive than B. cinerea, or than isolates with both transposons present. Two clades were resolved within B. pseudocinerea, only one of which has been reported from European vineyards. Phylogenetic diversity within B. cinerea in New Zealand was similar to that known from Europe, including isolates that appear to match Botrytis ‘Group S’. The taxonomic implications of this genetic diversity are discussed.     

The Role of Saprotrophy and Virulence in the Population Dynamics of Botrytis cinerea in Vineyards

ABSTRACT Change in relative frequencies of the three main genetic types of Botrytis cinerea (Group I, Group II vacuma, and Group II transposa) were monitored over time from 1998 to 2000 in three Bordeaux vineyards not treated with fungicides. During 2000, Group I isolates, detected mostly at flowering comprised only 2.5% of the entire population. Within Group II, the complementary frequencies of vacuma and transposa isolates differed significantly depending on grapevine phenological stages and organs. Every year and at all sites, including one noble rot site, transposa isolates dominated at every stage, particularly on overwintering canes and at harvest (greater than 86.7% on berries). The complementary frequency of vacuma isolates reached a maximum on senescing floral caps (between 23.5 and 71.4%) and then decreased significantly until harvest on leaves and berries. In pathogenicity tests on grape berries, transposa isolates were significantly more virulent than were vacuma isolates. Mycelial growth rate was negatively correlated with virulence, notably on leaves in transposa and with double resistance to the fungicides carbendazim and vinclozolin. In vacuma, this double resistance was positively correlated with virulence on leaves. Change in the vacuma and transposa frequencies was most likely caused by differences in saprotrophic and pathogenic fitness. Possible interactions between fungicide resistance profiles and fitness are discussed.     

浙江省果蔬灰霉病菌对嘧菌酯的抗药性研究

采用菌丝生长速率法,连续监测了2010—2012年间浙江省果蔬灰霉病菌对QoI类杀菌剂嘧菌酯的敏感性变化。 结果表明:病菌群体中的低敏感性亚群体的比例明显上升,EC50值>5 mg/L 菌株的比例分别为12.5%、15.8%和28.3%;在菌丝生长阶段和孢子萌发阶段,旁路氧化在灰霉病菌对嘧菌酯敏感性中的平均相对贡献值(F)分别为2.91±0.89和5.72±2.82;嘧菌酯抗药性菌株的菌丝生长速率、产孢量、产菌核数和致病力与敏感菌株相比无显著差异。抗药性分子机制研究表明,灰霉病菌中存在2种类型的cyt b基因:Ⅰ型cyt b基因在第143位密码子后紧跟内含子;Ⅱ型cyt b基因在第143位密码子后没有紧跟内含子。大多数的灰霉病菌菌株属于Ⅱ型。Ⅰ型菌株均为嘧菌酯敏感菌株,Ⅱ型菌株为嘧菌酯敏感菌株或抗性菌株。抗性菌株的cyt b 基因的第143位密码子由甘氨酸(GGC)突变为了丙氨酸(GCC),抗药性机制为G143A。     

Possible Role of Colonization and Cell Wall-Degrading Enzymes in the Differential Ability of Three Ulocladium atrum Strains to Control Botrytis cinerea on Necrotic Strawberry Leaves

ABSTRACT Ulocladium atrum (strain 385) consistently reduced Botrytis cinerea sporulation on necrotic fragments of strawberry leaves. On these tissues, two strains of U. atrum (isolates 18558 and 18559) showed lower antagonistic activities than the reference strain 385. Colonization of strawberry leaflets by the three U. atrum strains appeared similar in the absence of B. cinerea, whether quantified by chitin or immunological assays. The second method (based on anti-U. atrum antibodies) revealed that strawberry leaflet colonization by U. atrum 385 was better than by the other U. atrum strains in the presence of B. cinerea. An immunoassay using anti-B. cinerea antibodies revealed that the colonization of B. cinerea in tissues was lower in the presence of U. atrum 385 than with the two other U. atrum strains. The enzymatic activities produced by U. atrum 385 during the colonization phases of necrotic tissues were compared to B. cinerea and U. atrum strains 18558 and 18559. U. atrum 385 had the highest lipase, pectate lyase, and cellobiase activities while B. cinerea had the highest endo-beta-1,4-glucanase activity. The study of lytic activities hydrolyzing the fungal cell wall revealed higher beta-1,3-glucanase activity with U. atrum 385, which was stimulated by B. cinerea on necrotic strawberry leaflets. These results suggest that plant and fungal cell wall-degrading enzymes produced by U. atrum 385 may play a complementary role in the competitive colonization of dead strawberry leaves against B. cinerea.     

灰葡萄孢对腐霉利的抗性分子机制及快速检测技术

为明确灰葡萄孢Botrytis cinerea对腐霉利的抗性现状,于2017—2018年采用单孢分离法从浙江省5个地区的草莓大棚共分离获得200个菌株.通过区分剂量法测定了其对腐霉利的抗性,对抗药性菌株的分子机制进行了分析,并根据抗药性分子机制,建立了B.cinerea腐霉利高抗基因型的环介导等温扩增(loop-med...     

Molecular characterization of pyraclostrobin resistance and structural diversity of the cytochrome b gene in Botrytis cinerea from apple

Botrytis cinerea isolates obtained from apple orchards were screened for resistance to the quinone outside inhibitor (QoI) pyraclostrobin. Of the 220 isolates tested, 43 (19.5%) were resistant to pyraclostrobin. Analysis of partial sequences of the cytochrome b gene (cyt b) in five pyraclostrobin-resistant (PR) and five pyraclostrobin-sensitive (PS) isolates showed that PR isolates harbored the point mutation leading to the substitution of glycine by alanine at codon position 143 in cyt b (G143A). Two pairs of allele-specific primers were designed based on this point mutation, and allele-specific polymerase chain reaction analysis with these primers showed that all 73 PR isolates (including 30 collected from decayed apple fruit) harbored the G143A mutation but PS isolates did not. Six pairs of primers were designed to analyze the presence of various introns in cyt b. There were six types (I to VI) of cyt b present in 247 isolates of B. cinerea collected from various apple-production areas in Washington State. Of the 247 isolates, 23 had type I cyt b containing all four introns (Bcbi-67/68, Bcbi-131/132, Bcbi-143/144, and Bcbi-164), 176 had type II cyt b containing three introns (Bcbi-67/68, Bcbi-131/132, and Bcbi-164), six had type III cyt b containing two introns (Bcbi-67/68 and Bcbi-131/132), one had type IV cyt b containing two introns (Bcbi-131/132 and Bcbi-164), one had type V cyt b containing only the Bcbi-131/132 intron, and 40 had type VI cyt b containing no introns. This is the first report of types III to VI cyt b present in B. cinerea. All 73 PR isolates did not carry the Bcbi-143/144 intron in cyt b. Of the 247 isolates tested, >90% did not carry the Bcbi-143/144 intron in cyt b, suggesting that B. cinerea populations from apple pose a high inherent risk for the development of resistance to QoIs because the presence of this intron in cyt b prevents the occurrence of G143A-mediated resistance. Analysis of genetic background based on three microsatellite primers showed that PR isolates originated from different lineages, and there was no correlation between cyt b types (I, II, and III) and the genetic background of the isolates; however, isolates carrying type VI cyt b might originate from the same lineage.     

The use of Trichoderma species to control strawberry fruit rots

The effect of temperature on the growth and antagonistic properties of Trichoderma species against Botrytis cinerea and Mucor mucedo (strawberry fruit pathogens) was studied. Five strongly antagonistic isolates were further used in field experiments. The incidence of pre-harvest rots caused by B. cinerea and the rate of post-harvest spoilage were similarly reduced when strawberry flowers were sprayed either with the fungicide dichlofluanid or with spores of selected Trichoderma isolates.     

Hypovirulence and Double-Stranded RNA in Botrytis cinerea

ABSTRACT Twenty-one strains of Botrytis cinerea isolated from 13 species of plants grown in China were compared for pathogenicity on Brassica napus, mycelial growth on potato dextrose agar, and presence of double-stranded (ds)RNA. The results showed that the strain CanBc-1 was severely debilitated in pathogenicity and mycelial growth, compared with the 20 virulent strains. A dsRNA of approximately 3.0 kb in length was detected in CanBc-1 and 4 hypovirulent single-conidium (SC) isolates of CanBc-1, but was not detected in the 20 virulent strains of B. cinerea and 4 virulent SC isolates of CanBc-1. Results of the horizontal transmission experiment showed that the hypovirulent trait of CanBc-1 was transmissible and the 3.0-kb dsRNA was involved in the transmission of hypovirulence. Analysis of a 920-bp cDNA sequence generated from the 3.0-kb dsRNA of CanBc-1 indicated that the dsRNA element was a mycovirus, designated as B. cinerea debilitation-related virus (BcDRV). Further analyses showed that BcDRV is closely related to Ophiostoma mitovirus 3b infecting O. novo-ulmi, the causal agent of Dutch elm disease. Mitochondria and cytoplasm in hyphal cells of CanBc-1 became degenerated, compared with the virulent isolate CanBc-1c-66 of B cinerea. This is the first report on the occurrence of Mitovirus-associated hypovirulence in B. cinerea.