黑龙江省水稻纹枯病菌的致病力分化与AFLP分析

为了明确黑龙江省水稻纹枯病菌遗传多样性,为水稻抗病育种和水稻纹枯病的综合防治提供依据。本文对采自13个水稻种植地区的29个水稻纹枯病菌菌株进行了致病力测定和AFLP分析。结果表明9对AFLP引物对供试菌株扩增出396条带,其中多态性带187条,占总扩增带数的47.22%。黑龙江省水稻纹枯病菌的遗传距离变化在0.50~0.92之间,平均为0.71,群体遗传多样性较为丰富。UPGMA法可以将供试菌株分成4个AFLP聚类组群(Ⅰ、Ⅱ、Ⅲ和Ⅳ),相同地理来源的菌株基本上聚集在同一组群内,表明AFLP类群划分与菌株的地理来源有较强的相关性。黑龙江省水稻纹枯病菌致病性分化较为明显,并且AFLP类群划分与菌株的致病性鉴定之间存在一定相关性。     

继代培养对水稻纹枯病菌菌丝生长和致病力的影响

用水稻纹枯病菌3株菌株为材料,采取马铃薯蔗糖培养基试管斜面培养的方式进行菌丝继代培养,以离体接种法分析这些病菌菌株的致病力。结果表明,经过60次的继代培养,该病菌的菌丝生长和致病力2个性状均没有出现明显的变异,但菌株间的菌丝生长和致病力存在明显的差异。初步发现,该病菌的菌丝生长情况与致病力相关。     

水稻纹枯病寄主-病原物互作鉴别品种与菌株的筛选

 经过近十年的病原菌接种试验,从国内外数百份水稻种质中,筛选出了抗病水平不同的5个代表品种Lemont、武育粳3号、Jasmine85、C418和YSBR1,作为水稻纹枯病菌致病力的鉴别品种。从江苏、广东、广西、海南、云南、湖南、福建等省采集、分离的水稻纹枯病菌中,初步筛选出致病力有差异的30个菌株,于温室苗期接种上述5个品种,确定了不同致病力的5个代表菌株C30、GD-118、E67、YN-7和YN-3。将选出的品种和菌株在江苏扬州和浙江富阳进行大田成株期抗性和致病力分析,结果表明:品种的抗病性和菌株的致病力都存在极显著差异,分别属于5个和3个不同的显著性级别,均可暂时作为鉴定材料使用。据以上结果,初步建立了一套可供水稻纹枯病菌致病力检测和寄主抗病性鉴定的鉴别体系。本文还讨论了建立水稻纹枯病鉴别体系的必要性、可行性和需要进一步开展的工作。     

水稻纹枯病菌强拮抗菌B34的分离与鉴定

 从水稻植株上分离到1株对水稻纹枯病菌有强烈拮抗作用编号为B34的菌株,在PDA平板上表现有对水稻纹枯病菌较强作用的抑菌带,其发酵菌液对纹枯病菌丝有强烈的致畸作用,抑菌率达到71.5%,使菌核的菌丝萌发率降低69.0%。离体防效和田间小区防效分别高达81%和48.74%,高于清水和井冈霉素处理。经生理生化检测及细胞壁化学成分分析,确定其为致黄假单胞菌(Pseudomonas aureofaciens)。     

解淀粉芽胞杆菌YU-1对水稻纹枯病的防治作用

本文旨在筛选对水稻纹枯病有生防作用的菌株,并初步探索其生防作用机理。收集水稻、甘蓝、黄瓜等不同植物根际土壤,采用稀释分离和对峙培养法筛选对水稻纹枯病菌Rhizoctonia solani Kühn有抑菌作用的菌株;通过离体接种防效、盆栽防效、抑菌谱、对水稻纹枯病菌菌核萌发及形成的抑制作用等方面评价其生防潜力,并对生防菌株进行鉴定。结果表明,从采集的37份根际土壤中共分离获得细菌297株,其中4株对纹枯病菌具有较好的拮抗作用,菌株YU-1对水稻纹枯病菌菌丝生长的抑制率达89.8%;对西瓜枯萎病菌、草莓灰霉病菌、番茄早疫病菌、油菜菌核病菌、小麦赤霉病菌菌丝生长的抑制率均在85%以上;对水稻纹枯病的离体和盆栽防效分别为73.1%和66.3%;对水稻纹枯病菌菌核萌发的抑菌率在92%以上;经生理生化和分子鉴定为解淀粉芽胞杆菌Bacillus amyloliquefaciens。由此可看出,菌株YU-1对水稻纹枯病的防治具有较强的应用价值,具有进一步开发成生物农药的潜力。     

中国东北地区水稻纹枯病病原菌种类及融合群的分子鉴定

为明确中国东北地区水稻纹枯病病原菌种类及融合群的归属情况, 2015－2017年从黑龙江省、吉林省和辽宁省的17个水稻主产区采集水稻纹枯病标样, 分离获得水稻纹枯病菌214株, 运用水稻纹枯病菌的不同病原菌及融合群的特异性引物对214株水稻纹枯病菌进行病原菌种类和融合群鉴定, 并利用rDNA内转录间隔区(ITS)序列, 对供试水稻丝核菌的融合群归属进行了分析。结果表明:供试214株水稻纹枯病菌分属于茄丝核菌Rhizoctonia solani和水稻丝核菌Rhizoctonia oryzae-sativae, 菌株数分别为198株和16株, 占比分别为92.52%和7.48%。茄丝核菌菌株分属于2个融合群, 分别为AG1-IA和AG4, 菌株数分别为191株和7株, 占比分别为96.46%和3.54%。水稻丝核菌菌株均属于AG-Bb融合群, 菌株数为16株。不同年份水稻纹枯病的病原菌种类及融合群出现的频率和地域分布无明显变化, 而不同地域间水稻纹枯病病原菌的种类及融合群具有明显的分化特征, AG1-IA融合群在中国东北三省各个水稻产区均有分布且均为优势融合群, AG4融合群在辽宁省盘锦市出现频率最高, 水稻丝核菌AG-Bb融合群在吉林省吉林市、通化市和梅河口市出现频率最高。     

哈茨木霉防治水稻纹枯病研究

为测定哈茨木材霉菌株ＴＣ３和ＮＦ９对水稻纹枯病的防治效果及防治过程中不同因素对防治效果原影响,于中国水稻研究所富阳基地的温室和网室内通过人工接种诱发水稻纹枯病,分别用茨木霉菌株ＴＣ３和ＢＮ和ＮＦ９进行防治。研究结果表明,菌株ＴＣ３和ＮＦ９对水稻纹枯病均具有一定的防治效果,最高防效分别为７７．５６％和８３．０９％,同时还分析了哈获准木霉防治水稻纹枯病的施用方法、施用适、施用数量及其对不同水稻品种苗期     

水稻恶苗病抗病性筛选方法的初步研究

１９９１－１９９２年对水稻恶苗病进行不同鉴定方法，单一菌株与混合菌株接种，抗生分级标准，品种抗病性鉴定及Ｆ２代抗，感分离比率的研究。结果表明，水稻芽期浸菌接种法能明显区分不同品种（系）的抗感水平，且简便易行，适用于大量品种的筛选；采用不同地区混合菌株接种可扩大致病谱，其平均致病率也接近单一菌株的平均值，划分出抗生分级标准０－９级；据对２０４份品种（系）的鉴定，水稻品种（系）间抗性有明显差异，苗期抗     

水稻抗纹枯病苗期快速鉴定技术研究

 以抗感反应不同的5个水稻品种为试验材料,在人工气候箱、控温室中进行水稻苗期抗纹枯病接种试验,并与田间相应的成株期抗性试验进行比较,研究水稻苗期快速鉴定技术。结果表明:85%的相对湿度为纹枯病菌侵染危害水稻苗期植株的适宜湿度;苗期5个水稻品种对纹枯病抗性差异极显著,可将其分为相对感病(Lemont、武育粳3号)和相对抗病(YSBR1、Jasmine85、特青)2大类;接种叶龄对发病程度有显著的影响,5个品种在四叶期接种时的平均病级显著高于五叶期接种的平均病级;苗期水稻品种间抗感差异小于田间鉴定试验结果,但两者间品种抗感趋势基本一致。苗期快速鉴定技术可用于大规模水稻品种(组合)的抗性筛选或初步鉴定。     

田间水稻单个病斑中稻瘟病菌的遗传多样性

根据93个单病斑中分离的227个单孢菌株样品的分析，研究了来自同一定殖系统(同一病斑)中稻瘟病菌不同单孢菌株(同源菌株)的遗传变异。在致病性分析的样品中，仅有5个病斑分离的10个单孢菌株在所有鉴别品种上表现同源菌株有完全相同的致病反应，并且有完全相同的DNA指纹图谱带型；另有13个病斑上分离的32个单孢菌株的同源菌株不仅属于不同的生理小种，而且在1～3个单基因品种上也有不同的致病反应。用rep—PCR指纹图谱和致病型分析67个病斑的157个单孢样品中的同源菌株具有完全相同的DNA指纹图谱；13个病斑中分离的27个单孢样品中同源菌株具有显著差异的指纹图谱；8个病斑分离的27个单孢菌株样品中同一来源样品中部分样品有相同的指纹图谱带型，部分样品却有不同的指纹图谱。同源菌株之间有完全相同致病性其指纹图谱亦相同或有很小的差异；通常，致病性差异很大的同源菌株其指纹图谱差异也很大。作者认为来自同一定殖系统中的不同单孢菌株存在的遗传变异与病菌的异核现象或自然状态下不同遗传背景菌株同时侵染造成同一病斑有关。     

Phenotype Instability in Botrytis cinerea in the Absence of Benzimidazole and Dicarboximide Fungicides

ABSTRACT Stability of phenotypes of isolates of Botrytis cinerea that were sensitive or resistant to benzimidazole and dicarboximide fungicides was examined in the absence of fungicides in laboratory and growth room experiments. Twelve greenhouse isolates of B. cinerea were subcultured on potato dextrose agar (PDA) for 20 generations and on geranium seedlings for 15 generations. Three isolates of each of the following four phenotypes were used: sensitive to the fungicides thiophanate-methy1 (a benzimidazole) and vinclozolin (a dicarboximide) (S(T)S(V)), resistant to both fungicides (R(T)R(V)), resistant to thiophanate-methy1 and sensitive to vinclozolin (R(T)S(V)), and sensitive to thiophanate-methy1 and resistant to vinclozolin (S(T)R(V)). In three trials on PDA, 36 populations were subcultured; 8 populations changed phenotypes by the end of 20 generations, as determined by conidium germination on fungicide-amended medium. Five of the eight initially were S(T)R(V); the resulting phenotypes were S(T)S(V), R(T)S(V), and R(T)R(V). Populations from eight other isolates exhibited temporary changes in phenotype during intermediate generations on PDA but reverted to initial phenotypes by the twentieth generation; five of these populations changed to phenotype R(T)R(V). In two geranium seedling trials, each of the 12 greenhouse isolates was inoculated onto a set of three seedlings for each generation, and diseased tissue that developed was used to initiate the next generation. Therefore, a total of 72 populations of B. cinerea were subcultured in the two trials; 5 of these populations changed phenotype at the end of 15 generations. Three of the five initially were S(T)R(V); these changed to phenotypes S(T)S(V) or R(T)R(V). In each of the two trials on geranium seedlings, a population subcultured from one S(T)S(V) isolate changed phenotype one to phenotype R(T)R(V) and one to phenotype R(T)S(V). In all trials, no population resistant to thiophanate-methy1 changed to a thiophanate-methy1-sensitive phenotype, and no population changed to phenotype S(T)R(V). Random amplified polymorphic DNA (RAPD) fingerprints were generated with the 12 initial isolates and 49 isolates subcultured on PDA or geranium seedlings. Cluster analyses of RAPD markers showed that subcultured isolates exhibiting the same phenotype clustered together and that subcultured isolates derived from a common greenhouse isolate but with different phenotypes were in different clusters. Some populations that did not change phenotype exhibited considerable differences in RAPD marker patterns. The results of this study indicate that, in the absence of fungicides, sensitive populations of B. cinerea can develop resistance to thiophanate-methy1 and vinclozolin, and this resistance can be maintained in populations through multiple generations. Populations resistant only to vinclozolin (S(T)R(V)) exhibited a high frequency of phenotype change, and populations resistant to both fungicides (R(T)R(V)) were stable.     

Stability and fitness of pyraclostrobin- and boscalid-resistant phenotypes in field isolates of Botrytis cinerea from apple

Phenotype stability, fitness, and competitive ability of pyraclostrobin- and boscalid-resistant isolates of Botrytis cinerea from apple were investigated. Stability of resistance was determined after consecutive transfers on potato dextrose agar (PDA) or being cycled on apple fruit. In vitro fitness components mycelial growth, osmotic sensitivity, conidial germination, and sporulation were evaluated on agar media. Pathogenicity, virulence and sporulation on apple fruit were evaluated at both 20 and 0°C. Competition between fungicide-resistant and -sensitive isolates on apple fruit also was evaluated. Resistance to the two fungicides was retained at levels similar to that of the initial generation after 20 and 10 transfers on PDA and five and three disease cycles on apple fruit at 20 and 0°C, respectively. Great variability in individual fitness components tested was observed among isolates within the same phenotype groups either sensitive or resistant to the fungicides but, when compared as phenotype groups, there were no significant differences in the mean values of these fitness components between resistant and sensitive phenotypes except that the phenotype resistant only to boscalid produced fewer conidia in vitro than sensitive isolates. Resistant isolates were as pathogenic and virulent on apple fruit as sensitive isolates. There was no significant correlation between the values of individual fitness components tested and the level of resistance to pyraclostrobin or boscalid, except that virulence at 20°C positively correlated with the level of resistance to the two fungicides. The final frequency of pyraclostrobin-resistant individuals in the populations was significantly decreased compared with the initial generation and no boscalid-resistant individuals were detected after four disease cycles on apple fruit inoculated with a pair mixture of a dual-sensitive isolate and one isolate each of the three phenotypes resistant to pyraclostrobin, boscalid, or both. The results suggest that resistance of B. cinerea to pyraclostrobin and boscalid was stable in the absence of the fungicides and that resistance to the two fungicides did not significantly impair individual fitness components tested. However, both pyraclostrobin- and boscalid-resistant isolates exhibited competitive disadvantage over the dual-sensitive isolate on apple fruit.     

生防木霉菌对嘧霉胺抗药性变异及其相关蛋白诱导

选育对杀菌剂具有抗性的生防菌株是生物防治研究的重要内容之一.通过药剂选择培养基及紫外光诱导获得对嘧霉胺具抗性的突变体KTm-1、KTm-2.研究结果显示,与亲本菌株相比,抗性突变体的耐药性提高20倍以上.抗性菌株在无药培养基上连续转代8次,耐药性未见下降.活体拮抗测定表明,耐药菌株的抑菌能力略高于亲本菌株.PAGE显示抗性突变体KTm-1的可溶性蛋白谱带比其亲本菌株多4条,其中Rf=0.64的蛋白谱带是两个耐药菌株的特异性谱带,可视其为耐药相关蛋白.经Sephadex G-100柱层析纯化,SDS-PAGE测定耐药蛋白分子量为44.1KDa.     

The Dynamics of Mefenoxam Insensitivity in a Recombining Population of Phytophthora capsici Characterized with Amplified Fragment Length Polymorphism Markers

ABSTRACT Recent findings from Michigan suggest that recombination may play a role in the survival and evolution of sensitivity to the fungicide mefenoxam in populations of Phytophthora capsici on cucurbit hosts. In 1998, 63 mefenoxam insensitive isolates were recovered from a squash field in which mefenoxam had been applied. Additional isolates were recovered from untreated squash fields planted at this location in 1999 (200 isolates) and the spring of 2000 (34 isolates). Isolates from 1998 and 1999 were characterized using fluorescent amplified fragment length polymorphism (AFLP) markers and all isolates were screened for compatibility type and mefenoxam sensitivity. In 1998 and 1999, 92 and 71% of the isolates, respectively, had unique multilocus AFLP genotypes with no identical isolates recovered between years. Seventy-two identical AFLP markers were clearly resolved in both the 1998 and 1999 sample sets, and fixation indices for the 37 polymorphic AFLP loci indicate little differentiation between years. There was no decrease in the frequency of resistant isolates during the 2 years without mefenoxam selection. We conclude that oospores play a key role in overwintering and that the frequency of mefenoxam insensitivity may not decrease in an agriculturally significant time period (2 years) once mefenoxam selection pressure is removed.     

水稻品种构成对稻曲病菌遗传结构影响的初步研究

由稻曲病菌（Ustilaginoidea virens）引起的病害是当前限制我国水稻优质高产的重要病害之一。家畜食用被稻曲病菌厚垣孢子污染的谷物可导致中毒。近20年来，稻曲病防治主要以喷施化学农药为主。研究品种布局对稻曲病菌群体结构的影响，有助于了解病害的发生规律及提出有效的病害防治策略。本文初步分析了稻曲病菌群体结构与水稻品种构成的关系。     

灰葡萄孢多药抗性菌株的筛选和鉴定

从浙江杭州市售草莓上分离得到83个灰葡萄孢菌株,测定了这些菌株对苯醚菌酯、多菌灵和异菌脲的敏感性,筛选出对这3种药剂同时产生了抗性的两个菌株HZ021和HZ054,其在马铃薯葡萄糖琼脂(PDA)平板上的菌丝生长和产孢量与敏感菌株相比无显著差异,在黄瓜叶片上均表现出很强的致病力。结果表明,HZ021和HZ 054有很高的适合度。通过对抗性菌株中细胞色素b (CYT b)、双组份组氨酸激酶(OS-1)和β-微管蛋白(TUB 2)基因序列进行分析发现,HZ021和HZ054对多种药剂的抗性是由于其药剂靶标基因上的点突变所致。     

Investigating the Spatiotemporal Genetic Structure of Phytophthora capsici in Michigan

ABSTRACT Phytophthora capsici isolates were recovered from pepper and cucurbit hosts at seven locations in Michigan from 1998 to 2000. Isolates were characterized for compatibility type (CT), mefenoxam sensitivity (MS), and amplified fragment length polymorphism (AFLP) marker profiles. In total, 94 AFLP bands were resolved. Individual populations were highly variable. Within populations, 39 to 49% of the AFLP bands were polymorphic and estimated heterozygosities ranged from 0.16 to 0.19. Of the 646 isolates fingerprinted, 70% (454) had unique AFLP profiles. No clones were recovered between years or locations. Pairwise F statistics (Phi(ST)) between populations from different locations ranged from 0.18 to 0.40. A tree based on unweighted pair-group method with arithmetic average cluster analysis indicates discrete clusters based on location. Isolates from the same location showed no clustering based on the year of sampling. Analysis of molecular variance partitioned variability among (40%) and within populations (60%). The overall estimated Phi(ST) was 0.34 (SD = 0.03). A1/A2 CT ratios were approximately 1:1, and MS frequencies were similar between years for the two locations sampled over time. These data suggest that P. capsici persists in discrete outcrossing populations and that gene flow among locations in Michigan is infrequent.     

番茄叶霉病菌对嘧菌酯的抗性检测及抗性风险评估

利用孢子萌发法测定了从河北、浙江、江苏、山东、四川等省田间采集的97个番茄叶霉病菌菌株对嘧菌酯的敏感性。结果表明,田间番茄叶霉病菌已对嘧菌酯产生不同程度的抗药性。97个菌株可分为敏感菌株(EC50值50值5.4~54 μg/mL),中抗菌株(EC50值54~270 μg/mL)、高抗菌株(EC50值270~1 350 μg/mL)、特高抗菌株(EC50值>1 350 μg/mL) 5种类型,分别占55.67%、14.43%、11.34%、10.31%和8.25%。其中从未用过甲氧基丙烯酸酯类杀菌剂的27个菌株的EC50平均值为(0.544±0.349) μg/mL,可作为敏感基残;高抗和特高抗菌株集中在常施用甲氧基丙烯酸酯类杀菌剂的河北定州。通过紫外线诱导和药剂驯化的方法获得了番茄叶霉病菌5个抗嘧菌酯突变体,其抗药性不稳定,在不含药PDA平板上继代培养后抗药性水平下降,甚至恢复至敏感状态;除突变体cf 21M1的产孢量高于其亲本外,抗药性突变体的菌落生长速率、产孢量、孢子萌发率均显著低于其亲本菌株,所有抗嘧菌酯突变体及其亲本菌株接种于番茄离体叶片后,突变体病情指数与亲本菌株之间没有显著差异。嘧菌酯与多菌灵、苯醚甲环唑之间不存在交互抗药性。田间抗药性检测及室内抗药性风险评估结果表明,番茄叶霉病菌对嘧菌酯具有较高的抗药性风险。     

Frequency distribution of sensitivity of Ustilaginoidea virens to four EBI fungicides, prochloraz, difenoconazole, propiconazole and tebuconazole, and their efficacy in controlling rice false smut in Anhui Province of China

False smut, caused by Ustilaginoidea virens, is an important emerging disease of rice (Oryza sativa L.) in China. Up to now, as most varieties with high yielding and good quality are susceptible or even highly susceptible to false smut in most rice-growing ecological regions, especially in Anhui Province, chemical control with fungicides would be an important measure for the control of this disease. The ergosterol biosynthesis inhibitor (EBI) fungicides, such as prochloraz, difenoconazole, propiconazole and tebuconazole, are extensively used in China for the control of rice diseases, such as rice sheath blight and rice blast. In this study, a total of 102 U. virens isolates (from Anhui Province of China) were tested for their sensitivity to these four EBI fungicides during the stage of mycelial growth. The EC₅₀ ranges of values for prochloraz, difenoconazole, propiconazole and tebuconazole inhibiting mycelial growth of the 102 U. virens isolates were 0.04–0.75, 0.04–1.08, 0.04–0.38 and 0.03–0.57 μg?ml^?1, with the average EC₅₀ values of 0.32?±?0.08, 0.45?±?0.08, 0.19?±?0.03 and 0.21?±?0.06 μg?ml^?1, respectively. These values suggested that the tested U. virens isolates were very sensitive to these four EBI fungicides. Results of field trials showed that two sprays of three of the fungicides exhibited greater control efficacy than a single spray for the control of rice false smut. Two sprays of each was better than a single spray for the control of rice sheath blight. Two sprays of 50% propiconazole EC at 300 g a.i. ha^?1 gave the best control of rice false smut at both two sites during the two consecutive years, 2010 and 2011, with the control efficacy ranging from 71.5 to 74.3%. Sensitivity of the field U. virens isolates to EBI fungicides should be monitored. Mixtures, as well as alternation with other fungicides with different modes of action, should be tested.