基于细胞色素氧化酶基因和激发素基因的大豆疫霉菌特异性PCR引物

引起大豆疫霉根腐病的大豆疫霉菌(Phytophthora sojae)是危害大豆的破坏性病原菌之一,也是我国重要的检疫性植物病原菌。简单、快速、准确的鉴定和检测技术是阻止大豆疫霉菌传入和病害早期诊断的有效工具。本研究从大豆疫霉菌细胞色素氧化酶基因Ⅱ(coxⅡ)序列和两个激发素(elicitin)家族基因EST序列中开发了3对大豆疫霉菌特异引物:Cox3-F/Cox3-R、PSEL1-F/PSEL1-R和PSEL2-F/PSEL2-R。这3对引物在大豆疫霉菌中分别扩增出450、289bp和370bp的特异性片段,其检测大豆疫霉菌基因组DNA的灵敏度分别为20、2pg/μL和2pg/μL。3对引物能够有效检测大豆疫霉菌侵染的大豆病株,可以用于病害诊断和鉴别。     

Morphological and molecular characterization of Colletotrichum spp. from citrus orchards affected by postbloom fruit drop in Brazil

Brazilian isolates of Colletotrichum spp. from citrus orchards affected by postbloom fruit drop were examined for colony colour, mycelial growth, benomyl-resistance, pathogenicity, and genetic variability by random amplified polymorphic DNA (RAPD) analysis. All isolates were obtained from flowers and persistent calyxes from different citrus hosts from Sao Paulo, Brazil. DNA polymorphisms detected after amplification with random 10-mer primers were used to classify the isolates into two groups. Group I isolates grew rapidly on potato-dextrose agar (PDA) and were sensitive to benomyl, and group II isolates grew slowly on PDA and were benomyl-resistant. Colletotrichum acutatum was analyzed by RAPD and had high genetic similarity with group II isolates of Colletotrichum from citrus. Probably, the group I is C. gloeosporioides and group II is C. acutatum.     

泰安樱桃根癌病致病菌分离及生物型鉴定

由致病性土壤杆菌侵染引起的根癌病严重影响了樱桃树的生长?利用选择性培养基, 以阿糖醇和赤藓糖醇作为不同碳源, 从山东泰安食用樱桃根部瘤状组织和感病根际土壤中分离到土壤杆菌100余株, 通过胡萝卜切片法和番茄茎部针刺接种法从中筛选到31株有致瘤活性的菌株?经16S rDNA和recA基因序列分析, 结合生物型检测鉴定出96%以上的致病菌属原生物Ⅱ型(Agrobacterium rhizogenes), 只有1株致病菌为原生物Ⅰ型(A. tumefaciens)?根据Ti质粒上致病相关基因保守序列设计3对引物, 12株有致瘤活性菌株中11株PCR扩增到目的条带?经分子生物学测定和高压纸电泳检测, 所有致病菌株的质粒均为胭脂碱型?建立了致病型土壤杆菌的分子生物学快速鉴定方法, 为定向选择生防菌株提供基础?     

Morphological and molecular identification and PCR amplification to determine the toxigenic potential of Fusarium spp. isolated from maize ears in southern Poland

The average amount of precipitation in spring and summer 2010 and 2011 coupled with relatively high temperatures caused massive Fusarium spp. infection of maize and yield losses in southern Poland. In order to examine the cause of this disease outbreak, Fusarium spp. were isolated and fungal strains were identified based on morphological characters and species-specific PCR assays. A total of 200 maize samples were processed, resulting in the obtention of 71 strains, which belonged to five Fusarium species, F. poae being the predominant one (74.56%). Other isolates were identified as F. graminearum, F. oxysporum, F. verticillioides and F. proliferatum. PCR-based detection of mycotoxin-synthesis-pathway genes was also used to determine the potential of the analyzed strains to produce trichothecenes (DON and NIV) and fumonisins (FUM). Only 14 isolates revealed the potential to produce DON (11 strains) and FUM (3 strains). HPLC analyses of grain samples revealed the presence of DON only – other mycotoxins were not detected. Moreover, 57.1% of potentially mycotoxin-producing isolates indicated the toxicity in a biological test.     

陕西苹果树腐烂病菌(Cytospora spp.)不同分离株的生物学特性与致病性研究

 本研究从陕西分离得到61株苹果树腐烂病菌(Cytospora spp.),选取在PDA培养基上菌落特征差异明显的7个分离株,对其生物学特性及致病性进行研究。结果表明:各分离株在PDA、PSA、PMA和PEMA培养基上菌落生长较好,但在PDA上的菌落颜色和产孢情况差异很大。各分离株均能在5~32℃生长,但对35℃以上高温的适应性差异较大;所有分离株均能在pH4~9的条件下生长,最适pH为5~6;光照对各分离株的菌落生长有明显的促进作用。各分离株均可在Cza-pek培养基及供试的其它碳、氮源培养基上生长,其中以葡萄糖、麦芽糖和酵母膏为最佳碳、氮源。采用烫伤接种的方法,分别以菌饼和分生孢子悬浮液接种苹果离体枝条,两者均可侵染发病,但各分离株致病力差异显著,其中菌落颜色为黄褐色的菌株致病性强。因此,根据菌落颜色、致病性和生物学特性可将这7个分离株划分为3个类群:Ⅰ型为黄褐色强致病类群,但各分离株的产孢量有差异;Ⅱ型为乳白色不产孢的弱致病类群;Ⅲ型为灰褐色易产孢弱致病类群。其余54株分离物均属于Ⅰ型,这些结果说明陕西省苹果树腐烂病菌具有多样性和致病性分化现象。     

Genetic diversity and population structure of Acidovorax avenae subsp. avenae isolated from sugarcane in Argentina

A significant increase in the occurrence of red stripe (caused by Acidovorax avenae subsp. avenae) has been observed in the last decade in Argentina. Considering that no extensive sampling of the main sugarcane-producing area in the country has been conducted to characterize the diversity and population structure of A. avenae subsp. avenae, molecular markers were employed to analyse 112 isolates from Tucumán. By using repetitive element polymorphism-based polymerase chain reaction (rep-PCR) almost all isolates were differentiated and grouped into 10 clusters, revealing a high genetic diversity. Using the amplified fragment length polymorphism (AFLP) technique, five pairs of isolates were discriminated that could not be distinguished with rep-PCR. Cluster analysis showed no clear association between isolate clustering, sugarcane host genotype, crop age, type of tissue sampled, fertilization, or year of sampling. Linkage equilibrium analysis by using rep-PCR data indicated that the population has some degree of clonality. Three housekeeping genes were also sequenced: ugpB and pilT sequences were highly similar to A. avenae subsp. avenae sequences from other Argentinian isolates, whereas the lepA sequence did not reveal significant similarity. An additional four housekeeping genes could not be amplified, suggesting the existence of differences in those regions. Subsequently, virulence of 14 A. avenae subsp. avenae isolates was evaluated under controlled conditions. Results showed a differential level of aggressiveness among the isolates on a resistant sugarcane variety. This study confirmed that rep-PCR is an adequate tool for genetic analysis and population structure characterization in bacteria, and revealed both high genetic diversity and clonal population structure of A. avenae subsp. avenae in Tucumán, Argentina.     

西南地区玉米纹枯病病菌融合群鉴定和UP-PCR分析

为明确西南地区玉米纹枯病病菌组成结构,从西南地区(四川、贵州、云南和重庆)玉米纹枯病样上分离获得285个疑似丝核菌菌株,根据培养性状、菌丝形态和细胞核染色对其进行鉴定,通过载玻片配对培养法对鉴定出的菌株进行菌丝融合群鉴定,并采用UP-PCR方法从各融合群中选取代表性菌株进行遗传变异分析。结果显示,供试疑似菌株中共鉴定出253个丝核菌,分别为224株立枯丝核菌Rhizoctonia solani和29株玉蜀黍丝核菌R.zeae。立枯丝核菌包括5个融合群,其中AG1-ⅠA共201株,出现频率为79.4%;AG1-ⅠB共2株,出现频率为0.8%;AG2-2ⅢB共3株,出现频率为1.2%;AG4-HGⅠ共10株,出现频率为3.9%;AG-5共8株,出现频率为3.2%。29株玉蜀黍丝核菌融合群均为WAG-Z,出现频率为11.5%。遗传变异分析中,当相似系数为0.78时,按照出现频率从立枯丝核菌和玉蜀黍丝核菌选取的不同融合群的20株菌株被划分为6个类群,与菌丝融合分析结果完全吻合,其中AG2-2ⅢB融合群首次从西南地区玉米上分离到。