Genetic variability of Phytophthora infestans isolates from south-western Colombia

Late blight caused by Phytophthora infestans is one of the most limiting diseases in solanaceous crops in the world. This pathogen is a main constraint in the highland Andes, where these plants are grown under high humidity conditions and continuous cropping. The aim of this research was to increase the available information on the biology of P. infestans, specifically on its level of genetic variation in south-western Colombia, an area where various solanaceous crops susceptible to this pathogen converge. The study was carried out by using AFLP molecular markers with the restriction enzymes EcoRI and MseI and different primer combinations. Results indicated a low level of genetic variation among the 26 isolates evaluated, with only 18 polymorphic bands out of 135 amplicons obtained (13.43%), a Nei's genetic diversity index of 0.04, and a Shannon's information index of 0.06.     

Genetic diversity of Phytophthora capsici isolates from pepper and pumpkin in Argentina

Phytophthora capsici is a soilborne oomycete plant pathogen that limits pepper production worldwide. The population structure varies significantly depending on the location (e.g. Peru vs. USA) and little is known about the diversity of P. capsici in Argentina. Our objective was to assess the diversity of P. capsici in Argentina at key pepper production areas. Forty isolates were recovered 2006-2009 from pepper and one isolate from pumpkin at 11 locations. Isolates were assessed for mating type, mefenoxam sensitivity and multilocus single nucleotide polymorphism (SNP) genotype profiles. Ten isolates with identical SNP profiles also were genotyped with amplified fragment length polymorphism (AFLP) markers. All 41 isolates had the A1 mating type and were sensitive to mefenoxam. Genotypic analysis using eight polymorphic SNP markers indicated 87% of the isolates had the same multilocus genotype, which is fixed for heterozygosity at seven of the eight SNP sites. AFLP analyses confirmed these findings, and overall it appears that clonal reproduction drives the population structure of P. capsici in Argentina. The implications for breeding resistant peppers and overall disease management are discussed.     

中国和美国大豆疫霉群体遗传结构的ISSR分析

为探究中国和美国大豆疫霉的遗传关系, 采用简单序列重复区间扩增多态性(ISSR)技术, 对来自中国黑龙江省、福建省和美国的3个大豆疫霉地理群体的遗传多样性进行了分析。通过13个ISSR引物对供试的111株大豆疫霉菌株进行扩增, 共得到102个ISSR条带, 其中多态性条带为88个, 占86%。遗传变异分析表明, 美国群体具有更高的遗传变异度; Nei’s 遗传相似性和主成分分析均显示, 中国福建群体与美国群体间的遗传相似性最高, 而福建群体与黑龙江群体间遗传相似性最低; 聚类分析显示, 供试菌株在88%的相似性水平上可区分为7个聚类组, 且美国群体分布于更多的聚类组中; Shannon-Wiener多样性指数也表明美国群体的遗传多样性最为丰富。综合分析表明, 本研究的结果不支持关于美国的大豆疫霉可能来源于中国的推测。     

Virulence of Phytophthora sojae in the Pampeana Subregion of Argentina from 1998 to 2004

Phytophthora root rot is one of the most serious diseases of soybeans in Argentina. Surveys of commercial fields and trial plots of soybean were conducted throughout the northern Pampeana subregion (Argentina) between 1998 and 2004. A total of 193 isolates of Phytophthora sojae were collected and classified into races or virulence formulae. Among the 173 isolates tested on 8 differentials, 42 different pathotypes were detected, including 18 described races. Races 1, 4, 5, 7, 9, 13, 23 and 24 were found in both plants and soils, whereas races 2, 3, 6, 8, 11, 14, 15, 17, 43 and 44 were only isolated from plants. An additional 19 pathotypes were described from 20 isolates tested in Canada on the expanded set of 14 differential cultivars. Currently, all Rps genes/alleles associated with resistance have been defeated, indicating an increased complexity of virulence within the P. sojae populations in the region. The great increase in virulence complexity found in this study is most likely a result of a long period of continuous production of soybean cultivars with Rps genes and the extensive adoption of the no‐tillage system.     

Genetic characterization of grapevine-infecting Botrytis cinerea isolates from Argentina

BackgroundBotrytis cinerea is an ascomycete with a high genetic diversity and complex population structure, as reported from several hosts and sites. However, nothing is known about its genetic diversity in Argentina.AimsThe aim of this work is to estimate the genetic diversity of a local population of B. cinerea isolates obtained from grapevine in Argentina.MethodsIn this work, 35 strains that had been isolated from grapevines were genotyped for the presence of transposable elements and PCR-based RFLP molecular markers. The obtained results were compared with those from a large French population of the fungus, and used to perform a population genetics analysis using the Genepop software.ResultsAll the analysed isolates were classified as Group II (according to the most recent proposed classification) and showed a high degree of genetic diversity, with 14 different haplotypes. A significant difference in allele frequency was recorded between the local and French populations.ConclusionsThese comparisons between fungal populations, led to the detection of a high level of diversity and the differentiation between local and French groups of isolates. This was confirmed by an Fst value of 0.3332, which was higher than that reported for other pairwise comparisons of populations. This work constitutes the first report on the genetic diversity of B. cinerea isolates and their population structure in Argentina.     

Genetic and physical mapping of Avr1a in Phytophthora sojae

The interaction between soybean and the phytopathogenic oomycete Phytophthora sojae is controlled by host resistance (Rps) genes and pathogen avirulence (Avr) genes. We have mapped the Avr1a locus in F(2) populations derived from four different P. sojae races. Four RAPD and nine AFLP markers linked to Avr1a were initially identified. Nine markers were used to compare genetic linkage maps of the Avr1a locus in two distinct F(2) populations. Distorted segregation ratios favoring homozygous genotypes were noted in both crosses. Segregation analysis of all the markers in one F(2) population of 90 progeny generated a map of 113.2 cM encompassing Avr1a, with one marker cosegregating with the gene. The cosegregating DNA marker was used to isolate P. sojae BAC clones and construct a physical map covering 170 kb, from which additional DNA markers were developed. Three markers occurring within the BAC contig were mapped in an enlarged population of 486 F(2) progeny. Avr1a was localized to a 114-kb interval, and an average physical to genetic distance ratio of 391 kb/cM was calculated for this region. This work provides a basis for the positional cloning of Avr1a.     

大豆疫霉菌的EMS化学诱变

以甲基磺酸乙酯(ethylmethane sulfonate,EMS)为诱变剂,通过其对大豆疫霉菌Phytophthora sojae休止孢萌发的影响,确定化学诱变条件。通过收集单卵孢子,建立了包含640个单卵孢子系的突变体库,其中约有50%的诱变菌系在培养性状和菌落形态方面发生了明显变化,菌落形态多样,表现出较紧密或松散,近圆形或不规则;气生菌丝减少,生长速度较慢或快;在卵孢子产量方面,8.13%的菌系有增加,20.41%的菌系减少,27.82%的菌系极少或者没有卵孢子产生,43.64%的菌系卵孢子产量类似野生型。以质膜氢离子泵蛋白基因PsPMA1(plasma membrane H+-ATPase1)为对象,通过TILLING技术,从320个大豆疫霉菌突变体中获得9个突变体,进一步确认了EMS对大豆疫霉菌的诱变效果,并且估算EMS对大豆疫霉菌的诱变频率至多每115kb发生一个核苷酸变异。新构建的突变体库为开展大豆疫霉病菌的功能基因组研究奠定了遗传材料基础。     

大豆疫霉菌的分离与鉴定

采用叶碟诱捕法从2007年进口的美国大豆携带的土壤和2006年从黑龙江感病大豆田采集的土壤中分离出2株疫霉菌菌株,并对病原菌进行了形态特征、致病性、分子检测。结果表明:形态观察为疫霉属真菌;接种大豆后出现典型的大豆疫病症状;采用大豆疫霉的特异性引物PCR检测,2个菌株均能扩增出分子量为330 bp的特异性条带。结合形态、致病性测定和分子检测,2株病菌鉴定为大豆疫霉菌(Phytophthora sojaeKauf-mann et Gerdemann)。     

安徽和黑龙江省大豆疫霉群体遗传结构的SSR分析

采用简单重复序列(SSR)的分析方法,对来自安徽和黑龙江省的大豆疫霉群体进行了遗传多样性分析。通过使用20对SSR引物对供试的83株大豆疫霉菌株进行PCR扩增,共得到109个SSR标记,全部为多态性标记,平均每对引物扩增出5.5条带。遗传变异与相似性分析表明,安徽群体具有更高的遗传变异度,安徽群体与黑龙江群体间遗传相似性较低;聚类分析显示,供试菌株在80%的相似性水平上可被区分为7个类群,且安徽群体分布于更多的聚类组中;Shannon-Wiener多样性指数也表明安徽群体的遗传多样性较黑龙江群体丰富。综合分析表明,本研究的结果不支持关于安徽的大豆疫霉可能来源于黑龙江的推测。     

Genetic characterization,pathogenicity and benomyl susceptibility of Lecanicillium fungal isolates from Argentina

The aim of this study was to investigate biological and molecular characteristics of Lecanicillium strains isolated from Hemipteran hosts in Argentina. Morphology‐based taxonomic characterization together with molecular taxonomy based on rRNA operon internal transcribed spacer (ITS), mitochondrial nad1 gene, and nuclear ef1a gene sequences resulted in the assignment of nine out of ten isolates to the Lecanicillium lecanii sensu lato complex. However, whereas several isolates were thus unequivocally characterized as Lecanicillium muscarium or Lecanicillium longisporum, species assignment was not possible for three isolates that might represent a new species within the L. lecanii s.l. complex. We found two group‐I introns on 18S and 28S rRNA gene on only one isolate. Pathogenicity tests were conducted against the peach aphid using conidial suspensions (1 × 10⁷ conidia/ml), and the Kaplan–Meier analysis was performed to evaluate the survival of Myzus persicae. Lecanicillium longisporum CEP 155 and L. muscarium CEP 182 were significantly more pathogenic to M. persicae than all the Lecanicillium isolates causing aphid mortalities >85%. Determination of susceptibility to the benzimidazole fungicide benomyl revealed important differences between Lecanicillium strains. The inhibitory effect of benomyl appeared less pronounced for the L. muscarium fungal isolates than for those belonging to a different taxon. Based in our results, the best candidate strain as microbial biological control agent against M. persicae is L. muscarium CEP 182. However, further research under field conditions in greenhouses should be done in order to confirm the compatibility of entomopathogenic fungi and fungicides within an IPM strategy.     

拟南芥与大豆疫霉菌的非寄主互作及一个感病突变体的遗传分析

非寄主抗病性是一种普遍的自然现象, 该文通过建立拟南芥-大豆疫霉菌(Arabidopsis thaliana-Phytophthora sojae)非寄主互作系统, 筛选对大豆疫霉菌感病的拟南芥突变体, 为研究植物对卵菌的非寄主抗病性遗传机制奠定基础。以大豆疫霉菌游动孢子接种拟南芥T-DNA插入突变体离体叶片, 从代表12 000个独立转化株系的40 000株T₃代T-DNA插入拟南芥突变体中获得一系列对大豆疫霉菌感病的突变体。其中突变体581-51感病性状表现稳定, 离体叶片接菌后3天内出现明显的水渍状病斑, 4–5天后产生大量卵孢子和/或孢子囊。细胞学观察发现有典型的吸器形成。Southern杂交和遗传分析结果表明, 581-51突变体含有4个T-DNA插入事件, 其感病性状可能由隐性单基因控制。     

拟南芥与大豆疫霉菌的非寄主互作及一个感病突变体的遗传分析

非寄主抗病性是一种普遍的自然现象,该文通过建立拟南芥．大豆疫霉菌（Arabidopsis thaliana—Phytophthora sojae）非寄主互作系统,筛选对大豆疫霉菌感病的拟南芥突变体,为研究植物对卵菌的非寄主抗病性遗传机制奠定基础。以大豆疫霉菌游动孢子接种拟南芥T—DNA插入突变体离体叶片,从代表12000个独立转化株系的40000株T3代T。DNA插入拟南芥突变体中获得一系列对大豆疫霉菌感病的突变体。其中突变体581-51感病性状表现稳定,离体叶片接菌后3天内出现明显的水渍状病斑,4—5天后产生大量卵孢子和／或孢子囊。细胞学观察发现有典型的吸器形成。Southern杂交和遗传分析结果表明,581—51突变体含有4个T-DNA插入事件,其感病性状可能由隐性单基因控制。     

Pathotype and SSR variation in Phytophthora sojae from the Argentinean Pampas

Genetic variation in Phytophthora sojae was compared between northern (NPS) and southern (SPS) Pampeana sub-regions, in the core soya bean region of Argentina. Ninety-three isolates from plants and soil were evaluated in the present study. The pathotype for each isolate was determined using the hypocotyl technique on a set of eight differentials. For neutral genetic diversity evaluation, eight SSRs markers were utilized. Thirty pathotypes were determined, including 19 in SPS and 23 in NPS. The number of isolates for each locality and region was unevenly distributed. Pathotype variation was high for P. sojae samples compared among fields and within isolates from single fields. SSR variation studies resulted in an elevated haplotype composition, partitioned at all, but the regional levels. P. sojae populations from Argentina have evolved towards a complex epidemiological scenario, with no evidence of geographic correlation at the pathotypic nor the neutral genetic level. It is likely that future works aiming at elucidating the evolution of pathogenesis in this species will need to include non-neutral markers, specifically associated to avr loci. The results of the present work are considered a useful contribution to the adequate and sustainable management of this pathogen in the main soya bean area of Argentina.     

用SSR标记分析抗疫霉根腐病大豆品种(系)的遗传多样性

利用50对SSR引物对抗疫霉根腐病大豆品种(系)进行遗传多样性分析。在166份品种(系)中,50个SSR座位共产生265个等位变异,平均每个座位5.3个。采用NTSYS-pc2.10计算品种(系)间遗传相似系数,平均相似系数为0.3124,表明抗疫霉根腐病大豆品种(系)间的遗传差异较大。用UPMGA进行聚类分析,166个品种(系)在相似系数为0.33时被聚为6类,地理来源相同的品种(系)大多聚类在一起。一些具有相同或相近抗病反应型的品种(系)被聚类在同一个类群中,表明这些抗病品种(系)的遗传关系较近,应有选择地利用。W illiam s和C lark抗疫霉根腐病近等基因系构成明显不同于中国大豆的基因源,可以用于拓宽我国大豆品种的遗传基础。     

Genetic diversity among isolates of Trichinella spiralis from the Province of Buenos Aires, Argentina

Random Amplified Polymorphic DNAs, (RAPDs) are used to study the occurrence of Trichinella britovi and T5 among domestic animals in the Province of Buenos Aires, Argentina and to assess the genetic diversity among isolates of T. spiralisfrom this area in a number of infected hosts. All the local isolates proved to be T. spiralis. Six of the eight primers used indicate that the Buenos Aires isolates are distinct from each other as they produce a considerable number of polymorphic bands. Our overall estimates are relatively higher than other intraspecific distances previously estimated within species of this genus and among T. spiralis isolates. Such high degrees of variability observed among local isolates and between isolates from Buenos Aires and Spain should be taken into account when defining isolates within this species, and considering differences in the epidemiology of T. spiralis.     

Soybean sterol composition and utilization by Phytophthora sojae

The sterol fraction of Glycine max (soybean) was found to contain a mixture of 13 major sterols which differed dramatically in composition between seeds and shoots. Typical C4-desmethyl Delta(5)-sterols, including sitosterol, predominate the sterol mixture of shoots, whereas C4-methyl sterol intermediates, cycloartenol and 24(28)-methylene cycloartanol, accumulate in seeds. The significance of modified sterol profile of shoot compared to seed was relevant to the physiology of Phytophthora sojae, a phytopathogen of soybean shown to be auxotrophic for sterol. Sterols native to the host plant containing a C4-methyl group, such as cycloartenol, were not utilized by the fungus. Alternatively, all Delta(5)-sterols added to the culture media of P. sojae supported normal growth and promoted viable oospore production. The results demonstrate the importance of sterols in plant-fungal interactions and offer the possibility of bioengineering the phytosterol pathway for resistance to phytopathogens which scavenge specific sterols of the host plant to complete the life cycle.     

Selection and Characterization of Inhibitor-Resistant Mutants of Phytophthora sojae

Bhat, R. G., and Schmitthenner, A. F. 1993. Selection and characterization of inhibitor-resistant mutants of Phytophthora sojae. Experimental Mycology 17, 109-121. Selectable markers, resistance to metalaxyl (MEX) and p -fluorophenylalanine (FPA), were induced in Phytophthora sojae races by treating zoospores with N -methyl-N′-nitro-N′-nitrosoguanidine. Calcium treatment enhanced the percentage encystment of zoospores. MEX-resistant (MEX^r) and FPA-resistant (FPA^r) mutants were selected on a lima bean agar medium containing MEX (10 μg/ml) and FPA (50 μg/ml), respectively. A number of single inhibitor-resistant mutants were obtained. There was variation in in vitro growth of mutants. Virulence of mutants was evaluated by hypocotyl inoculation of soybean cultivars with Rps 1 (Amsoy 71), Rps1-c (Williams 79), Rps1-k (Williams 82), or rps1 (Williams) alleles. It was observed that the 23% of the MEX^r and 33% of the FPA^r mutants had a different virulence pattern than the original cultures.