Population genetic analysis of Phytophthora infestans in northwestern China

Late blight caused by Phytophthora infestans is the most devastating disease of potato worldwide. To understand the P. infestans population structure and dynamics in northwestern China, 959 single‐lesion isolates were purified in three consecutive years (2009–2011) and were characterized for mating type, pathotype, mtDNA haplotype and molecular variation at eight SSR loci. The results showed that the distribution of mating types changed significantly over years, with self‐fertile isolates dominant in 2010 and 2011. SSR genotyping distinguished 959 isolates into 151 genotypes, and association analysis indicated that P. infestans populations in 2010 and 2011 were strictly asexual while in 2009 they showed signs of sexual reproduction. Population analysis showed that the majority of genetic variation was within P. infestans populations. Isolates sharing identical SSR genotypes were detected in distant regions, indicating that migration of P. infestans could have occurred between regions. Pathogenicity assays on a set of potato differential lines containing R1 to R11 resistance genes detected four pathotypes from 74 selected isolates, with the pathotype virulent against all 11 R genes being dominant. Three mtDNA haplotypes (Ia, IIa, IIb) were detected with Ia being dominant among 507 isolates examined. Phylogenetic analysis indicated that P. infestans populations in northwestern China are distant from European lineages including 13‐A2 (blue‐13) at the time of this survey. The results have implications for the trade of healthy seed tubers as a means of managing late blight.     

大豆疫霉多态性SSR标记开发及遗传多样性分析

 用FastPCR软件在大豆疫霉全基因组中搜索到1 234个含2~4个重复基元精确SSRs。选择260个SSRs设计引物,经对大豆疫霉5个分离物的基因组DNA检测,有212对(81.5%)有效扩增出SSR特征条带,112对(52.8%)扩增多态性。用18对多态性SSR引物分析了来自美国、中国黑龙江省和福建省大豆疫霉分离物的遗传多样性,在73个分离物中共扩增出112个等位变异,变异范围为4~9,平均为6.22个,表明选择的引物对具有高的多态性。在3个大豆疫霉群体中,黑龙江省和福建省分离物的遗传距离最近,美国和福建省分离物的遗传距离最远。UPGMA聚类将73个分离物划分为6组,其中8个美国分离物(72.73%)和53个中国分离物(85.48%)被聚类在一起,表明大豆疫霉中国分离物与美国分离物可能具有共同的祖先,中国分离物可能为外来种。     

Large subclonal variation in Phytophthora infestans populations associated with Ecuadorian potato landraces

The population of Phytophthora infestans on potato landraces in three provinces (Carchi, Chimborazo and Loja) of Ecuador was analysed. All isolates (n = 66) were of the A1 mating type. Simple sequence repeats (SSR) were used to assess the genetic diversity of the isolates. The P. infestans isolates from the potato landraces grouped in a single clade together with reference isolates belonging to the clonal lineage EC‐1. In the 66 SSR profiles obtained, 31 multilocus genotypes were identified. The 66 isolates constituted 49 different races according to the Solanum demissum differential set ( R1 to R11). The P. infestans population was complex and virulent on 4 to 11 R genes. Analysis showed that the subclonal variation in the Ecuadorian EC‐1 clone is increasing over time and is much larger than clonal variation in lineages in the Netherlands and Nicaragua, suggesting high mutation rates and little or no selection in Ecuador.     

Pineapple heart rot isolates from Ecuador reveal a new genotype of Phytophthora nicotianae

Pineapple heart rot disease, caused by Phytophthora nicotianae (syn. P. parasitica), is responsible for significant annual reductions in crop yield due to plant mortality. In Ecuador, new infections arise during the rainy season and increase production costs due to the need for biocontrol and fungicide applications. Studies of P. nicotianae population structure suggest that certain genetic groups are associated with host genera; however, it is not clear how many host‐specific lineages of the pathogen exist or how they are related. The objectives of this study were to determine the level of genetic variation in the P. nicotianae population causing heart rot disease of pineapple in Ecuador and compare the genotypes found on pineapple to those previously reported from citrus, tobacco and ornamentals. Thirty P. nicotianae isolates collected from infected pineapple leaves from four farms were genotyped using nine simple sequence repeat loci. In addition, the DNA sequences of mitochondrial loci cox2 + spacer and trnG‐rns were analysed. Together, these loci supported a single clonal lineage with two multilocus genotypes differing in a single allele and low mitochondrial diversity. This lineage was distinct but closely related to isolates collected from vegetables and ornamentals in Italy. The results support the hypothesis of host specialization of P. nicotianae in intensive cropping systems and contribute to the understanding of population structure of this important pathogen.     

Dominance of a single clonal lineage in the Phytophthora infestans population from northern Shaanxi,China revealed by genetic and phenotypic diversity analysis

Late blight caused by Phytophthora infestans is the most serious disease of potato worldwide. To understand the P. infestans population structure in northern Shaanxi, an emerging potato production region in China, 125 single‐lesion isolates were randomly collected from farmers' fields in 2009 and characterized phenotypically and genotypically. A mating type assay showed that 94 isolates were A1 mating type. Virulence determination of selected isolates on a set of differential potato lines containing R1 to R11, respectively, showed the presence of two pathotypes, of which the pathotype lacking avirulence genes Avr3, Avr4 and Avr10 was dominant. Isolates lacking all avirulence factors Avr1 to Avr11 were detected but at lower frequency (13·6%). Analysis for mtDNA haplotype showed all 61 examined isolates were IIa. A total of seven multilocus genotypes were distinguished among 125 isolates, as determined with seven polymorphic microsatellite markers. The genotype SG‐1 was dominant in the population with a frequency of 75·2% and was present throughout the region. Analysis of the phenotypic and genotypic structures of P. infestans populations indicated strict clonal reproduction of the pathogen and suggested that sexual reproduction probably does not occur. Potential implications for disease management are discussed.     

中国不同地区致病疫霉遗传多样性的RAPD分析

 本文应用RAPD技术检测了我国主要马铃薯产区致病疫霉的遗传分化情况及不同地区菌株间的亲缘关系。用筛选出的10个随机引物对1997-2001年间采自我国9省市的82株及3株来自日本的致病疫霉DNA进行了PCR扩增,获得了79条谱带,其中多态性标记75条,占95%。根据扩增结果,运用UPGMA分析,获得了表现菌株间亲缘关系的树状图。菌株间的最大遗传距离为0.5,以距离0.3为阈值,可将供试菌株划分为10个组(RG1-10)。结果发现:A1交配型菌株群体内的差异大于A1和A2菌株群体之间的;RAPD分组与菌株的地理来源、交配型及对甲霜灵的敏感性无明显相关性。研究结果显示,来自中国北方甘肃、内蒙、吉林、黑龙江地区的菌株与一些来自云南、四川等西南地区的菌株亲缘关系相近。病原菌随种薯的迁移可能是导致这种现象的原因之一。     

中国柑橘大实蝇遗传多样性分析

利用4对微卫星引物对采自中国7省市的18个柑橘大实蝇地理种群进行标记,并进行遗传多样性分析。结果表明,从供试柑橘大实蝇地理种群检测到的等位基因数为19~30个,平均为23.06个,且4个微卫星位点(Bp125、BcuF 1.6、Bcac 5.10、Bcac 6.10)的等位基因数分别为7、14、9、8个。对18个柑橘大实蝇种群的哈迪—温伯格平衡测试结果显示,其卡方值的变化范围为5.582~∞,各种群间差异显著(变化范围为0.000~0.694)。表明中国柑橘大实蝇种群的遗传多样性较高,且不同种群间存在较大的遗传分化。     

大豆疫霉根腐病菌单游动孢子的毒性遗传与变异

 采用离体叶柄伤口接种法测定大豆疫霉根腐病菌44号生理小种单游动孢子连续3代或4代分离后代的毒性,结果表明:从S1中选择与亲本相比毒性不发生变异的1号单游动孢子菌株(44号生理小种)和变异最大的30号单游动孢子菌株(1号生理小种)继续分离2代或3代,单游动孢子毒性变异趋势主要是从44号生理小种变异为3号生理小种,也有变异成毒性公式为7或1a,7的单游动孢子。大豆疫霉根腐病菌无性世代毒性变异几率很高,多数单游动孢子毒性在分离后代中都发生变异,产生不同的小种或毒性公式,并且毒性变异基本不能稳定遗传。     

Pathogenicity of 21 newly described Phytophthora species against seven Western Australian native plant species

The pathogenicity of some Phytophthora species recently described from Western Australia, together with P. cinnamomi as a control, was tested against seven Western Australian native plant species in the glasshouse. Host species were Banksia grandis, B. littoralis, B. occidentalis, Casuarina obesa, Corymbia calophylla, Eucalyptus marginata and Lambertia inermis. Twenty‐two Phytophthora species were grown on a vermiculite, millet seed and V8 substrate and used as soil inoculum when the plant hosts were approximately 3 months old. Pathogenicity was assessed after 6 weeks and plants were scored for death, root damage, and percentage reduction of shoot growth compared with control plants. The pathogenicity of P. cinnamomi was confirmed. Phytophthora niederhauserii was shown to be similar to P. cinnamomi in pathogenicity and of concern ecologically. Other species that killed one or more hosts were P. boodjera, P. constricta, P. elongata, P. moyootj and P. rosacearum, while P. condilina, P. gibbosa, P. gregata, P. litoralis and P. ‘personii’ caused significant reduction to shoot and/or root growth, but did not kill plants. Host species susceptible to the highest number of Phytophthora species were B. grandis, B. littoralis, B. occidentalis and E. marginata. No Phytophthora species tested killed C. calophylla.     

Temperature adaptation in Australasian populations of Puccinia striiformis f. sp. tritici

Stripe rust, caused by Puccinia striiformis f. sp. tritici, is one of the major fungal pathogens of wheat. A new pathotype was introduced to Australia in 2002 and several derivative pathotypes were detected in subsequent seasons. It has been suggested that the severity of stripe rust outbreaks in Australia since 2002 could be as a result of traits other than virulence in the pathogen population. This study was conducted to investigate the hypothesis that the stripe rust pathogen population dominant in Australia since 2002 was better adapted to warm temperature conditions compared to previous pathogen populations. Sixteen pathotypes were selected to examine the influence of two contrasting temperature regimes during the 24 h incubation (10°C and 15°C) and the subsequent post‐inoculation (17°C and 23°C) periods on latent period and infection efficiency on four susceptible wheat cultivars. In addition, the effect of two contrasting incubation temperatures on urediniospore germination was examined. The results indicated that pathotypes of P. striiformis f. sp. tritici detected after 2002 did not show evidence of adaptation to high temperatures, which suggests that other factors contributed to the observed increased aggressiveness.     

A Phytophthora conserved transposon‐like DNA element as a potential target for soyabean root rot disease diagnosis

A transposon‐like element, A3aPro, with multiple copies in the Phytophthora sojae genome, was identified as a suitable detection target for this devastating soyabean root rot pathogen. The PCR primers TrapF1/TrapR1 were designed based on unique sequences derived from the transposon‐like sequence. A 267‐bp DNA fragment was amplified using this primer pair, the specificity of which was evaluated against 118 isolates of P. sojae, 72 isolates of 25 other Phytophthora spp., isolates of Pythium spp. and isolates of true fungi. In tests with P. sojae genomic DNA, detection sensitivities of 10 pg and 10 fg DNA were achieved in standard PCR (TrapF1/TrapR1) and nested PCR (TrapF1/TrapR1 and TrapF2/TrapR2), respectively. Meanwhile, PCR with TrapF1/TrapR1 primers detected the pathogen at the level of a single oospore, and even one zoospore. These primers also proved to be efficient in detecting pathogens from diseased soyabean tissues, residues and soils. In addition, real‐time quantitative PCR (qPCR) assays coupled with the TrapF1/TrapR1 primers were developed to detect and quantify the pathogen. The results demonstrated that the TrapF1/TrapR1 and TrapF2/TrapR2 primer‐based PCR assay provides a rapid and sensitive tool for the detection of P. sojae in plants and in production fields.     

福建省大豆疫病病原鉴定及其核糖体DNA-ITS序列分析

 从福建省龙海大豆根腐病株上分离的疫霉菌株中,选取6个代表菌株,对病原菌进行了形态特征、致病性、寄主范围鉴定及核糖体DNA-ITS序列分析,结果表明,该菌为疫霉属真菌,在黑麦琼脂培养基上生长缓慢,菌丝致密、无隔,形成菌丝膨大体,近直角分支,分支处稍缢缩。水培后产生大量椭圆形孢子囊,不形成乳突,通过内层出方式产生新孢子囊,游动孢子在孢子囊内形成,同宗配合,藏卵器球形,雄器侧生;接种后可出现典型的大豆疫病症状;人工接种只侵染大豆、豇豆和菜豆等少数豆科植物。其核糖体DNA-ITS序列分析表明,分离菌株与GenBank中大豆疫霉的ITS序列的同源性均为99.8%,仅有2个碱基的差异,结合形态特征和致病性测定,将这些病原菌鉴定为Phytophthora sojae. 这是首次报道大豆疫霉菌在福建省存在。     

Genetic diversity within and between sulfonylurea‐resistant and susceptible populations of Schoenoplectus juncoides in Japan

To reveal the effects of herbicide selection on genetic diversity in the outcrossing weed species Schoenoplectus juncoides, six sulfonylurea‐resistant (SU‐R) and eight sulfonylurea‐susceptible (SU‐S) populations were analysed using 40 polymorphic inter‐simple sequence repeat loci. The plants were collected from three widely separated regions: the Tohoku, Kanto and Kyushu districts of Japan. Genetic diversity values (Nei's gene diversity, h) within each SU‐S population ranged from h = 0.125 to h = 0.235. The average genetic diversity within the SU‐S populations was H_S = 0.161, and the total genetic diversity was H_T = 0.271. Although the H_S of the SU‐R populations (0.051) was lower than that of the SU‐S populations, the H_T of the SU‐R populations (0.202) was comparable with that of the SU‐S populations. Most of the genetic variation was found within the region for both the SU‐S and SU‐R populations (88% of the genetic variation respectively). Two of the SU‐R populations showed relatively high genetic diversity (h = 0.117 and 0.161), which were comparable with those of the SU‐S populations. In contrast, the genetic diversity within four SU‐R populations was much lower (from h = 0 to 0.018) than in the SU‐S populations. The results suggest that selection by sulfonylurea herbicides has decreased genetic diversity within some SU‐R populations of S. juncoides. The different level of genetic diversity in the SU‐R populations is most likely due to different levels of inbreeding in the populations.     

Genetic variation and structure in native and invasive Solidago canadensis populations

Solidago canadensis is native to North America, but has become a noxious invasive plant in China. We know only a little about its invasion history and the effects of introductions on its genetic composition. Here, we investigated genetic variation and structure between 15 North American and 13 Chinese populations of S. canadensis using AFLP makers. Four AFLP loci suggested relatively high genetic diversity of this weed and similar genetic variation between the invasive range and the native range. Most genetic variation was within populations across two ranges, but the Chinese range had a higher degree of among‐population variation than the North American range. Multiple tests, including Bayesian assignment, UPGMA analysis, PCoA and analysis of ‘isolation by distance’, showed that the Chinese populations originated from at least two distinct native sources and that secondary introduction or dispersal should be common in China. Also, North American populations were possibly a single genetic group. Overall, S. canadensis in China was probably founded from multiple introductions and then spread through long‐distance dispersal associated with human activities. Genetic variability in the species in the invaded range appears to have favoured establishment and spread and may well provide a challenge to successful control.     

Low genetic variability in Sclerotinia sclerotiorum populations from common bean fields in Minas Gerais State,Brazil, at regional,local and micro‐scales

Sclerotinia sclerotiorum, causal agent of white mould, is the most destructive and widely distributed soilborne pathogen of common bean during the autumn–winter season in Brazil. Nevertheless, little is known about the genetic structure of the pathogen population. Microsatellite (SSR) markers and mycelial compatibility groups (MCGs) were used to characterize 118 isolates collected from 20 bean fields located in the most important growing regions of Minas Gerais State (MG). Additionally, the genetic variability among 10 isolates obtained from a single sclerotium was investigated in 10 different sclerotia. Seventy SSR haplotypes and 14 MCGs were identified among the 118 isolates. The genetic differences within bean growing areas accounted for most of the genetic variation (72%). Despite the relatively high genotypic diversity, the SSR loci were at linkage disequilibrium. Moreover, 70% of the isolates were assigned to only two MCGs, and haplotypes of a given MCG were closely related. The discriminant analysis of principal components revealed five groups. There was strong genetic differentiation between isolates collected in one municipality in southern MG when compared to other regions. Common bean resistance to white mould should be assessed with representative isolates of the five genetic groups and, if possible, of the different MCGs detected in the present study. One to five haplotypes were detected among the 10 isolates obtained from a single sclerotium. Therefore, in order to ensure genetic identity of an isolate, hyphal tip or monoascosporic isolates should be used.     

Phytophthora pachypleura sp. nov., a new species causing root rot of Aucuba japonica and other ornamentals in the United Kingdom

Isolates of an unknown Phytophthora species from the ‘Phytophthora citricola complex’ have been found associated with mortality of Aucuba japonica in the UK. Based on morphological characteristics, growth–temperature relationships, sequences of five DNA regions and pathogenicity assays, the proposed novel species is described as Phytophthora pachypleura. Being homothallic with paragynous antheridia and semipapillate sporangia, P. pachypleura resembles other species in the ‘P. citricola complex’ but can be discriminated by its distinctively thick‐walled oospores with an oospore wall index of 0·71. In the phylogenetic analysis based on three nuclear (ITS, β‐tubulin, EF‐1α) and two mitochondrial (cox1, nadh1) DNA regions, P. pachypleura formed a distinct clade within the ‘P. citricola complex’ with P. citricola s. str., P. citricola E and P. acerina as its closest relatives. Phytophthora pachypleura is more aggressive to A. japonica than P. plurivora and P. multivora and has the potential to affect other ornamental species.     

Phytophthora infestans populations in central,eastern and southern African countries consist of two major clonal lineages

Limited knowledge is available on Phytophthora infestans populations in Sub‐Saharan Africa (SSA). Therefore, and in response to recent severe late blight epidemics, P. infestans isolates from potato, tomato and Petunia × hybrida from eight SSA countries were characterized. Isolates were characterized with ‘old’ markers, including mating type (176 isolates), mitochondrial DNA haplotype (mtDNA) (281 isolates), glucose‐6‐phosphate isomerase (Gpi) (70 isolates), restriction fragment length polymorphism analysis with probe RG‐57 (49 isolates), and by metalaxyl sensitivity (64 isolates). Most isolates belonged to the US‐1 genotype or its variants (US‐1.10 and US‐1.11). The exceptions were genotype KE‐1 isolates (A1 mating type, mtDNA haplotype Ia, Gpi 90/100 and unique RG‐57 genotype), identified in two fields in Kenya, which are related to genotypes previously identified in Rwanda (RW‐1 and RW‐2), Ecuador and Europe. Metalaxyl‐resistant P. infestans isolates from potato were present in all the countries except Malawi, whereas all the isolates from tomato were sensitive. Genotyping of 176 isolates with seven simple sequence repeat (SSR) markers, including locus D13 that was difficult to score, revealed 79 multilocus genotypes (MLGs) in SSA. When this locus was excluded, 35 MLGs were identified. Genetic differentiation estimates between regional populations from SAA were significant when locus D13 was either excluded (P = 0·05) or included (P = 0·007), but population differentiation was only low to moderate (F_ST = 0·044 and 0·053, respectively).     

大豆疫霉菌单孢分离物生物学性状的遗传变异研究

 本文研究了大豆疫霉菌单游动孢子无性分离物和自交单卵孢子分离物的菌丝生长速率、菌落形态、同宗配合性状、产孢量以及对甲霜灵敏感性的遗传变异。结果表明:菌落形态、生长速率和同宗配合性状在单游动孢子后代和自交后代可稳定遗传,控制上述性状的遗传因子是纯合的;大豆疫霉菌的游动孢子产生能力和对甲霜灵的敏感性在单游动孢子后代和自交后代中均发生连续性变异,表明这两种性状可能是数量遗传性状,也可能控制这两种性状的基因为杂合基因或细胞质遗传因子。     

Diversity of Avr‐vnt1 and AvrSmira1 effector genes in Polish and Norwegian populations of Phytophthora infestans

The oomycete Phytophthora infestans, the cause of late blight, is one of the most important potato pathogens. During infection, it secretes effector proteins that manipulate host cell function, thus contributing to pathogenicity. This study examines sequence differentiation of two P. infestans effectors from 91 isolates collected in Poland and Norway and five reference isolates. A gene encoding the Avr‐vnt1 effector, recognized by the potato Rpi‐phu1 resistance gene product, is conserved. In contrast, the second effector, AvrSmira1 recognized by Rpi‐Smira1, is highly diverse. Both effectors contain positively selected amino acids. A majority of the polymorphisms and all selected sites are located in the effector C‐terminal region, which is responsible for their function inside host cells. Hence it is concluded that they are associated with a response to diversified target protein or recognition avoidance. Diversification of the AvrSmira1 effector sequences, which existed prior to the large‐scale cultivation of plants containing the Rpi‐Smira1 gene, may reduce the predicted durability of resistance provided by this gene. Although no isolates virulent to plants with the Rpi‐phu1 gene were found, the corresponding Avr‐vnt1 effector has undergone selection, providing evidence for an ongoing ‘arms race’ between the host and pathogen. Both genes remain valuable components for resistance gene pyramiding.