Analysis of genetic diversity in <Emphasis Type="Italic">Monilinia fructicola</Emphasis> from the Ebro Valley in Spain using ISSR and RAPD markers

The genetic diversity of Spanish and French field populations of Monilinia fructicola, a quarantine fungal pathogen in Europe, was compared with that of Californian, Uruguayan, and New Zealand M. fructicola populations using inter simple sequence repeat (ISSR) and random amplified polymorphic DNA (RAPD) markers. Unweighted pair-group method with arithmetic average (UPGMA) cluster analysis and principal component analysis (PCA) of the ISSR data set revealed that the Spanish and French M. fructicola isolates were more closely related between themselves than to the non-European isolates. The levels of genetic diversity in the Spanish and French isolates are lower than those of the non-European isolates, indicating that M. fructicola is a recently introduced pathogen. UPGMA cluster analysis and PCA of the combined ISSR + RAPD data set of the European M. fructicola populations revealed that the Spanish isolates were more closely related among themselves than with the French isolates. Analysis of molecular variance partitioned the genetic variance to among the two regions (Spain and France) (20%), among the regional populations (35%), and within the populations in each region (45%) suggesting restricted gene flow between the three European populations. The observed index of association (I_A) in each European M. fructicola populations indicates that the French and Spanish populations of M. fructicola are mainly asexually reproducing, with the Sudanell population potentially having a teleomorphic stage. The present finding of low genetic diversity in the Spanish and French M. fructicola populations is probably due to founder effects and genetic drift.     

Genetic Variability and Population Structure of the Wheat Foot Rot Fungus, Fusarium culmorum, in Tunisia

The random amplified polymorphic DNA (RAPD) method was used to investigate the genetic variability and population structure of Fusarium culmorum isolated from wheat stem bases. A total of 108 isolates, representing seven geographically distinct populations, was collected from five climatic regions in Tunisia. Pseudo-allelic frequencies were estimated at each of the 25 putative RAPD loci analyzed by scoring for the presence or absence of amplified fragments; 92 haplotypes were found among the 108 strains. The analysis of the population structure did not reveal any trend with regard to geographic origin. Total gene diversity (H_T ^* = 0.318) was mostly attributable to diversity within populations (H_S ^* = 0.308). Analysis of molecular variance confirmed that most of the genetic variability was within populations. Genetic differentiation among populations was low to moderate (G_ST ^* ranged from 0 to 0.190 and averaged 0.041 over all loci). Cluster analysis with UPGMA using genetic distances did not reveal any spatial clustering of the isolates collected from the different geographic regions. Based on these results, we conclude that the F. culmorum isolates recovered from different regions in Tunisia might be part of a single population pool.     

Genetic structure of <Emphasis Type="Italic">Fusarium verticillioides</Emphasis> populations isolated from maize in Argentina

Fusarium verticillioides (sexual stage Gibberella moniliformis) is a common fungal pathogen of maize worldwide that also produces fumonisin mycotoxins. Populations of this fungus can be diverse with respect to neutral and selectable genetic markers. We used vegetative compatibility groups (VCGs) and amplified fragment length polymorphisms (AFLPs) to evaluate the genetic structure of three F. verticillioides populations from commercial maize fields in Argentina. Based on work with similar populations from outside South America, we expected individuals within the populations to be genetically diverse, that genotypic variation would be distributed in a manner consistent with random mating, and that populations from different locations would be genetically indistinguishable from one another. We analysed 62 AFLP loci for 133 fungal isolates. All three populations were genotypically diverse but genetically similar and potentially part of a larger, randomly mating population, with significant genetic exchange occurring between the three subpopulations. There was no evidence for linkage disequilibrium at P = 0.05. The low values of G _ST , the lack of frequent private alleles, and the lack of a systemic pattern of linkage disequilibrium all suggest that sexual reproduction is sufficiently common in F. verticillioides and that the dispersal of strains is sufficiently efficient for the population of F. verticillioides in the main maize growing region to be a single randomly mating population with no detectable genetic subdivision. Thus differences in disease and/or toxin production observed in this region are best attributed to differences other than the genetic composition of the population.     

Molecular analysis of Spanish populations of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">dianthi</Emphasis> demonstrates a high genetic diversity and identifies virulence groups in races 1 and 2 of the pathogen

Fusarium wilt, caused by Fusarium oxysporum f. sp. dianthi (Fod), is the most important carnation disease worldwide. The knowledge of the diversity of the soil population of the pathogen is essential for the choice of suitable resistant cultivars. We examined the genetic diversity of Fod isolates collected during the period 1998–2008, originating from soils and carnation plants in the most important growing areas in Spain. Additionally, we have included some Fod isolates from Italy as a reference. Random amplified polymorphic DNA (RAPD) fragments generated by single-primer PCR were used to compare the relationship between isolates. UPGMA analysis of the RAPD data separated Fod isolates into three clusters (A, B, and C), and this distribution was more related to aggressiveness than to the race of the isolates. The results obtained in PCR amplifications using specific primers for race 1 and race 2, and SCAR primers developed in this work, correlated with the molecular groups previously determined from the RAPD analysis, and provided new molecular markers for the precise identification of the isolates. Results from successive pathogenicity tests showed that molecular differences between isolates of the same race corresponded with differences in aggressiveness. Isolates of races 1 and 2 in cluster A (R1I and R2I isolates) and cluster C (R1-type isolates) were all highly aggressive, whereas isolates of races 1 and 2 in cluster B (R1II and R2II isolates) showed a low aggressiveness profile. The usefulness of the molecular markers described in this study has been proved in double-blind tests with Fod isolates collected in 2008. Results from this work indicate a change in the composition of the Spanish Fod population over time, and this temporal variation could be related to the continuous change in the commercial carnation cultivars used by growers. This is the first report of genetic diversity among Fod isolates in the same race.     

The genetic structure of populations of <Emphasis Type="Italic">Turnip mosaic virus</Emphasis> in Kyushu and central Honshu,Japan

The genetic structure of the populations of Turnip mosaic virus in Kyushu and central Honshu, Japan was assessed. The host specificity of isolates was determined, and their gene sequences compared utilizing a population genetic approach. Phylogenetic analysis of partial sequences revealed that 32 of 49 Honshu isolates (65%) collected during 1997–2001 belonged to the basal-BR group as did 23 of 64 isolates from Kyushu. All these basal-BR isolates infected both Brassica and Raphanus plants. However, analyses of the positions of recombination sites in five regions of the genome (one third of the full sequence) showed that at least four intra-lineage recombinants were present in these populations. These analyses showed that Kyushu and Honshu shared none of these subpopulations, and genetically distinct basal-BR populations were present in the two districts. We conclude that different basal-BR subpopulations had expanded into those districts. The nucleotide sequences are deposited in the DDBJ/EMBL/GenBank databases under accession numbers AB267281-AB267376.     

Molecular and Pathotype Analysis of the Rice Blast Fungus in North Vietnam

Rice blast caused by Pyricularia oryzae is a devastating disease worldwide. In Vietnam, rice blast is especially severe in the Red River Delta in the North. The genetic diversity of 114 P. oryzae isolates collected from rice in 2001 in the Red River Delta and nine additional Vietnamese P. oryzae isolates was analysed using Amplified Fragment Length Polymorphism (AFLP). DNA similarity and cluster analysis based on 160 polymorphic AFLP markers showed twelve different AFLP genetic groups among the 123 field isolates. Isolates collected from japonica hosts clustered separately from indica host isolates with at least 60% dissimilarity with little evidence for gene flow between the two populations. In the 2001 population originating from indica hosts, three genetic groups were predominant and represented 99% of the isolates sampled. One predominant clonal lineage represented 59% of the 2001 indica host population and was found in eleven provinces in the Red River Delta of North Vietnam. Significant genotype flow could be demonstrated between the indica population south of Red river and the indica population north of Red river. There was significant linkage disequilibrium between the AFLP loci within the indica population, indicating that this is not a random mating population. Pathogenicity tests of 25 isolates selected from the 12 AFLP groups on a set of 29 differential rice lines revealed two avirulent isolates and 23 pathotypes. Different combinations of known resistance genes were found to have potential for blast resistance breeding for North Vietnam. First two authors contributed equally     

Population structure of the rice sheath blight pathogen <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> AG-1 IA from India

The population structure of Rhizoctonia solani AG-1 IA causing rice sheath blight from India was evaluated for 96 isolates using seven RFLP loci. Nineteen of the isolates did not hybridise to R. solani AG-1 IA RFLP probes and rDNA analyses subsequently confirmed that they were either Ceratobasidium oryzae-sativae isolates or another Rhizoctonia sp. The population structure of the remaining 77 R. solani AG-1 IA Indian isolates was similar to that of a previously characterized Texas population. Clonal dispersal of R. solani AG-1 IA in India was moderate within fields and no clones were shared among field populations. Low levels of population subdivision and small genetic distances among populations were consistent with high levels of gene flow. Frequent sexual reproduction was indicated by the fact that most populations were in Hardy–Weinberg equilibrium (HWE). The two loci (R68 and R111) that deviated significantly from HWE showed an excess of heterozygosity. Although Texas and Indian populations were geographically very distant, they exhibited only moderate population subdivision, with an F_ST value of 0.193.     

Relation between pathogenicity and genetic variation within <Emphasis Type="Italic">Plasmodiophora brassicae</Emphasis>

The relation between diversity of pathogenicity on clubroot-resistant (CR) cultivars of Chinese cabbage (Brassica rapa subsp. pekinensis) bred in Japan and DNA polymorphisms in 17 populations of Plasmodiophora brassicae from cruciferous plants was examined by inoculation tests and random amplified polymorphic DNA (RAPD) analysis using 18 arbitrary primers. Four pathotypes (A–D) were identified after inoculation of six CR cultivars of Chinese cabbage in the 17 populations from cruciferous crops. A relatively high level of genetic diversity was also detected among these populations in the RAPD analysis. Although the four pathotypes could not be clearly differentiated using the RAPD data, most populations of three pathotypes had a consistent location on the dendrogram. All pathotype B (virulent on five cultivars except Utage 70) and D (avirulent on all cultivars) populations, which were common in incompatible interactions with cv. Utage 70, were located in a single subcluster. All five pathotype C populations (virulent only on cv. Utage 70) except for one population grouped in another single subcluster. Because four pathotype A populations (virulent on all six cultivars, races 4 and 9) fell in different subclusters, the populations may be genetically polyphyletic. Populations from cruciferous weed Cardamine flexuosa differed remarkably from those from cruciferous crops in pathogenicity on common cultivars of Chinese cabbage and turnip and C. flexuosa, but they grouped in a single cluster with all race 9 populations from crops. Race 9 populations from crops may thus be closely related to populations from the weed rather than to races 1 and 4 from crops.     

Intraspecific diversity within avocado field isolates of <Emphasis Type="Italic">Rosellinia necatrix</Emphasis> from south-east Spain

Fifty-five isolates of Rosellinia necatrix, the cause of common avocado white root rot disease, were collected from south-east Spain and characterised according to their virulence behaviour and their molecular patterns to assess broader levels of genetic diversity. Virulence properties were revealed by in vitro inoculation on avocado plants. Differences in reaction types showed variability among these isolates. No sequence differences were observed when the internal transcribed spacer 1 (ITS1) and ITS2 regions and DNA fragments of the β-tubulin, adenosine triphosphatase and translation elongation factor 1 genes were explored in representive isolates from five virulence groups. Random amplified polymorphic DNA (RAPD) amplifications were also performed for each isolate using 19 random primers. Four of these primers revealed polymorphism among isolates and repetitive and discriminative bands were used to build an unweighted pair group with arithmetic mean tree. However, RAPD clustering showed low stability, and no correlation between RAPD and virulence groups was observed, possibly indicating high levels of sexual recombination.     

Sequence analysis within the RNA 3 of seven Spanish tomato isolates of Parietaria mottle virus (PMoV-T) reveals important structural differences with the parietaria isolates (PMoV)

The genomic regions encoding the putative movement protein (MP), coat protein (CP) and intergenic region (IGR) of seven Spanish isolates of the Parietaria mottle virus which infects tomato plants (PMoV-T) were sequenced. Values for the genetic diversity of the PMoV-T isolates were 0.056, 0.047 and 0.013 for the CP, MP genes and IGR, respectively. Nucleotide and amino acid sequence comparison of the seven PMoV-T isolates with those of PMoV revealed significant differences. All of them had a cytosine deletion at position 1366, also confirmed in an Italian tomato isolate, which involves a start codon for the CP gene different from that for the PMoV sequence, resulting in a CP 16 amino acids shorter than the PMoV CP. The certainty of a cytosine deletion only associated to the tomato isolates or the possibility of a mistake in the PMoV published sequence are the two hypotheses that could explain this difference. Structural motifs highly conserved in Ilarviruses were identified in PMoV-T MP and CP. A stable hairpin structure is proposed for IGR, by the initiation site for subgenomic RNA 4 synthesis. Phylogenetic analysis of CP and MP amino acid sequences showed that Spanish PMoV-T isolates form a separate group from PMoV and other members of the Ilarvirus genus. Comparative analysis with different PMoV isolates including tomato isolates from other regions and isolates from different hosts are necessary to confirm this differentiation.     

西南地区稻瘟病菌群体遗传多样性分析

为明确西南地区稻瘟病菌Magnaporthe grisea(Hebert)Barr群体遗传结构及其多样性水平,选用13对SSR引物对来自18个县(市)的221个稻瘟病菌单孢菌株进行PCR扩增,利用最长距离法和生物学软件进行聚类分析和群体遗传多样性分析。结果显示,13对SSR引物均能扩增出一条大小相同且清晰的条带,多态位点百分率高达100%。221个菌株在0.16相异水平上可划分为13个遗传宗谱,宗谱SCL01含205个菌株,占总菌株数的92.76%,为优势宗谱;宗谱SCL02~SCL013为劣势宗谱,差异极大。在群体水平上,菌源丰富的8个区域稻瘟病菌群体的Nei’s基因多样性指数为0.2133,Shannon信息指数为0.3588,具有丰富的遗传多样性,且群体间差异较大;这8个种群基于UPGMA法大都聚为一类,种群遗传谱系与地理区域分布呈一定相关性,群体遗传多样性均值为0.2518,存在一定的遗传分化,且群体内多样性大于群体间,总遗传变异的59.37%存在于群体内。总体上,西南地区稻瘟病菌群体结构既有明显的优势宗谱,又存在许多复杂多变的特异性小宗谱,具有丰富的遗传多样性,与地理分布关系较为密切。     

Cultural characteristics, pathogenicity and genetic diversity of Fusarium oxysporum isolates from tobacco fields in Spain

Several formae speciales of Fusarium oxysporum are capable to produce disease in tobacco plants. Different authors have classified those isolates as a forma specialis or a race within on the basis of the severity of disease and host specificity. Fusarium wilt of tobacco plant in Extremadura (central Spain) tobacco fields have been recorded in the last years and F. oxysporum was isolated from symptomatic plants. The aim of our study was to characterize these F. oxysporum populations. For this purpose, the in vitro spore production and growth and the virulence (severity of disease) have been tested. Although all isolates behaved as pathogen, the virulence of isolates was different. The differences in growth could not be correlated with other characteristics but the two isolates with scarce spore production have also behaved as the weakest pathogen. We have analyzed intergenic spacer (IGS) region polymorphism of ribosomal DNA and random amplified polymorphic DNA (RAPD) markers to assess the genetic diversity within F. oxysporum isolates. These molecular analyses showed two major groups with different physiological capabilities that could reflect two different lineages. One group was characterized by medium–high sporulation, high virulence and the same IGS-RFLP pattern. The other group was more heterogeneous featuring low–medium sporulation and variable virulence and growth. This first experimental approach to pathogen population could be a good starting point for further studies including non-pathogenic isolates and a larger number of pathogen that could clarify if there are two or more genetic lineages.     

Genetic variation within and between southern Ontario populations of Sclerotinia homoeocarpa

Restriction fragment length polymorphisms (RFLP) of the intergenic spacer region (IGS) of rDNA and random amplified polymorphic DNA (RAPD) markers were used to survey genetic variability among 181 isolates of Sclerotinia homoeocarpa from Ontario and 10 isolates from Japan. RAPD and IGS-RFLP analyses revealed polymorphisms within and between populations of S . homoeocarpa , distinguishing 151 genotypes. Both types of markers gave similar results in phenetic analysis of genetic distances between populations. Cluster analysis showed that Japanese isolates of S. homoeocarpa were genetically distinct from Ontario isolates, demonstrating significant intraspecific differentiation. An average genetic similarity of 0.66 was found between Japanese isolates. Among Ontario isolates, average genetic similarity was 0.86, and genotypic diversity analysis showed that 49.3% of the total genetic variation observed within Ontario populations occurred among individuals within populations compared to 50.7% between populations. Gametic linkage disequilibrium analysis within Ontario populations revealed an average 15.6% significant nonrandom associations between putative RAPD loci, and that half of the populations showed signs of significant linkage disequilibrium. These results suggest that both clonal propagation and recombination events occurred in local populations of S. homoeocarpa . The high level of genetic similarity between populations and the low levels of intraspecific genetic variation may reflect a small founding population for southern Ontario isolates of S. homoeocarpa .     

Extent and pattern of genetic differentiation within and between European populations of Phelipanche ramosa revealed by amplified fragment length polymorphism analysis

The extent and pattern of genetic differentiation between Phelipanche ramosa populations colonising tobacco in different European regions were investigated by amplified fragment length polymorphism (AFLP) analysis, in order to determine levels of variation for tobacco resistance breeding and management programmes. Four different AFLP primer pairs amplified a total of 1050 clear and reproducible bands, of which 962 (91.62%) were polymorphic among the 35 individuals taken from four P. ramosa populations collected in Spain, Italy, Bulgaria and Germany. Cluster analysis based on the AFLP data categorised the plants into distinct groups, in line with their geographical origin, denoting clear genetic differentiation among the four populations. This differentiation was supported by both high bootstrap values and significant results of the analysis of molecular variance. The most divergent population was the one from Bulgaria. The majority of the genetic diversity was attributable to differences among populations (77.80%), as expected from the predominant autogamous behaviour of this species. Populations differed significantly in within-population diversity, as measured by Shannon's information index. The German population presented the lowest genetic diversity and the Italian population harboured the highest level of within-population genetic diversity. There are significant differences in genetic diversity level among the studied P. ramosa populations and clear population-specific genetic diversity structures. These need to be taken into account, together with the potential differences in parasite aggressiveness, when planning breeding and management strategies for P. ramosa control in tobacco cultivation.     

Genetic Diversity in Relation to Sexual and Asexual Reproduction in Populations of Melampsora larici-epitea

Genetic diversity of Melampsora larici-epitea leaf rust from three cultivated stands of the willow Salix viminalis was studied using AFLP polymorphisms at 60 loci. One population was located in Northern Ireland and two in Sweden. Analysis of molecular variance (AMOVA) showed that most of the genetic variation was distributed on a fine scale within the field in all populations. Both Swedish populations displayed a very high genotypic diversity (normalized Shannon's indices of 0.95 and 1.00) and random association among loci. These results suggested that sexual reproduction had an important influence on the Swedish populations. The occurence of the alternate host (larch) adjacent to one of the Swedish rust populations did not affect the genetic diversity. However, severe rust attacks started earlier in the season in this population. The M. larici-epitea population in Northern Ireland was characterized by a low genotypic diversity (normalized Shannon's index = 0.54) and non-random association among loci was indicated by test of multilocus association and by pairwise tests among loci. These results suggested that asexual reproduction had a major effect on the genetic structure of this population, probably because of the overwintering of asexual spores and/or a population bottleneck associated with the annual sexual phase.     

Molecular genetic variability of Italian binucleate <Emphasis Type="Italic">Rhizoctonia</Emphasis> spp. isolates from strawberry

Fifty-eight binucleate Rhizoctonia isolates were collected over six years from strawberry plants displaying symptoms of black root rot in Italy. Almost all isolates were able to produce necrosis on strawberry roots, most of them also showed this ability on faba bean and, with lower frequency, on a crucifer and a cereal crop used in rotation with strawberry in Italy. The sequence alignment of Internal Transcribed Spacer (ITS) regions of 51 binucleate Rhizoctonia were analyzed and compared with a set of eight sequences representative of Rhizoctonia isolate Anastomosis Groups (AG) already found to be pathogenic on strawberry (AG-A, AG-G, AG-I and AG-F). The neighbour-joining tree, based on ITS region sequences, divided Italian strawberry Rhizoctonia isolates into two main clusters corresponding to AG-A and AG-G. The results were confirmed by hyphal anastomosis tests. The clustering obtained with the phylogenetic tree was also confirmed using PCR-Restriction Fragment Length Polymorphism of 28S rDNA to compare some isolates, defined as AG-A and AG-G on the basis of ITS region sequence analysis, with representative AG isolates pathogenic on strawberry. The AG-A and AG-G Rhizoctonia spp. were widespread in Italian strawberry-growing areas, although with different relative frequencies: AG-G was most frequent in northern (latitude 44°N) and AG-A in southern (latitude 39–40°N) Italy. Analysis of MOlecular VAriance, based on geographic location, showed that Rhizoctonia molecular variations between northern and southern Italy accounted for 36.6% of the total, but most of the variations (61%) occurred within each of the four geographical regions from where the isolates originated.     

中国及欧洲各国的苹果蠹蛾种群遗传多样性分析

为研究不同国家和地区苹果蠹蛾Cydia pomonella种群的遗传多样性,对采自我国新疆维吾尔自治区和黑龙江省,以及德国、法国和西班牙等欧洲国家的11个种群共60个苹果蠹蛾个体进行基因组重测序,有效测序样本数为56个,共获得8 455 843个群体单碱基突变（single nucleotide polymorphism,SNP）位点。利用SNP数据进行了苹果蠹蛾群体遗传进化树的构建、主成分分析以及遗传结构分析。结果显示,入侵我国黑龙江省的苹果蠹蛾与德国BD种群更为接近,我国新疆维吾尔自治区与黑龙江省苹果蠹蛾种群有一定的分化,前者种群遗传多样性更低。表明我国东北地区和西北地区的苹果蠹蛾种群具有不同的遗传多样性特征,入侵我国的苹果蠹蛾种群可能来源于欧洲。     

Genetic diversity and differentiation of <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> populations in sunflower

Ninety-six isolates of sunflower Sclerotinia sclerotiorum (Lib.) de Bary from Inner Mongolia (IM) in China, from Canada and the United Kingdom (UK) were sampled to investigate the genetic diversity and structure using Sequence-Related Amplified Polymorphism. A total of 123 polymorphic bands were obtained, ranging in size from 100 to 500 base pairs. The five populations of S. sclerotiorum isolated from the three countries showed various levels of genetic variability. The percentage of polymorphic loci varied from 30.89% in the UK population to 97.56% in the Middle IM population. The values of Shannon index (i) varied from 0.1876 in the UK population to 0.5301 in the West IM population. The heterozygosity of the five geographic populations obtained by estimating allele frequency varied from 12.91% in the UK population to 35.44% in the West IM population. The genetic identity, as indicated by the Nei unbiased identity index, ranged from 0.9744 between populations from Canada and East IM to 0.6477 between populations from West IM and UK. UPGMA cluster analysis using Nei’s genetic distance gave distances ranging from 0.0259 to 0.4343. The rates of gene flow among five geographic populations ranged from 1.5406 between West IM and UK populations to 18.4149 between West IM and Middle IM populations. The four populations from West IM, Middle IM, East IM and Canada were clustered into one subgroup in which the isolates from West and Middle IM belonged to one population, whereas those from East IM and Canada essentially were another population. The isolates from the UK formed a population that was significantly distinct from other populations.