Phylogenetic relationships between the lettuce root rot pathogen Fusarium oxysporum f. sp. lactucae races 1, 2, and 3 based on the sequence of the intergenic spacer region of its ribosomal DNA

The genetic relationship between the vegetative compatibility groups (VCGs) and between physiological races of Fusarium oxysporum f. sp. lactucae (FOL), the causal pathogen of lettuce root rot, was determined by analyzing the intergenic spacer (IGS) region of its ribosomal DNA. A total of 29 isolates containing a type strain were tested: 24 Japanese isolates, 2 Californian isolates, and 3 Italian isolates. Three races (races 1, 2, and 3) were found in Japan, and race 1 was also distributed in California and Italy. Races 1, 2, and 3 each belonged to a distinct VCG: VCG-1, VCG-2, and VCG-3 (VCG-3-1, VCG-3-3), respectively. Phylogenetic (neighbor-joining) analysis of the IGS sequences revealed that races 1, 2, and 3 coincided with three phylogenetic groups (PG): PG-1, PG-2, and PG-3, respectively. These results indicate that the three races are genetically quite different and have a strong correlation with VCGs and phylogenetic groupings. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession no. AB195218     

Phylogenetic analysis of Verticillium dahliae vegetative compatibility groups

The evolutionary relationships among Verticillium dahliae vegetative compatibility (VCG) subgroups VCG1A, VCG1B, VCG2A, VCG2B, VCG4A, VCG4B, and VCG6 were investigated by parsimony analysis of amplified fragment length polymorphism (AFLP) fingerprints and sequences of six DNA regions (actin, beta-tubulin, calmodulin, and histone 3 genes, the ITS 1 and 2 regions of the rDNA, and a V. dahliae-specific sequence), using 101 isolates of diverse host and geographic origin. Polymorphisms in gene sequences among isolates of different VCGs were very low and individual gene genealogies provided very little resolution at the VCG level. The combined analysis of all DNA regions differentiated all VCG subgroups except for isolates in VCG1A and VCG1B. VCG clonal lineages in V. dahliae and evolutionary relationships among them were resolved independently by analyses of AFLP fingerprints, multiple gene genealogies, and the combined data set of AFLP fingerprinting and multiple gene genealogies. Two main lineages (I and II) were identified with lineage II comprising two closely related subgroups of VCGs. Lineage I included VCG1A, VCG1B, and VCG2B334; and lineage II included, VCG2A and VCG4B (subclade 1); and VCG2B824, VCG4A, and VCG6 (subclade 2). VCG subgroups were monophyletic except for VCG2B that appeared polyphyletic. Limiting the parsimony analysis either to AFLP fingerprints or DNA sequences would have obscured intra-VCG differentiation. Therefore, the dual approach represented by the independent and combined analyses of AFLP fingerprints and DNA sequences was a highly valuable method for the identification of phylogenetic relationships at the intraspecific level in V. dahliae.     

Mitochondrial haplotype analysis for differentiation of isolates of Phytophthora cinnamomi

Although Phytophthora cinnamomi is heterothallic, there are few instances of successful crossing in laboratory experiments, and analysis of field populations indicates a clonally reproducing population. In the absence of sexual recombination, the ability to monitor mitochondrial haplotypes may provide an additional tool for identification of clonal isolates and analysis of population structure. To determine mitochondrial haplotypes for this species, seven mitochondrial loci spanning a total of 6,961 bp were sequenced for 62 isolates representing a geographically diverse collection of isolates with A1 and A2 mating type. Three of the regions were primarily intergenic regions between trnG and rns, rns and nad3, and nad6 and cox1, while the remaining loci spanned cox2, nad9, rps10, and secY coding regions and some of the flanking spacer regions. In total, 45 mitochondrial haplotypes were identified (75% of the total isolates examined) with differences due to single-nucleotide polymorphisms (SNPs, totaling 152 bp) and length mutations (17 indels >2 bp representing a total of 910 bp in length). SNPs were the predominate mutation in the four coding regions and their flanking intergenic regions, while both SNPs and length mutations were observed in the three primarily intergenic regions. Some of the length mutations in these regions were due to addition or loss of unique sequences while others were due to variable numbers of subrepeats (in the trnG-rns region, there were 3 to 12 copies of a 24-bp subrepeat sequence that differentiated 17 haplotypes). Network analysis of the haplotypes identified eight primary clades, with the most divergent clade representing primarily A1 isolates collected from Papua New Guinea. The isolate grouping in the network corresponded to mating type and previously published isozyme classifications, with three exceptions: a haplotype representing an A1 mating type (H29) was placed well within the A2 mating type haplotype grouping, one haplotype (H26) had isolates with two isozyme classifications, and one isozyme group was represented on separate network clades, suggesting that recombination has occurred in the past. Among the 62 isolates examined, several examples were identified of isolates recovered from different geographic regions having the same mitochondrial haplotype, suggesting movement of isolates via plant material. Analysis of the data set to determine whether fewer loci could be sequenced to classify haplotypes indicated that the trnG-rns and rns-nad6 loci would classify 87% of the haplotypes identified in this study, while additional sequencing of the nad9 or secY loci would further differentiate the remaining six haplotypes. Based on conservation of gene order in Phytophthora spp., the trnG-rns locus should be useful for mitochondrial haplotype classification in other species, as should the cox2, nad9, rps10, and secY loci. However, the rns-nad3 and nad6-cox1 loci span regions that can have a different gene order in some Phytophthora spp.     

Molecular Variability Within and Among Verticillium dahliae Vegetative Compatibility Groups Determined by Fluorescent Amplified Fragment Length Polymorphism and Polymerase Chain Reaction Markers

ABSTRACT A degree of genetic diversity may exist among Verticillium dahliae isolates within vegetative compatibility groups (VCGs) that bears phytopathological significance and is worth investigating using molecular tools of a higher resolution than VCG characterization. The molecular variability within and among V. dahliae VCGs was studied using 53 artichoke isolates from eastern-central Spain, 96 isolates from cotton, 7 from cotton soil, and 45 from olive trees in countries of the Mediterranean Basin. Isolates were selected to represent the widest available diversity in cotton- and olive-defoliating (D) and -nondefoliating (ND) pathotypes, as well as for VCG. The VCG of 96 cotton and olive isolates was determined in this present study. Molecular variability among V. dahliae isolates was assessed by fluorescent amplified fragment length polymorphism (AFLP) analysis and by polymerase chain reaction (PCR) assays for DNA fragments associated with the D (462 bp) and ND (824 bp) pathotypes, as well as a 334-bp amplicon associated with D pathotype isolates but also present in some VCG2B isolates. Isolates from cotton were in VCG1A, VCG1B, VCG2A, VCG2B, and VCG4B and those from olive trees were in VCG1A, VCG2A, and VCG4B. Artichoke isolates included representatives of VCG1A, VCG2A, VCG2B (including a newly identified VCG2Ba), and VCG4B. AFLP data were used to generate matrixes of genetic distance among isolates for cluster analysis using the neighbor-joining method and for analysis of molecular variance. Results demonstrated that V. dahliae isolates within a VCG subgroup are molecularly similar, to the extent that clustering of isolates correlated with VCG subgroups regardless of the host source and geographic origin. VCGs differed in molecular variability, with the variability being highest in VCG2B and VCG2A. For some AFLP/VCG subgroup clusterings, V. dahliae isolates from artichoke grouped in subclusters clearly distinct from those comprising isolates from cotton and olive trees. In addition, VCG2B isolates from artichoke formed two distinct clusters that correlated with PCR markers of 334 bp (VCG2B(334)) or 824 bp (VCG2B(824)). Artichoke isolates in the VCG2B(334)/2beta(334) cluster were molecularly similar to isolates of VCG1A. The molecular difference found among artichoke isolates in VCG2B correlates with virulence of isolates to artichoke and cotton cultivars demonstrated in a previous study.     

Origin of Race 3 of Fusarium oxysporum f. sp. lycopersici at a Single Site in California

ABSTRACT Thirty-nine isolates of Fusarium oxysporum were collected from tomato plants displaying wilt symptoms in a field in California 2 years after F. oxysporum f. sp. lycopersici race 3 was first observed at that location. These and other isolates of F. oxysporum f. sp. lycopersici were characterized by pathogenicity, race, and vegetative compatibility group (VCG). Of the 39 California isolates, 22 were in VCG 0030, 11 in VCG 0031, and six in the newly described VCG 0035. Among the isolates in VCG 0030, 13 were race 3, and nine were race 2. Of the isolates in VCG 0031, seven were race 2, one was race 1, and three were nonpathogenic to tomato. All six isolates in VCG 0035 were race 2. Restriction fragment length polymorphisms (RFLPs) and sequencing of the intergenic spacer (IGS) region of rDNA identified five IGS RFLP haplotypes, which coincided with VCGs, among 60 isolates of F. oxysporum from tomato. Five race 3 isolates from California were of the same genomic DNA RFLP haplotype as a race 2 isolate from the same location, and all 13 race 3 isolates clustered together into a subgroup in the neighbor joining tree. Collective evidence suggests that race 3 in California originated from the local race 2 population.     

Region-wide analysis of genetic diversity in Verticillium dahliae populations infecting olive in southern Spain and agricultural factors influencing the distribution and prevalence of vegetative compatibility groups and pathotypes

Severity of Verticillium wilt in olive trees in Andalusia, southern Spain is associated with the spread of a highly virulent, defoliating (D) Verticillium dahliae pathotype of vegetative compatibility group 1A (VCG1A) but the extent of this spread and the diversity of the pathogen population have never been documented. VCG typing of 637 V. dahliae isolates from 433 trees in 65 orchards from five olive-growing provinces in Andalusia indicated that 78.1% were of VCG1A, 19.8% of VCG2A, 0.6% of VCG2B, 1.4% of VCG4B, and one isolate was heterokaryon self-incompatible. A single VCG prevailed among isolates within most orchards but two and three VCGs were identified in 12 and 3 orchards, respectively, with VCG1A+VCG2A occurring in 10 orchards. VCG1A was the predominant VCG in the three most important olive-growing provinces, and was almost as prevalent as VCG2A in another one. Molecular pathotyping of the 637 isolates using specific polymerase chain reaction assays indicated that VCG1A isolates were of the D pathotype whereas isolates of VCG2A, -2B, and -4B were of the less virulent nondefoliating (ND) pathotype. The pathotype of isolates correlated with the disease syndrome affecting sampled trees. Only three (seq1, seq2, and seq4) of the seven known sequences of the V. dahliae-specific 539- or 523-bp amplicon were identified among the 637 isolates. Distribution and prevalence of VCGs and seq sequences among orchards indicated that genetic diversity within olive V. dahliae in Andalusia is higher in provinces where VCG1A is not prevalent. Log-linear analysis revealed that irrigation management, source of irrigation water, source of planting stock, and cropping history of soil were significantly associated with the prevalence of VCG1A compared with that of VCG2A. Multivariate analyses using a selected set of agricultural factors as variables allowed development of a discriminant model for predicting the occurrence of D and ND pathotypes in the area of the study. Blind tests using this model correctly indentified the V. dahliae pathotype occurring in an orchard. The widespread occurrence and high prevalence of VCG1A/D pathotype in Andalusia have strong implications for the management of the disease.     

Genetic and Virulence Diversity in Verticillium dahliae Populations Infecting Artichoke in Eastern-Central Spain

ABSTRACT Severe Verticillium dahliae attacks have occurred in artichoke crops in the Comunidad Valenciana region of eastern-central Spain since the late 1990s. Knowledge of genetic and virulence diversity in the pathogen population is a key factor for the management of the disease through disease risk assessment as well as development and use of resistant cultivars. V. dahliae isolates from artichoke (109 isolates) and cotton (three isolates) in that region were characterized by vegetative compatibility grouping (VCG), and specific polymerase chain reaction assays using three sets of primer pairs that differentiate the cotton-defoliating (D) and -nondefoliating (ND) V. dahliae pathotypes. In all, 35 and 39 V. dahliae isolates representative of the identified VCGs and geographic origins were tested for virulence to artichoke cvs. Nun 6374 and Nun 9444, and cotton cv. Acala SJ-2, respectively. Four VCGs were identified among 107 artichoke isolates, and 2 isolates were heterokaryon self-incompatible: VCG1A (one isolate), VCG2A (31 isolates), VCG2B (72 isolates), and VCG4B (three isolates). The three cotton isolates were VCG1A. Isolates in VCG2B were distributed across the region and were the most prevalent isolates in the northern part. Conversely, 83.9% of isolates in VCG2A were recovered from the southern part of the region. Two subgroups of isolates were identified in VCG2B based on heterokaryon compatibility with either international or local tester isolates, which further showed diversity in the amplification of 334- and 824-bp DNA fragments which are markers of the D and ND pathotypes, respectively. Virulence of isolates to artichoke and cotton correlated with VCG but the pattern of correlation varied with the host. VCG1A isolates from artichoke and cotton induced defoliation in cotton but not in artichoke. Collectively, isolates of VCG2B and VCG4B were the most virulent and isolates of VCG1A or HSI were the least virulent to artichoke; but isolates of VCG1A were more virulent to cotton than those of any other VCG. Also, molecular subgrouping in VCG2B determined by amplification of the 334- and 824-bp markers correlated with virulence of isolates to the two hosts tested.     

A PCR-based 'molecular tool box' for in planta differential detection of Verticillium dahliae vegetative compatibility groups infecting artichoke

A multiplex-nested-PCR procedure was developed for in planta detection of Verticillium dahliae isolates infecting artichoke and assessment of their vegetative compatibility groups (VCGs). PCR markers were identified and assigned to V. dahliae VCGs, including: i) a 334 bp marker amplified from VCG1A or VCG2B³³⁴ isolates; ii) a 688 bp marker amplified from VCG2A or VCG4B isolates; and iii) a 688 bp and a 964 bp PCR marker amplified from VCG2B⁸²⁴ isolates. The infecting V. dahliae VCGs were identified in artichoke tissues according to specific patterns of amplified markers after two rounds of PCR. The PCR-based 'molecular tool box' was first optimized using DNA extracted from artichoke plants artificially inoculated with isolates representative of known VCGs. Thereafter, the efficiency of the molecular procedure was tested using DNA extracted from naturally-infected artichoke plants showing a range of symptom severity as well as from symptomless plants. The novel multiplex-nested-PCR assay was clearly superior in detecting the pathogen compared to conventional isolation procedures, and in addition was informative about the VCGs. Moreover, the PCR method allowed the detection and VCG identification of V. dahliae infections in symptomless but infected plants, which had yielded false negatives when checked by microbiological isolation procedures. This 'molecular tool box' has uncovered the presence of several V. dahliae VCGs infecting the same artichoke plants in the Comunidad Valenciana Region. In addition, it is useful for genetic and pathogenicity diversity studies of V. dahliae populations infecting artichoke, and may help in predicting the severity of verticillium wilt epidemics.     

Vegetative Compatibility Groupings of Verticillium dahliae from Cotton in Mainland China

One hundred and fourteen isolates of Verticillium dahliae obtained from cotton and eggplant in mainland China were successfully assigned to two vegetative compatibility groups (VCGs) except for one self-incompatible isolate. Eleven isolates were strongly compatible with T9, the tester strain of the cotton defoliating pathotype, forming a linear growth of wild type with abundant microsclerotia and dense mycelia between compatible nitrate-nonutilizing mutants. The remaining 102 isolates were grouped into the non-defoliating VCG2, although the strength of the reaction varied; some isolates were strongly compatible with the tester strain while others were only slightly compatible. All VCG1 isolates including T9 showed the same defoliating symptom in greenhouse inoculation tests. This study confirmed the presence of the defoliating pathotype (VCG1) of V. dahliae in mainland China.     

Aggressiveness of Verticillium dahliae isolates from different vegetative compatibility groups to potato and tomato

Aggressiveness of Verticillium dahliae isolates from three vegetative compatibility groups (VCGs) was tested on potato and tomato. VCG4B was the most aggressive to potato and VCG2A was the most aggressive to tomato; VCG2B was the least aggressive to both potato and tomato. In potato, disease incidence, symptom severity and colonization index of stem segments were significantly higher in plants inoculated with VCG4B isolates than in those inoculated with VCG2B and VCG2A isolates. Inoculation with VCG4B and VCG2A decreased plant height and fresh weight more than inoculation with VCG2B. In tomato, VCG2A caused significantly more severe symptoms than either VCG4B or VCG2B. The colonization index in tomato plants inoculated with VCG2A was also significantly higher than in those inoculated with VCG4B and VCG2B. Similar patterns of relative aggressiveness were observed in potato and tomato when the pathogenicity of isolates of various VCGs, each originating from a specific host (cotton, potato or eggplant), was compared.     

Genetic Relationships Among Verticillium dahliae Isolates from Cotton in Greece Based on Vegetative Compatibility

Vegetative compatibility groups of a collection of 71 Greek Verticillium dahliae isolates obtained from cotton plants were tested. Nit mutants were generated from single spore wild strains by selecting chlorate-resistant sectors on minimal medium amended with potassium chlorate, 25g/l. These mutants were tested against tester strains from the USA and Greece of the previously described VCGs 1, 2, 3 and 4. Forty-six of 71 isolates belonged to VCG2, because they were able to anastomose with the testers of this group, two isolates belonged to VCG4 and one to VCG1, while the 22 remaining strains could not be assigned to any of the identified VCGs. Our data demonstrated that wilt of cotton is caused only by V. dahliae in Greece, and VCG2 is the most commonly detected VCG. Some strains were found to be more virulent to cotton than other strains from the same VCG. This is the first report of VCG1 of Verticillium in Greece.     

Vegetative Compatibility Groups of Verticillium dahliae in Israel: Their Distribution and Association with Pathogenicity

A collection of 565 isolates of Verticillium dahliae, recovered between 1992 and 1997 from 13 host plant species and soil at 47 sites in Israel, was tested for vegetative compatibility using nitrate-nonutilizing (nit) mutants. Three vegetative compatibility groups (VCGs) were found and identified as VCG2A (28 isolates), VCG2B (158 isolates), and VCG4B (378 isolates) by using international reference strains. One isolate was heterokaryon self-incompatible. Of the VCG2B isolates, 92% were recovered from the northern part of Israel and 90% of VCG4B isolates were recovered from the south, with some overlap in the central region. Isolates of the minor group VCG2A were geographically scattered among the two major VCGs. Isolates of the same VCG resembled one another more than isolates from different VCGs based on colony and microsclerotial morphology, temperature responses, and, partially, pathogenicity. Different pathotypes were defined among 60 isolates tested, using cotton (cv. Acala SJ-2) and eggplant (cv. Black Beauty) as differentials. All isolates in VCG2A and 86% of the isolates in VCG4B, irrespective of their origin, induced weak to moderate symptoms on cotton and moderate to severe symptoms on eggplant and were similar to the previously described cotton nondefoliating patho-type. In contrast, all cotton isolates in VCG2B caused severe foliar symptoms, stunting, and often death, but little or no defoliation of inoculated cotton plants. These were defined as a cotton defoliating-like pathotype and induced only weak to moderate symptoms on eggplant. We concluded that vegetative compatibility grouping of V. dahliae in Israel is closely associated with specific pathogenicity and other phenotypic traits.     

Host Range Specificity in Verticillium dahliae

ABSTRACT Verticillium dahliae isolates from artichoke, bell pepper, cabbage, cauliflower, chili pepper, cotton, eggplant, lettuce, mint, potato, strawberry, tomato, and watermelon and V. albo-atrum from alfalfa were evaluated for their pathogenicity on all 14 hosts. One-month-old seedlings were inoculated with a spore suspension of about 10(7) conidia per ml using a root-dip technique and incubated in the greenhouse. Disease incidence and severity, plant height, and root and shoot dry weights were recorded 6 weeks after inoculation. Bell pepper, cabbage, cauliflower, cotton, eggplant, and mint isolates exhibited host specificity and differential pathogenicity on other hosts, whereas isolates from artichoke, lettuce, potato, strawberry, tomato, and watermelon did not. Bell pepper was resistant to all Verticillium isolates except isolates from bell pepper and eggplant. Thus, host specificity exists in some isolates of V. dahliae. The same isolates were characterized for vegetative compatibility groups (VCGs) through complementation of nitrate nonutilizing (nit) mutants. Cabbage and cauliflower isolates did not produce nit mutants. The isolate from cotton belonged to VCG 1; isolates from bell pepper, eggplant, potato, and tomato, to VCG 4; and the remaining isolates, to VCG 2. These isolates were also analyzed using the random amplified polymorphic DNA (RAPD) method. Forty random primers were screened, and eighteen of them amplified DNA from Verticillium. Based on RAPD banding patterns, cabbage and cauliflower isolates formed a unique group, distinct from other V. dahliae and V. albo-atrum groups. Minor genetic variations were observed among V. dahliae isolates from other hosts, regardless of whether they were host specific or not. There was no correlation among pathogenicity, VCGs, and RAPD banding patterns. Even though the isolates belonged to different VCGs, they shared similar RAPD profiles. These results suggest that management of Verticillium wilt in some crops through crop rotation is a distinct possibility.     

Vegetative compatibility groups within Verticillium dahliae isolates from different hosts in Greece

Forty-four isolates of Verticillium dahliae obtained from different diseased hosts were tested by vegetative compatibility group (VCG) analysis to investigate their genetic relatedness and correlate the results with four VCGs (1, 2, 3, 4) previously described. Based on complementarity of nit mutants, only three VCGs were identified from the Greek isolates. Seventeen isolates were assigned to VCG 2 (A or B), two to VCG 3 and eight to VCG 4 (A or B). The 17 remaining isolates could not be grouped to any of the three VCGs. All isolates belonging to a distinct VCG complemented strongly with at least one of the two tester strains of that group, or with several strains of the Greek collection belonging to that VCG.     

Evidence for Natural Selection in the Mitochondrial Genome of Mycosphaerella graminicola

ABSTRACT Pathogenicity assays were combined with restriction fragment length polymorphism (RFLP) markers in the mitochondrial and nuclear genomes to compare Mycosphaerella graminicola populations adapted to bread wheat (Triticum aestivum) and durum wheat (T. turgidum) in the Mediterranean Basin. The majority of isolates had unique nuclear DNA fingerprints and multilocus haplotypes. Only six mitochondrial DNA (mtDNA) haplotypes were identified among 108 isolates assayed. There were minor differences in frequencies of alleles at nuclear RFLP loci between the two host-adapted populations, but differences in the frequencies of mtDNA haplotypes were highly significant (P < 0.0001). mtDNA haplotype 1 dominated on the isolates adapted to bread wheat, and its frequency was twice as high as for the isolates adapted to durum wheat. mtDNA haplotype 4, which contained a unique approximately 3-kb insertion, was detected only in isolates showing specificity toward durum wheat and was the dominant haplotype on this species. We propose that the low mitochondrial diversity in this pathogenic fungus is due to a selective sweep and that differences in the frequencies of mtDNA haplotypes between the two host-adapted populations were due to natural selection according to host species.     

Mitochondrial DNA Variability in Fusarium Proliferatum (Gibberella Intermedia)

Restriction fragment length polymorphisms (RFLP) were used to assess genetic diversity of mitochondrial DNA (mtDNA) among 184 isolates of Fusarium proliferatum recovered from maize, asparagus, palms and reed. All strains were cross-fertile with standard mating type tester strains of Gibberella intermedia. Sixteen mitochondrial haplotypes were identified following digestion of DNAs with HaeIII, with seven, seven, five and six different haplotypes from maize, asparagus, palms and reed, respectively. Four haplotypes (I, III, IV and VII) were found on more than one host. Of these four, haplotype I was dominant on maize, representing 71% of the isolates. The banding patterns for haplotypes III and IV were >90% similar to the banding pattern of haplotype I. Haplotypes I, III and IV accounted for 87% of the isolates from maize, but were less common on the other hosts, accounting for 70%, 52% and 33% of the isolates from asparagus, palms and reed, respectively. Thirteen of the 16 haplotypes were recovered from only a single host plant species. When comparing the banding patterns and frequencies of these haplotypes, at least five were recovered at a higher frequency from one host relative to the others. Our results suggest that mtDNA RFLP analysis is a useful indicator of genetic divergence in Fusarium proliferatum.     

Vegetative compatibility groups and parasexual segregation in Colletotrichum acutatum isolates infecting different hosts

Heterokaryosis is an important mechanism which provides genetic variability increase in filamentous fungi. In order to assess the diversity of vegetative compatibility reactions existing among Colletotrichum acutatum isolates derived from different hosts, complementary nit mutants of each isolate were obtained and paired in all possible combinations. Vegetative compatibility groups (VCG) were identified among the isolates according to their ability to form viable heterokaryons. Seven VCGs were identified among the isolates, one of which contained isolates from different hosts. VCGs 2 and 6 contained two and three members, respectively; VCG-3 contained four members, and four VCGs (1, 4, 5, and 7) contained a single one. This study shows, for the first time, the isolation and the parasexual segregation of a heterozygous diploid sector derived from the heterokaryon formed with nit mutants from VCG-6. Diploid, named DE-3, showed nit+ phenotype and growth rate similar to the parental wild isolate. When inoculated in the presence of the haploidizing agent benomyl, the diploid strain produced parasexual haploid segregants exhibiting the nit phenotypes of the crossed mutants. Since viable heterokaryons and diploid may be formed among vegetative compatible isolates of C. acutatum, this study suggests that the parasexual cycle may be an alternative source of genetic variability in C. acutatum isolates.     

Genetic variation and population structure of Fusarium oxysporum f.sp. vasinfectum in Australia

Genetic variation among 348 isolates of Fusarium oxysporum f.sp. vasinfectum (Fov) collected from diseased cotton plants in 31 fields in six cotton-growing regions in New South Wales and Queensland in 2002 and 2004 was analysed using amplified fragment length polymorphisms (AFLPs). Twenty-eight haplotypes were identified based on 146 polymorphic bands generated with four Eco RI and Mse I and four Hind III and Mse I primer combinations. The haplotypes separated into two distinct groups (37% similarity), with 21 in group I and seven in group II. The two unique vegetative compatibility groups of Fov known to occur in Australia (VCG 01111 and VCG 01112) were correlated to the two AFLP groups, with both VCG 01111 reference isolates being included in group I and both VCG 01112 reference isolates in group II. Group I was widespread, occurring in all regions sampled and all but one of the fields, while group II was limited to three fields in the Boggabilla region. Group I was further divided into two subgroups. The two haplotypes in subgroup I-B (I-20 and I-21) may represent the emergence of a new form of Fov based on their marked genetic discrimination from the subgroup I-A haplotypes. No spatial population differentiation was discernible at the national level, as only 3·9% of total genetic variation was attributed to differences among regions ( P =  0·4868). When each region was analysed separately, clear differentiation was found in the Boggabilla region, with 86·3% of total genetic variation resulting from differences among fields ( P <  0·0001).     

Genetic and pathogenic characterization of <Emphasis Type="Italic">Verticillium dahliae</Emphasis> isolates from eggplant in Turkey

During 2005 to 2007, eggplant fields in 19 provinces from three different regions (western, southern and southeastern Anatolia regions) of Turkey were surveyed for Verticillium wilt. Sixty-seven isolates of Verticillium dahliae from wilted eggplants were collected and used for vegetative compatibility analysis using nitrate non-utilizing mutants and reference tester strains of vegetative compatibility groups (VCGs) 1A, 2A, 2B, 3, 4A and 4B. Among all isolates, 33 (12 from western, 15 from southern and six from southeastern Anatolia) were assigned to VCG2B, 23 (four from western, eight from southern and 11 from southeastern Anatolia) to VCG2A, six (four from southern, one from western, and one from southeastern Anatolia) to VCG4B and five (one from western, one from southern and three from southeastern Anatolia) to VCG1A, whereas VCG3 and VCG4A were not defined among isolates. In order to test if there is a correlation between VCG and pathogenicity in V. dahliae, pathogenicity of 30 isolates, representing the four multimember VCGs, were tested on Solanum melongena cvs. ‘Kemer’ and ‘Aydın Siyahı’ in an unheated greenhouse. All isolates were found to be pathogenic on both cultivars and there was no difference in susceptibility between the two cultivars. VCG4B isolates collectively led to higher vascular discoloration index (VDI) on both cultivars and higher disease severity index (DSI) on ‘Kemer’ compared with other VCGs. Similarly, VCG1A caused lower VDI on both cultivars and lower DSI on ‘Kemer’. Isolates within each of VCGs 1A, 2A and 4B caused similar VDI on both cultivars. Isolates of VCG2B were found to vary in their VDI values on both cultivars. To the best of our knowledge, the present study is the first report of natural infections of eggplant by VCG1A.     

Population Genetic Analysis Corroborates Dispersal of Fusarium oxysporum f. sp. radicis-lycopersici from Florida to Europe

ABSTRACT Fusarium oxysporum isolates from tomato plants displaying crown and root rot symptoms were collected in central and southern Florida and analyzed using vegetative compatibility grouping (VCG) and nuclear restriction fragment length polymorphism (RFLP) data. VCG 0094 of F. oxysporum f. sp. radicis-lycopersici, previously known only from northwestern Europe, was predominant among 387 isolates assessed. In addition, two newly described VCGs (0098 and 0099) were detected at low frequencies. Floridian VCG 0094 isolates displayed a continuum of compatibilities, which is in contrast to the three distinct subgroups previously identified among European VCG 0094 isolates. RFLP haplotypes were constructed using one repetitive and three low-copy probes. Population subdivision of VCG 0094 from various Floridian counties and from northwestern Europe (Belgium, the Netherlands, and the United Kingdom) was evaluated by analysis of molecular variance. A "natural" population structure was revealed, differentiating populations from the east and west coasts of Florida. In addition, isolates from Europe were statistically indistinguishable from the Palm Beach County, FL, population. Furthermore, gene diversity among Palm Beach County VCG 0094 isolates was more than five times greater than among European isolates. Results from both VCG and RFLP analyses strongly support the inference that the European VCG 0094 constitutes a founder population that resulted from intercontinental migration of a few isolates from Palm Beach County, FL.