Globally invading populations of the fungal plant pathogen Verticillium dahliae are dominated by multiple divergent lineages.

The spread of aggressive fungal pathogens into previously non-endemic regions is a major threat to plant health and food security. Analyses of the spatial and genetic structure of plant pathogens offer valuable insights into their origin, dispersal mechanisms and evolution, and have been useful to develop successful disease management strategies. Here, we elucidated the genetic diversity, population structure and demographic history of worldwide invasion of the ascomycete Verticillium dahliae, a soil-borne pathogen, using a global collection of 1100 isolates from multiple plant hosts and countries. Seven well-differentiated genetic clusters were revealed through discriminant analysis of principal components (DAPC), but no strong associations between these clusters and host/geographic origin of isolates were found. Analyses of clonal evolutionary relationships among multilocus genotypes with the eBURST algorithm and analyses of genetic distances revealed that genetic clusters represented several ancient evolutionary lineages with broad geographic distribution and wide host range. Comparison of different scenarios of demographic history using approximate Bayesian computations revealed the branching order among the different genetic clusters and lineages. The different lineages may represent incipient species, and this raises questions with respect to their evolutionary origin and the factors allowing their maintenance in the same areas and same hosts without evidence of admixture between them. Based on the above findings and the biology of V. dahliae, we conclude that anthropogenic movement has played an important role in spreading V. dahliae lineages. Our findings have implications for the development of management strategies such as quarantine measures and crop resistance breeding.

Verticillium dahliae race 2-specific PCR reveals a high frequency of race 2 strains in commercial spinach seed lots and delineates race structure.

Two pathogenic races of Verticillium dahliae have been described on lettuce and tomato. Host resistance to race 1 is governed by plant immune receptors that recognize the race 1-specific fungal effector Ave1. Only partial resistance to race 2 exists in lettuce. Although polymerase chain reaction (PCR) assays are available to identify race 1, no complementary test exists to positively identify race 2, except for lengthy pathogenicity assays on host differentials. Using the genome sequences of two isolates of V. dahliae, one each from races 1 and 2, we identified potential markers and PCR primers to distinguish the two races. Several primer pairs based on polymorphisms between the races were designed and tested on reference isolates of known race. One primer pair, VdR2F-VdR2R, consistently yielded a 256-bp amplicon in all race 2 isolates exclusively. We screened DNA from 677 V. dahliae isolates, including 340 from spinach seedlots, with the above primer pair and a previously published race 1-specific primer pair. DNA from isolates that did not amplify with race 1-specific PCRs amplified with the race 2-specific primers. To validate this, two differential lines of lettuce were inoculated with 53 arbitrarily selected isolates from spinach seed and their pathogenicity and virulence were assessed in a greenhouse. The reactions of the differential cultivars strongly supported the PCR data. V. dahliae race structure was investigated in crops in coastal California and elsewhere using primers specific to the two races. All artichoke isolates from California were race 1, whereas nearly all tomato isolates were race 2. Isolates from lettuce, pepper, and strawberry from California as well as isolates from spinach seed from two of four countries comprised both races, whereas only race 2 was observed in cotton, mint, olive, and potato. This highlights the importance of identifying resistance against race 2 in different hosts. The technique developed in this study will benefit studies in ecology, population biology, disease surveillance, and epidemiology at local and global scales, and resistance breeding against race 2 in lettuce and other crops.

Sources of Verticillium dahliae affecting lettuce.

ABSTRACT Since 1995, lettuce in coastal California, where more than half of the crop in North America is grown, has consistently suffered from severe outbreaks of Verticillium wilt. The disease is confined to this region, although the pathogen (Verticillium dahliae) and the host are present in other crop production regions in California. Migration of the pathogen with infested spinach seed was previously documented, but the geographic sources of the pathogen, as well as the impact of lettuce seed sparsely infested with V. dahliae produced outside coastal California on the pathogen population in coastal California remain unclear. Population analyses of V. dahliae were completed using 16 microsatellite markers on isolates from lettuce plants in coastal California, infested lettuce seed produced in the neighboring Santa Clara Valley of California, and spinach seed produced in four major spinach seed production regions: Chile, Denmark, the Netherlands, and the United States (Washington State). California produces 80% of spinach in the United States and all seed planted with the majority infested by V. dahliae comes from the above four sources. Three globally distributed genetic populations were identified, indicating sustained migration among these distinct geographic regions with multiple spinach crops produced each year and repeated every year in coastal California. The population structure of V. dahliae from coastal California lettuce plants was heavily influenced by migration from spinach seed imported from Denmark and Washington. Conversely, the sparsely infested lettuce seed had limited or no contribution to the Verticillium wilt epidemic in coastal California. The global trade in plant and seed material is likely contributing to sustained shifts in the population structure of V. dahliae, affecting the equilibrium of native populations, and likely affecting disease epidemiology.

Population biology of fungal plant pathogens.

Studies of the population genetics of fungal and oomycetous phytopathogens are essential to clarifying the disease epidemiology and devising management strategies. Factors commonly associated with higher organisms such as migration, natural selection, or recombination, are critical for the building of a clearer picture of the pathogen in the landscape. In this chapter, we focus on a limited number of experimental and analytical methods that are commonly applied in population genetics. At first, we present different types of qualitative and quantitative traits that could be identified morphologically (phenotype). Subsequently, we describe several molecular methods based on dominant and codominant markers, and we provide our assessment of the advantages and shortfalls of these methods. Third, we discuss various analytical methods, which include phylogenies, summary statistics as well as coalescent-based methods, and we elaborate on the benefits associated with each approach. Last, we develop a case study in which we investigate the population structure of the fungal phytopathogen Verticillium dahliae in coastal California, and assess the hypotheses of transcontinental gene flow and recombination in a fungus that is described as asexual.

Analysis of Verticillium dahliae Suggests a Lack of Correlation Between Genotypic Diversity and Virulence Phenotypes.

Verticillium dahliae causes severe wilt and recurring losses in numerous agricultural and ornamental hosts worldwide. Two virulence phenotypes (races) have been identified based on the Ve resistance gene and its homologs but their distribution and evolutionary history are unknown. Sequence analyses of the intergenic spacer of the ribosomal DNA and amplified fragment length polymorphism markers suggested an absence of correlation between genotypic diversity and virulence phenotypes. Additionally, both race 1 and 2 phenotypes were isolated in various geographic regions and hosts. Sustained levels of migration of both virulence phenotypes among various geographic regions were evident, and the study also suggested that both virulence phenotypes infect a variety of hosts, regardless of the availability of resistant cultivars. Given the high genotypic diversity observed in V. dahliae, more than the two known virulence phenotypes may be present in nature but not yet identified because of the current lack of sources of resistance other than the Ve gene and its homologs. The inclusion of various genotypes exhibiting the same virulence phenotype may greatly improve the long-term effectiveness of resistance to race 2 of V. dahliae regardless of the host. This study also confirms the transcontinental gene flow and high genotypic diversity of V. dahliae affecting lettuce in coastal California regardless of the molecular markers employed.

Population analyses of the vascular plant pathogen Verticillium dahliae detect recombination and transcontinental gene flow.

The fungal pathogen Verticillium dahliae has resulted in significant losses in numerous crops in coastal California, but lettuce remained unaffected until the mid-1990s. Since then outbreaks have decimated entire fields, but the causes of this sudden susceptibility of lettuce remain elusive. The population structure of V. dahliae isolated from coastal California (n=123) was investigated with 22 microsatellite markers, and compared with strains from tomato in central California (n=60), spinach seed imported from Washington State and Northern Europe (n=43), and ornamentals from Wisconsin (n=17). No significant differentiation was measured among hosts in coastal California or with the spinach and Wisconsin ornamental sampling groups. In contrast, the tomato sampling group was significantly differentiated. Significant gene flow was measured among the various geographic and host sampling groups, with the exception of tomato. Evidence of recombination in V. dahliae was identified through gametic disequilibrium and an exceedingly high genotypic diversity. The high incidence of V. dahliae in spinach seed and high planting density of the crop are sources of recurrent gene flow into coastal California, and may be associated with the recent outbreaks in lettuce.

Diversity, pathogenicity, and management of verticillium species.

The genus Verticillium encompasses phytopathogenic species that cause vascular wilts of plants. In this review, we focus on Verticillium dahliae, placing emphasis on the controversy surrounding the elevation of a long-spored variant as a new species, recent advances in the analysis of compatible and incompatible interactions, highlighted by the use of strains expressing fluorescent proteins, and the genetic diversity among Verticillium spp. A synthesis of the approaches to explore genetic diversity, gene flow, and the potential for cryptic recombination is provided. Control of Verticillium wilt has relied on a panoply of chemical and nonchemical strategies, but is beset with environmental or site-specific efficacy problems. Host resistance remains the most logical choice, but is unavailable in most crops. The genetic basis of resistance to Verticillium wilt is unknown in most crops, as are the subcellular signaling mechanisms associated with Ve-mediated, race-specific resistance. Increased understanding in each of these areas promises to facilitate management of Verticillium wilts across a broad range of crops.

Timing Fungicide Applications for Managing Sclerotinia Stem Rot of Potato.

Fungicides were applied to potato foliage at row closure (between rows) and at full bloom of primary inflorescences to control Sclerotinia stem rot during replicated trials in 2003, 2004, and 2005. Application at row closure followed labeled recommendations from manufacturers. Incidence of Sclerotinia stem rot did not vary significantly among fungicides when full labeled rates of thiophanate-methyl, fluazinam, and boscalid were applied at full bloom of primary inflorescences. Incidence of Sclerotinia stem rot was significantly less when fungicides thiophanatemethyl, fluazinam, or boscalid were applied to potato foliage at full bloom of primary inflorescences than at row closure or when fungicides were not applied in 2004 and 2005, and when thiophanate-methyl or fluazinam was applied to potato foliage at full bloom of primary inflorescences than at row closure or when fungicides were not applied in 2003. Mean percentage of control for the fungicides combined, relative to the nontreated control, was 43, 48, and 20% in 2003, 2004, and 2005, respectively, when application was made at row closure; whereas, it was 77, 83, and 80% in 2003, 2004, and 2005, respectively, when application was at full bloom of primary inflorescences. Mean disease incidences of infected stem were significantly less when fluazinam was applied at 100% bloom of primary inflorescences than at 20% drop of blossoms from primary inflorescences in 2004 and 2005. In summary, control of Sclerotinia stem rot was significantly better when fungicides were applied at full bloom of primary inflorescences than at row closure during all 3 years of the study.