The vascular wilt fungi Verticillium dahliae and V. albo-atrum infect over 200 plant species, causing billions of dollars in annual crop losses. The characteristic wilt symptoms are a result of colonization and proliferation of the pathogens in the xylem vessels, which undergo fluctuations in osmolarity. To gain insights into the mechanisms that confer the organisms' pathogenicity and enable them to proliferate in the unique ecological niche of the plant vascular system, we sequenced the genomes of V. dahliae and V. albo-atrum and compared them to each other, and to the genome of Fusarium oxysporum, another fungal wilt pathogen. Our analyses identified a set of proteins that are shared among all three wilt pathogens, and present in few other fungal species. One of these is a homolog of a bacterial glucosyltransferase that synthesizes virulence-related osmoregulated periplasmic glucans in bacteria. Pathogenicity tests of the corresponding V. dahliae glucosyltransferase gene deletion mutants indicate that the gene is required for full virulence in the Australian tobacco species Nicotiana benthamiana. Compared to other fungi, the two sequenced Verticillium genomes encode more pectin-degrading enzymes and other carbohydrate-active enzymes, suggesting an extraordinary capacity to degrade plant pectin barricades. The high level of synteny between the two Verticillium assemblies highlighted four flexible genomic islands in V. dahliae that are enriched for transposable elements, and contain duplicated genes and genes that are important in signaling/transcriptional regulation and iron/lipid metabolism. Coupled with an enhanced capacity to degrade plant materials, these genomic islands may contribute to the expanded genetic diversity and virulence of V. dahliae, the primary causal agent of Verticillium wilts. Significantly, our study reveals insights into the genetic mechanisms of niche adaptation of fungal wilt pathogens, advances our understanding of the evolution and development of their pathogenesis, and sheds light on potential avenues for the development of novel disease management strategies to combat destructive wilt diseases.
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.
Sclerotinia sclerotiorum, the causal agent of potato stem rot, is prevalent and poorly managed on potatoes in the Columbia Basin of Washington. Because of the ubiquitous nature of the fungus and high crop diversity within the Columbia Basin, understanding the population structure and the potential for outcrossing of the pathogen would be helpful in developing disease management strategies. The population structure of S. sclerotiorum in the Columbia Basin from potato was examined using microsatellite markers and mycelial compatibility. Analysis of molecular variance revealed that 92% of the variability among 167 isolates was found within subpopulations, with limited, yet statistically significant impact of the collection date, but not the year or location of collection. Linkage disequilibrium and index of association analyses noted a potential for outcrossing in two locations, which was substantiated by the discovery of recombinant ascospores in three field-generated apothecia from the 12 apothecia examined. Microsatellite haplotypes were not correlated with mycelial compatibility groups. This high haplotypic diversity did not seem to impact pathologically important phenotypes. Greenhouse inoculations of potato plants exhibited no significant differences in aggressiveness on potato stems. Moreover, in vitro studies of response to fungicides and temperature stimuli yielded no significant differences among studied isolates. These findings illustrate the potential for outcrossing in warm temperate regions of North America, where a diversity of crops are planted simultaneously and in neighboring fields. This study also indicates that the unsatisfactory management of potato stem rot is likely not directly attributable to genetic factors, but to gaps in agricultural practices.
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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.
Potato early dying (PED), also known as Verticillium wilt, caused by Verticillium dahliae, is a seasonal yield-limiting disease of potato worldwide, and PED-resistant cultivars currently represent only a small percentage of potato production. In this study, we developed a real-time quantitative polymerase chain reaction (Q-PCR) approach to detect and quantify V. dahliae. The efficiency of the designed primer pair VertBt-F/VertBt-R, derived from the sequence of the beta-tubulin gene, was greater than 95% in monoplex Q-PCR and duplex (using Plexor technology) procedures with primers PotAct-F/PotAct-R, obtained from the sequence of the actin gene, designed for potato. As few as 148 fg of V. dahliae DNA were detected and quantified, which is equivalent to five nuclei. Q-PCR detected V. dahliae in naturally infected air-dried potato stems and fresh stems of inoculated plants. Spearman correlations indicated a high correlation (upward of 80%) between V. dahliae quantifications using Q-PCR and the currently used plating assays. Moreover, Q-PCR substantially reduced the variability compared with that observed in the plating assay, and allowed for the detection of V. dahliae in 10% of stem samples found to be pathogen free on the culture medium. The described Q-PCR approach should provide breeders with a more sensitive and less variable alternative to time-consuming plating assays to distinguish response of breeding lines to colonization by V. dahliae.
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.
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