Frequency of Verticillium Species in Commercial Spinach Fields and Transmission of V. dahliae from Spinach to Subsequent Lettuce Crops.

Verticillium wilt caused by V. dahliae is a devastating disease of lettuce in California (CA). The disease is currently restricted to a small geographic area in central coastal CA, even though cropping patterns in other coastal lettuce production regions in the state are similar. Infested spinach seed has been implicated in the introduction of V. dahliae into lettuce fields but direct evidence linking this inoculum to wilt epidemics in lettuce is lacking. In this study, 100 commercial spinach fields in four coastal CA counties were surveyed to evaluate the frequency of Verticillium species recovered from spinach seedlings and the area under spinach production in each county was assessed. Regardless of the county, V. isaacii was the most frequently isolated species from spinach followed by V. dahliae and, less frequently, V. klebahnii. The frequency of recovery of Verticillium species was unrelated to the occurrence of Verticillium wilt on lettuce in the four counties but was related to the area under spinach production in individual counties. The transmission of V. dahliae from infested spinach seeds to lettuce was investigated in microplots. Verticillium wilt developed on lettuce following two or three plantings of Verticillium-infested spinach, in independent experiments. The pathogen recovered from the infected lettuce from microplots was confirmed as V. dahliae by polymerase chain reaction assays. In a greenhouse study, transmission of a green fluorescence protein-tagged mutant strain of V. dahliae from spinach to lettuce roots was demonstrated, after two cycles of incorporation of infected spinach residue into the soil. This study presents conclusive evidence that V. dahliae introduced via spinach seed can cause Verticillium wilt in lettuce.

Clonal expansion of Verticillium dahliae in lettuce.

Few studies in population biology have documented how structure and diversity of pathogens evolve over time at local scales. With the historical samples of Verticillium dahliae available from lettuce, we investigated the structure and diversity of this pathogen in time and space. Three hundred twenty-nine V. dahliae isolates from lettuce fields collected over 18 years were characterized with polymorphic microsatellite markers and polymerase chain reaction tests for race and mating type. Genetic variation within and among commercial lettuce fields in a single season was also investigated using an additional 146 isolates. Sixty-two haplotypes (HTs) were observed among the 329 isolates. A single HT was frequently observed over multiple years and locations (61.40%). Genetic diversity, allelic richness, and private allelic richness suggested a relatively recent clonal expansion. Race 1 (93.63%) and MAT1-2-1 (99.69%) were overwhelmingly represented among the isolates. Linkage disequilibrium was significant (P < 0.001) for all populations, suggesting limited sexual recombination in the sampled populations from lettuce. Populations from 2006, 2009, and 2010 had higher numbers of unique HTs, implying a recent introduction of novel HTs. We conclude that V. dahliae population from lettuce evaluated in this study is expanding clonally, consistent with an asexually reproducing pathogen, and the movement of clonal genotypes locally occurs over time.

Comparative pathogenicity, biocontrol efficacy, and multilocus sequence typing of Verticillium nonalfalfae from the invasive Ailanthus altissima and other hosts.

Verticillium wilt, caused by Verticillium nonalfalfae, is currently killing tens of thousands of highly invasive Ailanthus altissima trees within the forests in Pennsylvania, Ohio, and Virginia and is being considered as a biological control agent of Ailanthus. However, little is known about the pathogenicity and virulence of V. nonalfalfae isolates from other hosts on Ailanthus, or the genetic diversity among V. nonalfalfae from confirmed Ailanthus wilt epicenters and from locations and hosts not associated with Ailanthus wilt. Here, we compared the pathogenicity and virulence of several V. nonalfalfae and V. alfalfae isolates, evaluated the efficacy of the virulent V. nonalfalfae isolate VnAa140 as a biocontrol agent of Ailanthus in Pennsylvania, and performed multilocus sequence typing of V. nonalfalfae and V. alfalfae. Inoculations of seven V. nonalfalfae and V. alfalfae isolates from six plant hosts on healthy Ailanthus seedlings revealed that V. nonalfalfae isolates from hosts other than Ailanthus were not pathogenic on Ailanthus. In the field, 100 canopy Ailanthus trees were inoculated across 12 stands with VnAa140 from 2006 to 2009. By 2011, natural spread of the fungus had resulted in the mortality of >14,000 additional canopy Ailanthus trees, 10,000 to 15,000 Ailanthus sprouts, and nearly complete eradication of Ailanthus from several smaller inoculated stands, with the exception of a few scattered vegetative sprouts that persisted in the understory for several years before succumbing. All V. nonalfalfae isolates associated with the lethal wilt of Ailanthus, along with 18 additional isolates from 10 hosts, shared the same multilocus sequence type (MLST), MLST 1, whereas three V. nonalfalfae isolates from kiwifruit shared a second sequence type, MLST 2. All V. alfalfae isolates included in the study shared the same MLST and included the first example of V. alfalfae infecting a non-lucerne host. Our results indicate that V. nonalfalfae is host adapted and highly efficacious against Ailanthus and, thus, is a strong candidate for use as a biocontrol agent.

Global population structure and migration patterns suggest significant population differentiation among isolates of Pyrenophora tritici-repentis.

The global population structure and migration patterns of foliar wheat pathogen Pyrenophora tritici-repentis (PTR) were determined using 12 microsatellite loci. Analysis of 439 single-spore isolates of PTR from five continents (18 wheat-producing countries) showed high level of genetic diversity, and moderate to high population differentiation between continents. A high level of gene diversity (H(S)=0.31 to 0.56) was observed within each population. Allelic richness indicated the European and the North American population have a high effective population size. Bayesian analyses showed five clusters where the inferred clusters did not represent geographical populations. Corrected standardized fixation index (G(ST)(″)) estimates ranged from 0.042 to 0.265 between populations, indicating low to high genetic differentiation exists between populations. We found migration (gene flow) between old world (Europe) and new world (Americas) population; however, little migration was observed among other continents. The European population was the major source of immigrants for the North American, South American, Australian and the Asian populations. Significant (P<0.001) linkage disequilibrium (LD) was detected in the Australian and the South American populations. In contrast, non-significant (P<0.001) LD values were observed in the Asian, European and the North American populations. Overall, our findings demonstrate the population differentiation exits among the global populations and strict quarantine measures should be applied to prevent the accelerated global spread of this pathogen.

First Report of Verticillium Wilt Caused by Verticillium nonalfalfae on Tree-of-Heaven (Ailanthus altissima) in Ohio.

Verticillium wilt of the highly invasive tree-of-heaven [Ailanthus altissima (Mill.) Swingle], caused by Verticillium nonalfalfae Interbitzin et al. (1), formerly classified as V. albo-atrum Reinke and Berthold, has been reported in the United States from two states: Pennsylvania (2) and Virginia (3). Infected A. altissima in both states exhibited similar symptoms of wilt, premature defoliation, terminal dieback, yellow vascular discoloration, and mortality. In June 2012, the second author observed dead and dying A. altissima trees in southern Ohio (Pike County) that exhibited symptoms similar to those on diseased A. altissima trees in Pennsylvania and Virginia. Samples were collected from stems of three symptomatic A. altissima trees and sent to Penn State for morphological and molecular identification. Immediately upon arrival, samples were surface-disinfected and plated onto plum extract agar (PEA), a semi-selective medium for Verticillium spp., amended with neomycin and streptomycin (2). The samples yielded six isolates, two from each of the three symptomatic trees, all of which were putatively identified as V. nonalfalfae based on the presence of verticillate conidiophores and formation of melanized hyphae. DNA was extracted from three isolates and molecular analyses performed using known primers (1) coding for elongation factor 1-alpha (EF), glyceraldehyde-3-phosphate dehydrogenase (GPD), and tryptophan synthase (TS). A BLAST search generated sequences that revealed 100% similarity to V. nonalfalfae for all three protein coding genes among the three Ohio isolates and reference sequences from Ailanthus, including isolates VnAaPA140 (GenBank Accession Nos. KC307764, KC307766, and KC307768) and VnAaVA2 (KC307758, KC307759, and KC307760), as well as isolate PD592 from potato (JN188227, JN188163, and JN188035), thereby confirming taxonomic placement of the Ohio Ailanthus isolates among those recovered from Ailanthus in Pennsylvania and Virginia. Aligned sequences from one representative isolate, VnAaOH1, were deposited into GenBank as accessions KC307761 (EF), KC307762 (GPD), and KC307763 (TS). In August 2012, the pathogenicity of all six isolates was confirmed by root-dipping 10 healthy 3-week-old A. altissima seedlings (seeds collected in University Park, PA) into conidial suspensions of 1 × 107 cfu/ml, wherein all inoculated seedlings wilted and died within 4 and 9 weeks, respectively. V. nonalfalfae was reisolated from all inoculated seedlings; control seedlings inoculated with distilled water remained asymptomatic. Ohio is the third state from which V. nonalfalfae has been reported to be pathogenic on A. altissima. If V. nonalfalfae proves to be widespread, it may represent a natural biocontrol for the invasive A. altissima. Also, since USDA APHIS evaluates and regulates new potential biocontrol agents on a state-by-state basis, it is important to document each state in which V. nonalfalfae is killing A. altissima, so that in-state inoculum can be used for biocontrol efforts, simplifying the regulatory process. References: (1) P. Inderbitzin et al. 2011 PLoS ONE, 6, e28341, 2011. (2) M. J. Schall and D. D. Davis. Plant Dis. 93:747, 2009. (3) A. L. Snyder et al. Plant Dis. 96:837, 2013.