The Genome of the Saprophytic Fungus Verticillium tricorpus Reveals a Complex Effector Repertoire Resembling That of Its Pathogenic Relatives

Vascular wilts caused by Verticillium spp. are destructive plant diseases affecting hundreds of hosts. Only a few Verticillium spp. are causal agents of vascular wilt diseases, of which V. dahliae is the most notorious pathogen, and several V. dahliae genomes are available. In contrast, V. tricorpus is mainly known as a saprophyte and causal agent of opportunistic infections. Based on a hybrid approach that combines second and third generation sequencing, a near-gapless V. tricorpus genome assembly was obtained. With comparative genomics, we sought to identify genomic features in V. dahliae that confer the ability to cause vascular wilt disease. Unexpectedly, both species encode similar effector repertoires and share a genomic structure with genes encoding secreted proteins clustered in genomic islands. Intriguingly, V. tricorpus contains significantly fewer repetitive elements and an extended spectrum of secreted carbohydrate- active enzymes when compared with V. dahliae. In conclusion, we highlight the technical advances of a hybrid sequencing and assembly approach and show that the saprophyte V. tricorpus shares many hallmark features with the pathogen V. dahliae.

First Report of Verticillium Wilt on Lettuce (Lactuca sativa) in Washington Caused by Verticillium tricorpus

Symptoms of Verticillium wilt were observed on lettuce (Lactuca sativa L.) harvested from high tunnel and open field experimental plots in annual, consecutive spring plantings in western Washington from 2010 to 2012. Leaves had v-shaped, chlorotic lesions, and yellow or brown vascular tissue was noted in the crowns. Total disease incidence increased from 0.2% in 2010 to 1.9% in 2011 and to 14.4% in 2012. Verticillium spp. obtained from infected crown tissues and cultured on half-strength potato dextrose agar medium produced yellow pigment, black microsclerotia, white mycelia, tan chlamydospores, and uniseptate conidia averaging 10.6 × 3.7 μm. Isolates were identified tentatively as Verticillium tricorpus I. (3). Three isolates, Vt.Ls.2010, Vt.Ls.2011-1, and Vt.Ls.2011-2, were evaluated for pathogenicity on 4-week-old ‘Coastal Star’ seedlings in two greenhouse trials. In Trial I, four replicates of two duplicate plants per each isolate, and in Trial II, five replicates of one plant per each isolate were inoculated with conidial suspensions adjusted to 2.0 × 106 and 5.0 × 106 conidia/ml, respectively. Additionally, in each trial, two sets of control treatments of five plants each were inoculated with either an isolate of V. dahliae at the same conidial concentration or with sterile water. Root tips were cut and exposed to the suspensions for 5 s, then seedlings were transplanted into Sunshine Mix #1 (SunGro Horticulture Distribution Inc., Bellevue, WA), and kept in a greenhouse at 17.7 ± 3.4°C. Plants were harvested 8 to 9 weeks post-inoculation, and symptoms were rated visually. Vt.Ls.2010, Vt.Ls.2011-1, and Vt.Ls.2011-2 caused chlorosis and vascular discoloration on 25, 13, and 13% of the plants in Trial I; and 40, 60, and 20% of plants in Trial II, respectively. V. dahliae caused similar symptoms on 25 and 40% of the plants in the two trials, respectively, but these plants had greater intensity and length of vascular discoloration compared with the three test isolates. None of the water control plants were symptomatic. All V. tricorpus isolates were recovered from inoculated plants, and colony morphologies were similar to the original isolates. The internal transcribed spacer (ITS) rDNA of isolate Vt.Ls.2010 was amplified with ITS4 and ITS6 primer sets. ITS rDNA sequences between Vt.Ls.2010 and two isolates of V. tricorpus in GenBank (Accession Nos. FJ900211 and AB353343) were 100% identical. V. tricorpus is considered a weak pathogen of lettuce crops in California (2), but authors in Japan recently reported pathogenic isolates of V. tricorpus on lettuce (4). To our knowledge, this is the first report of Verticillium wilt caused by V. tricorpus in Washington. Lettuce is the number two crop grown in high tunnels in the United States (1), and cropping lettuce continuously in them can increase the risk of this and other soilborne pathogens. References: (1) E. E. Carey et al. HortTechnology 19:37, 2009. (2) Q.-M. Qin et al. Plant Dis. 92:69, 2008. (3) H. C. Smith. N. Z. J. Agric. Res. 8:450, 1965. (4) T. Usami et al. J. Gen. Plant Pathol. 77:17, 2010.

Characterization of Verticillium dahliae and V. tricorpus Isolates from Lettuce and Artichoke

Verticillium isolates collected from lettuce and artichoke were characterized for morphology, growth and pathogenicity. Several isolates were identified as Verticillium tricorpus based on morphological and cultural characteristics, including the production of dark resting mycelia, chlamydospores, microsclerotia, and yellow to orange pigmentation in culture. Compared with isolates of V. dahliae, these isolates also produced microsclerotia and conidia that were significantly larger and exhibited a distinct growth pattern at varying temperatures. Using database sequence information, primers were developed from the internal transcribed spacer region to produce a diagnostic 337-bp product specific to V. tricorpus and used to confirm the identification of isolates. Pathogenicity tests indicated that isolates of V. tricorpus were weak pathogens, causing a median disease severity (DS) of <1 (0-to-5 scale) on lettuce and artichoke. In contrast, isolates of V. dahliae consistently caused severe wilt with a median DS of >3.5 on lettuce and 5.0 on artichoke. Although lettuce and artichoke inoculated with isolates of V. tricorpus exhibited reduced height and fresh foliar and root weight, the reductions were not statistically significant, unlike in plants inoculated with isolates of V. dahliae. Lettuce co-inoculated with isolates of V. tricorpus and V. dahliae exhibited reduced symptoms of Verticillium wilt and improved growth relative to those inoculated with V. dahliae alone. The early introduction of V. tricorpus in soil-drench inoculations appeared to provide better relief from subsequent V. dahliae inoculation than when the two species were co-inoculated simultaneously using the root-dip method, suggesting competitive exclusion as a plausible mechanism. A spore-polymerase chain reaction assay developed using cultured spores directly as template and primers specific to V. tricorpus confirmed the presence of V. tricorpus on inoculated roots. This work demonstrates the potential use of V. tricorpus to directly reduce the effect of V. dahliae on lettuce and artichoke and, to our knowledge, is the first reported characterization of V. tricorpus isolates collected from lettuce and artichoke.