Krishna V. Subbarao

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.

Tomato immune receptor Ve1 recognizes effector of multiple fungal pathogens uncovered by genome and RNA sequencing

Fungal plant pathogens secrete effector molecules to establish disease on their hosts, and plants in turn use immune receptors to try to intercept these effectors. The tomato immune receptor Ve1 governs resistance to race 1 strains of the soil-borne vascular wilt fungi Verticillium dahliae and Verticillium albo-atrum, but the corresponding Verticillium effector remained unknown thus far. By high-throughput population genome sequencing, a single 50-Kb sequence stretch was identified that only occurs in race 1 strains, and subsequent transcriptome sequencing of Verticillium-infected Nicotiana benthamiana plants revealed only a single highly expressed ORF in this region, designated Ave1 (for Avirulence on Ve1 tomato). Functional analyses confirmed that Ave1 activates Ve1-mediated resistance and demonstrated that Ave1 markedly contributes to fungal virulence, not only on tomato but also on Arabidopsis. Interestingly, Ave1 is homologous to a widespread family of plant natriuretic peptides. Besides plants, homologous proteins were only found in the bacterial plant pathogen Xanthomonas axonopodis and the plant pathogenic fungi Colletotrichum higginsianum, Cercospora beticola, and Fusarium oxysporum f. sp. lycopersici. The distribution of Ave1 homologs, coincident with the presence of Ave1 within a flexible genomic region, strongly suggests that Verticillium acquired Ave1 from plants through horizontal gene transfer. Remarkably, by transient expression we show that also the Ave1 homologs from F. oxysporum and C. beticola can activate Ve1-mediated resistance. In line with this observation, Ve1 was found to mediate resistance toward F. oxysporum in tomato, showing that this immune receptor is involved in resistance against multiple fungal pathogens.

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.

Phylogenetics and taxonomy of the fungal vascular wilt pathogen Verticillium, with the descriptions of five new species.

Knowledge of pathogen biology and genetic diversity is a cornerstone of effective disease management, and accurate identification of the pathogen is a foundation of pathogen biology. Species names provide an ideal framework for storage and retrieval of relevant information, a system that is contingent on a clear understanding of species boundaries and consistent species identification. Verticillium, a genus of ascomycete fungi, contains important plant pathogens whose species boundaries have been ill defined. Using phylogenetic analyses, morphological investigations and comparisons to herbarium material and the literature, we established a taxonomic framework for Verticillium comprising ten species, five of which are new to science. We used a collection of 74 isolates representing much of the diversity of Verticillium, and phylogenetic analyses based on the ribosomal internal transcribed spacer region (ITS), partial sequences of the protein coding genes actin (ACT), elongation factor 1-alpha (EF), glyceraldehyde-3-phosphate dehydrogenase (GPD) and tryptophan synthase (TS). Combined analyses of the ACT, EF, GPD and TS datasets recognized two major groups within Verticillium, Clade Flavexudans and Clade Flavnonexudans, reflecting the respective production and absence of yellow hyphal pigments. Clade Flavexudans comprised V. albo-atrum and V. tricorpus as well as the new species V. zaregamsianum, V. isaacii and V. klebahnii, of which the latter two were morphologically indistinguishable from V. tricorpus but may differ in pathogenicity. Clade Flavnonexudans comprised V. nubilum, V. dahliae and V. longisporum, as well as the two new species V. alfalfae and V. nonalfalfae, which resembled the distantly related V. albo-atrum in morphology. Apart from the diploid hybrid V. longisporum, each of the ten species corresponded to a single clade in the phylogenetic tree comprising just one ex-type strain, thereby establishing a direct link to a name tied to a herbarium specimen. A morphology-based key is provided for identification to species or species groups.

Characterization of Verticillium dahliae Isolates and Wilt Epidemics of Pepper

Epidemics of Verticillium wilt in pepper fields of the central coast of California and isolates of Verticillium dahliae associated with these epidemics were characterized. The mean incidence of wilted plants per field ranged from 6.3 to 97.8% in fields with Anaheim, jalapeno, paprika, or bell peppers. In general, incidence of wilt in jalapeno and bell pepper crops was lower than in crops of other types of pepper. Inoculum density of V. dahliae in the surveyed pepper fields ranged from 2.7 to 66.6 microsclerotia g-1 dry soil, and the correlation between disease incidence and density of microsclerotia was high (r = 0.81, P < 0.01). Distribution of Verticillium wilt was aggregated in a majority of the pepper fields surveyed, but the degree of aggregation varied. Vegetative compatibility group (VCG) characterization of 67 isolates of V. dahliae indicated that 67% belonged to VCG 2, 22% to VCG 4, and 11% to a new group, designated VCG 6. The pathogenicity of isolates of V. dahliae from bell pepper and tomato plants was tested by inoculating 1-month-old bell pepper (cv. Cal Wonder) and tomato (cv. EP 7) seedlings and incubating the inoculated plants in the greenhouse. Seedlings of bell pepper were susceptible only to the isolates of V. dahliae from pepper, whereas seedlings of tomato were susceptible to both pepper and tomato isolates. Pepper isolates belonging to VCG 2, VCG 4, and VCG 6 were highly pathogenic to bell pepper and chili pepper. Temperatures between 15 and 25°C were optimal for mycelial growth of a majority of isolates of V. dahliae. Molecular characterization of pepper isolates of V. dahliae using a polymerase chain reaction (PCR)-based random amplified polymorphic DNA (RAPD) technique revealed minor variation among these isolates, but unique polymorphic banding patterns were observed for isolates belonging to VCG 6. Verticillium wilt of pepper is a major production constraint in the central coast of California. More aggressive isolates of V. dahliae may have been selected in this region as a result of intensive cropping practices.

Colonization of Resistant and Susceptible Lettuce Cultivars by a Green Fluorescent Protein-Tagged Isolate of Verticillium dahliae

Interactions between lettuce and a green fluorescent protein (GFP)-expressing, race 1 isolate of Verticillium dahliae, were studied to determine infection and colonization of lettuce cultivars resistant and susceptible to Verticillium wilt. The roots of lettuce seedlings were inoculated with a conidial suspension of the GFP-expressing isolate. Colonization was studied with the aid of laser scanning confocal and epi-fluorescence microscopes. Few differences in the initial infection and colonization of lateral roots were observed between resistant and susceptible cultivars. Hyphal colonies formed on root tips and within the root elongation zones by 5 days, leading to the colonization of cortical tissues and penetration of vascular elements regardless of the lettuce cultivar by 2 weeks. By 8 to 10 weeks after inoculation, vascular discoloration developed within the taproot and crown regions of susceptible cultivars well in advance of V. dahliae colonization. Actual foliar wilt coincided with the colonization of the taproot and crown areas and the eruption of mycelia into surrounding cortical tissues. Advance colonization of stems, pedicels, and inflorescence, including developing capitula and mature achenes was observed. Seedborne infection was limited to the maternal tissues of the achene, including the pappus, pericarp, integument, and endosperm; but the embryo was never compromised. Resistant lettuce cultivars remained free of disease symptoms. Furthermore, V. dahliae colonization never progressed beyond infected lateral roots of resistant cultivars. Results indicated that resistance in lettuce may lie with the plants ability to shed infected lateral roots or to inhibit the systemic progress of the fungus through vascular tissues into the taproot.

Evaluation of Broccoli Residue Incorporation into Field Soil for Verticillium Wilt Control in Cauliflower

Wilt incited by Verticillium dahliae has recently become an important disease on cauliflower in the Salinas Valley. Although broccoli is closely related to cauliflower, wilt has not occurred on this host and V. dahliae isolates from cauliflower were weakly pathogenic on broccoli in greenhouse inoculations. In this study, the effectiveness of broccoli residues on propagule attrition in soil and wilt incidence on cauliflower was determined in a commercial field infested with V. dahliae microsclerotia. The treatments were broccoli residue with tarp, broccoli residue without tarp, chloropicrin, metham sodium, control with tarp, control without tarp, cauliflower followed by cauliflower, and cauliflower followed by fallow. Approximately 200 kg of chopped broccoli was uniformly disk incorporated into the corresponding plots (36 m2). Densities of V. dahliae microsclerotia were determined at 0, 30, 90, and 145 days after treatment in 1993 and 0, 30, 74, 109, and 140 days after treatment in 1994 using the modified Anderson sampler technique. Cauliflower cultivar White Rock was planted in all plots. Plant height, number of marketable heads, head weight, and wilt severity were determined at maturity. Even though the pre-treatment number of V. dahliae propagules in broccoli-treated and control plots were similar, by the end of the cauliflower cropping season numbers either were the same or increased in control plots compared with a reduction in numbers in broccoli residue-treated plots during both seasons. There were no differences between tarped and non-tarped plots either in broccoli residue-treated or control plots. In fumigated plots, propagules declined initially but later returned to pre-treatment levels by the end of the cropping season. Continuous cauliflower or fallowing after one cauliflower crop resulted in stable or increased levels of microsclerotia. Verticillium wilt severity was lowest in metham sodium-treated plots, followed by broccoli residue-treated plots and chloropicrin in both years. Plant height, marketable heads, and head weight were higher in broccoli-treated than in control plots. These results suggest that broccoli residues reduce V. dahliae microsclerotia in soil and wilt of cauliflower as much as or more than chloropicrin and metham sodium, and that rotation with broccoli may be a feasible approach to manage Verticillium wilt in cauliflower and other susceptible crops.

Verticillium wilt of cauliflower in California.

Since 1990, commercial cauliflower in coastal California has been severely affected by a vascular wilt disease. Symptoms consist of chlorosis, defoliation, stunting, wilting, and vascular discoloration. Disease has been widespread and has caused significant damage in summer and fall crops. Verticillium dahliae was consistently isolated from xylem tissue in stems and roots of affected plants. Techniques tested for inoculation of cauliflower plants were dipping clipped or nonclipped roots into spore suspensions, injecting spore suspensions into cauliflower stems, and planting seedlings into soil along with an agar block colonized with microsclerotia. Only dipping roots into spore suspensions was consistently successful in causing Verticillium wilt

Effects of chitin and chitosan on the incidence and severity of Fusarium yellows of celery

The effects of chitin and chitosan on disease incidence and severity of Fusarium yellows of celery and on populations of Fusarium oxysporum were investigated between 1994 and 1996. Field experiments were conducted at two locations with a history of severe Fusarium yellows. Disease incidence and severity were significantly reduced by pre-plant chitin amendments to soil. Chitosan applied as a root dip alone did not reduce disease incidence but significantly (P < 0.05) reduced disease severity when used with a tolerant celery cultivar. Standard soil dilution methods were used to enumerate populations of soil microflora. Chitin increased bacterial and actinomycete populations in soil in 2 of the 3 years of study. The effects of potential biocontrol agents recovered from chitin-treated plots in 1995 were studied in 1996; enriching the transplant medium with isolates of bacteria and actinomycetes 4 weeks and 1 week prior to transplanting did not alter the established equilibrium in the field, and no biocontrol effect was observed. Chitin amendments to soil or chitosan treatment of transplants did not reduce soil populations of F. oxysporum. Whether these treatments affected the F. oxysporum f. sp. apii subpopulation within the F. oxysporum population could not be determined.

Effects of crop rotation and irrigation on Verticillium dahliae microsclerotia in soil and wilt in cauliflower.

ABSTRACT Experiments were conducted in field plots to evaluate the effects of broccoli residue on population dynamics of Verticillium dahliae in soil and on Verticillium wilt development on cauliflower under furrow and subsurface-drip irrigation and three irrigation regimes in 1994 and 1995. Treatments were a factorial combination of three main plots (broccoli crop grown, harvested, and residue incorporated in V.dahliae-infested plots; no broccoli crop or residue in infested plots; and fumigated control plots), two subplots (furrow and subsurface-drip irrigation), and three sub-subplots (deficit, moderate, and excessive irrigation regimes) arranged in a split-split-plot design with three replications. Soil samples collected at various times were assayed for V. dahliae propagules using the modified Anderson sampler technique. Incidence and severity of Verticillium wilt on cauliflower were assessed at 7- to 10-day intervals beginning a month after cauliflower transplanting and continuing until harvest. Number of propagules in all broccoli plots declined significantly (P < 0.05) after residue incorporation and continued to decline throughout the cauliflower season. The overall reduction in the number of propagules after two broccoli crops was approximately 94%, in contrast to the fivefold increase in the number of propagules in infested main plots without broccoli after two cauliflower crops. Disease incidence and severity were both reduced approximately 50% (P < 0.05) in broccoli treatments compared with no broccoli treatments. Differences between furrow and subsurface-drip irrigation were not significant, but incidence and severity were significantly (P < 0.05) lower in the deficit irrigation regime compared with the other two regimes. Abundance of microsclerotia of V. dahliae on cauliflower roots about 8 weeks after cauliflower harvest was significantly (P < 0.05) lower in treatments with broccoli compared with treatments without broccoli. Rotating broccoli with cauliflower and incorporating broccoli residues into the soils is a novel means of managing Verticillium wilt on cauliflower and perhaps on other susceptible crops. This practice would be successful regardless of the irrigation methods or regimes followed on the susceptible crops.