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Verticillium wilt, caused by the fungal pathogen Verticillium dahliae, is the major cause of disease-related yield losses in cotton ( Gossypium hirsutum). Despite these losses, the major cultivars of G. hirsutum remain highly susceptible to Verticillium wilt. The lack of understanding on the genetic basis for Verticillium wilt resistance may further hinder progress in deploying elite cultivars with proven resistance, such as the wilt resistant G. hirsutum cultivar Zhongzhimian No. 2. To help remedy this knowledge gap, we sequenced the whole genome of Zhongzhimian No. 2 and assembled it from a combination of PacBio long reads, Illumina short reads, and high-throughput chromosome conformation capture technologies. The final assembly of the genome was 2.33 Gb, encoding 95,327 predicted coding sequences. The GC content was 34.39% with 99.2% of the bases anchored to 26 pseudo-chromosomes that ranged from 53.8 to 127.7 Mb. This resource will help gain a detailed understanding of the genomic features governing high yield and Verticillium wilt resistance in this cultivar. Comparative genomics will be particularly helpful, since there are several published genomes of other Gossypium species. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

The arms race between fungal pathogens and plant hosts involves recognition of fungal effectors to induce host immunity. Although various fungal effectors have been identified, the effector functions of ribonucleases are largely unknown. Herein, we identified a ribonuclease secreted by Verticillium dahliae (VdRTX1) that translocates into the plant nucleus to modulate immunity. The activity of VdRTX1 causes hypersensitive response (HR)-related cell death in Nicotiana benthamiana and cotton. VdRTX1 possesses a signal peptide but is unlikely to be an apoplastic effector because its nuclear localization in the plant is necessary for cell death induction. Knockout of VdRTX1 significantly enhanced V. dahliae virulence on tobacco while V. dahliae employs the known suppressor VdCBM1 to escape the immunity induced by VdRTX1. VdRTX1 homologs are widely distributed in fungi but transient expression of 24 homologs from other fungi did not yield cell death induction, suggesting that this function is specific to the VdRTX1 in V. dahliae. Expression of site-directed mutants of VdRTX1 in N. benthamiana leaves revealed conserved ligand-binding sites that are important for VdRTX1 function in inducing cell death. Thus, VdRTX1 functions as a unique HR-inducing effector in V. dahliae that contributes to the activation of plant immunity.

Though Verticillium dahliae is an asexually reproducing fungus, it is considered heterothallic owing to the presence of only one of the two mating-type idiomorphs (MAT1-1 or MAT1-2) in individual isolates. But sexual reproduction has never been observed either in nature or in the laboratory. All of the genomic information in the literature thus far has therefore come from studies on isolates carrying only the MAT1-2 idiomorph. Herein, we sequenced and compared high-quality reference genomes of MAT1-1 strain S011 and MAT1-2 strain S023 obtained from the same sunflower field. The two genomic sequences displayed high synteny, and encoded similar number genes, a similarity especially notable among pathogenicity-related genes. Homolog analysis between these two genomes revealed that 80% of encoded genes are highly conserved (95% identity and coverage), but only 20% of the single copy genes were identical. These novel genome resources will support the analysis of the structure and function of the two idiomorphs and provide valuable tools to elucidate the evolution and potential mechanisms of sexual reproduction in V. dahliae.

Type 2C protein phosphatases (PP2Cs) regulate various biological processes in eukaryotes. However, their functions in Verticillium dahliae have not been characterized. In this study, homologs VdPtc1, VdPtc3, VdPtc5, VdPtc6 and VdPtc7 were identified in V. dahliae based on homologous comparison with those in Saccharomyces cerevisiae. VdPtc2 and VdPtc4 are missing in the genome of V. dahliae XJ592 strain. VdPtc3 is the homologs of Ptc2, Ptc3 and Ptc4 proteins in S. cerevisiae, implying that VdPtc3 may play versatile functions in V. dahliae. VdPtc3 promoted conidium development, melanin and microsclerotium formation in V. dahliae. The ΔVdPtc3 strains showed increased sensitivity to NaCl and sorbitol and augmented the phosphorylation of p38 MAPK Hog1 induced by osmotic stress. Besides, the ΔVdPtc3 strains also showed milder Verticillium wilt symptom on cotton. Furthermore, uniquely to filamentous fungi, VdPtc3 interacts with VdAtg1, which modulates melanin and microsclerotium formation as well as pathogenicity.

Lettuce (Lactuca sativa) is one of the most economically important vegetables in the United States, with approximately 50% of the domestic production concentrated in the Salinas Valley of California. Verticillium wilt, caused by races 1 and 2 of the fungal pathogen Verticillium dahliae, poses a major threat to lettuce production in this area. Although resistance governed by a single dominant gene against race 1 has previously been identified and is currently being incorporated into commercial cultivars, identification of resistance against race 2 has been challenging and no lines with complete resistance have been identified. In this study, we screened germplasm for resistance and investigated the genetics of partial resistance against race 2 using three mapping populations derived from crosses involving L. sativa × L. sativa and L. serriola × L. sativa. The inheritance of resistance in Lactuca species against race 2 is complex but a common quantitative trait locus (QTL) on linkage group 6, designated qVERT6.1 (quantitative Verticillium dahliae resistance on LG 6, first QTL), was detected in multiple populations. Additional race 2 resistance QTLs located in several linkage groups were detected in individual populations and environments. Because resistance in lettuce against race 2 is polygenic with a large genotype by environment interaction, breeding programs to incorporate these resistance genes should be aware of this complexity as they implement strategies to control race 2.

Verticillium dahliae is a widespread fungal pathogen that causes Verticillium wilt on many economically important crops and ornamentals worldwide. Populations of V. dahliae have been divided into two distinct races based upon differential host responses in tomato and lettuce. Recently, the contemporary race 2 isolates were further divided into an additional race in tomato. Herein, we provide a high-quality reference genome for the race 1 strain VdLs.16 isolated from lettuce in California, U.S.A. This resource will contribute to ongoing research that aims to elucidate the genetic basis of V. dahliae pathogenicity and population genomic diversity.

SENESCENCE-ASSOCIATED GENE 13 (SAG13) of Arabidopsis is a widely conserved gene of unknown function that has been extensively used as a marker of plant senescence. SAG13 induction occurs during plant cell death processes, including senescence and hypersensitive response, a type of programmed cell death that occurs in response to pathogens. This implies that SAG13 expression is regulated through at least two different signaling pathways affecting these two different processes. Our work highlights a contrasting role for SAG13 in regulating resistance against disease-causing biotrophic bacterial and necrotrophic fungal pathogens with contrasting infection strategies. We provide further evidence that SAG13 is not only induced during oxidative stress but also plays a role in protecting the plant against other stresses. SAG13 is also required for normal seed germination, seedling growth, and anthocyanin accumulation. The work presented here provides evidence for the role of SAG13 in regulating multiple plant processes including senescence, defense, seed germination, and abiotic stress responses. SAG13 is a valuable molecular marker for these processes and is conserved in multiple plant species, and this knowledge has important implications for crop improvement.

Verticillium dahliae is a widely distributed soilborne pathogen that causes vascular wilt in more than 200 plant species. Defoliating and nondefoliating symptoms caused by the disease that result in either the loss or retention of leaves in infected plants, respectively, in hosts such as cotton, olive, and okra, divide the causal agent into defoliating and nondefoliating pathotypes. Our goal in this current work was to generate genome resources for the defoliating strain XJ592 and the nondefoliating strain XJ511 of V. dahliae isolated from cotton in China.

Secreted small cysteine‐rich proteins (SCPs) play a critical role in modulating host immunity in plant–pathogen interactions. Bioinformatic analyses showed that the fungal pathogen Verticillium dahliae encodes more than 100 VdSCPs, but their roles in host–pathogen interactions have not been fully characterized. Transient expression of 123 VdSCP‐encoding genes in Nicotiana benthamiana identified three candidate genes involved in host–pathogen interactions. The expression of these three proteins, VdSCP27, VdSCP113, and VdSCP126, in N. benthamiana resulted in cell death accompanied by a reactive oxygen species burst, callose deposition, and induction of defence genes. The three VdSCPs mainly localized to the periphery of the cell. BAK1 and SOBIR1 (associated with receptor‐like protein) were required for the immunity triggered by these three VdSCPs in N. benthamiana. Site‐directed mutagenesis showed that cysteine residues that form disulphide bonds are essential for the functioning of VdSCP126, but not VdSCP27 and VdSCP113. VdSCP27, VdSCP113, and VdSCP126 individually are not essential for V. dahliae infection of N. benthamiana and Gossypium hirsutum, although there was a significant reduction of virulence on N. benthamiana and G. hirsutum when inoculated with the VdSCP27/VdSCP126 double deletion strain. These results illustrate that the SCPs play a critical role in the V. dahliae–plant interaction via an intrinsic virulence function and suppress immunity following infection., Small cysteine‐rich proteins secreted by Verticillium dahliae play critical roles in interactions with hosts via an intrinsic virulence function and can suppress immunity following infection.

The lettuce downy mildew pathogen, Bremia lactucae, is an obligate oomycete that causes extensive produce losses. Initial chlorotic symptoms that severely reduce the market value of the produce are followed by the appearance of white, downy sporulation on the abaxial side of the leaves. These spores become airborne and disseminate the pathogen. Controlling lettuce downy mildew has relied on repeated fungicide applications to prevent outbreaks. However, in addition to direct economic costs, heterogeneity and rapid adaptation of this pathogen to repeatedly applied fungicides has led to the development of fungicide-insensitivity in the pathogen. We deployed a quantitative PCR assay-based detection method using a species-specific DNA target for B. lactucae coupled with a spore trap system to measure airborne B. lactucae spore loads within three commercial fields that each contained experimental plots, designated EXP1 to EXP3. Based upon these measurements, when the spore load in the air reached a critical level (8.548 sporangia per m3 air), we advised whether or not to apply fungicides on a weekly basis within EXP1 to EXP3. This approach saved three sprays in EXP1, and one spray each in EXP2 and EXP3 without a significant increase in disease incidence. The reduction in fungicide applications to manage downy mildew can decrease lettuce production costs while slowing the development of fungicide resistance in B. lactucae by eliminating unnecessary fungicide applications.

The accumulation of reactive oxygen species (ROS) is a widespread defence mechanism in higher plants against pathogen attack and sometimes is the cause of cell death that facilitates attack by necrotrophic pathogens. Plant pathogens use superoxide dismutase (SOD) to scavenge ROS derived from their own metabolism or generated from host defence. The significance and roles of SODs in the vascular plant pathogen Verticillium dahliae are unclear. Our previous study showed a significant upregulation of Cu/Zn‐SOD1 (VdSOD1) in cotton tissues following V. dahliae infection, suggesting that it may play a role in pathogen virulence. Here, we constructed VdSOD1 deletion mutants (ΔSOD1) and investigated its function in scavenging ROS and promoting pathogen virulence. ΔSOD1 had normal growth and conidiation but exhibited significantly higher sensitivity to the intracellular ROS generator menadione. Despite lacking a signal peptide, assays in vitro by western blot and in vivo by confocal microscopy revealed that secretion of VdSOD1 is dependent on the Golgi reassembly stacking protein (VdGRASP). Both menadione‐treated ΔSOD1 and cotton roots infected with ΔSOD1 accumulated more O2‐ and less H2O2 than with the wildtype strain. The absence of a functioning VdSOD1 significantly reduced symptom severity and pathogen colonization in both cotton and Nicotiana benthamiana. VdSOD1 is nonessential for growth or viability of V. dahliae, but is involved in the detoxification of both intracellular ROS and host‐generated extracellular ROS, and contributes significantly to virulence in V. dahliae., Verticillium dahliae Cu/Zn superoxide dismutase detoxifies self‐ and host‐generated ROS to maximize virulence.

Two QTLs for resistance to lettuce drop, qLDR1.1 and qLDR5.1, were identified. Associated SNPs will be useful in breeding for lettuce drop and provide the foundation for future molecular analysis. Lettuce drop, caused by Sclerotinia minor and S. sclerotiorum, is an economically important disease of lettuce. The association of resistance to lettuce drop with the commercially undesirable trait of fast bolting has hindered the integration of host resistance in control of this disease. Eruption is a slow-bolting cultivar that exhibits a high level of resistance to lettuce drop. Eruption also is completely resistant to Verticillium wilt caused by race 1 of Verticillium dahliae. A recombinant inbred line population from the cross Reine des Glaces × Eruption was genotyped by sequencing and evaluated for lettuce drop and bolting in separate fields infested with either S. minor or V. dahliae. Two quantitative trait loci (QTLs) for lettuce drop resistance were consistently detected in at least two experiments, and two other QTLs were identified in another experiment; the alleles for resistance at all four QTLs originated from Eruption. A QTL for lettuce drop resistance on linkage group (LG) 5, qLDR5.1, was consistently detected in all experiments and explained 11 to 25% of phenotypic variation. On LG1, qLDR1.1 was detected in two experiments explaining 9 to 12% of the phenotypic variation. Three out of four resistance QTLs are distinct from QTLs for bolting; qLDR5.1 is pleiotropic or closely linked with a QTL for early bolting; however, the rate of bolting shows only a small effect on the variance in resistance observed at this locus. The SNP markers linked with these QTLs will be useful in breeding for resistance through marker-assisted selection.

Verticillium wilt caused by Verticillium spp., also called potato early dying disease, is one of the most serious soilborne diseases affecting potato production in China. The disease has been expanding into most potato production areas over the past few years. Information on host resistance against Verticillium wilt among the potato cultivars in China is scarce, but it is critical for sustainable management of the disease. This study, therefore, evaluated 30 commercially popular potato cultivars against Verticillium dahliae strain Vdp83 and Verticillium nonalfalfae strain Vnp24, two well-characterized strains causing Verticillium wilt of potato in China. Both strains were isolated from diseased potato plants, and they were previously proven to be highly virulent. Ten plants of each cultivar were inoculated with the V. dahliae strain and incubated on greenhouse benches. Symptoms were rated at weekly intervals, and the relative area under the disease progress curve was calculated. The experiment was repeated once, and nonparametric analysis was used to calculate the relative marginal effects and the corresponding confidence intervals. Five resistant cultivars and four susceptible cultivars identified from the analyses were then challenged with the V. nonalfalfae strain. Cultivar responses to V. nonalfalfae were like those exhibited against V. dahliae, except for one cultivar. This study showed that resistance among potato cultivars exists in China against Verticillium spp. and that the resistance to V. dahliae identified in potato is also effective against the other Verticillium species that cause Verticillium wilt with a few exceptions. Cultivars identified as resistant to Verticillium wilt can be deployed to manage the disease until the breeding programs develop new cultivars with resistance from the sources identified in this study.

Downy mildew on spinach is caused by Peronospora effusa, an oomycete pathogen that poses a challenge to spinach production worldwide, especially in organic production. Following infection, P. effusa produces abundant amounts of asexual sporangia. Sporangia become windborne and initiate new infections locally or distantly, leading to widespread epidemics. Oospores produced from the union of opposite mating types have been observed within infected leaves and seeds and may remain viable for many years. Sexual reproduction increases the genetic diversity of P. effusa through sexual recombination, and thus, the movement of oospores on seed has likely fueled the rapid explosion of new pathotypes in different regions of the world over the past 20 years. This review summarizes recent advances in spinach downy mildew research, especially in light of the findings of oospores in contemporary commercial spinach seed lots as well as their germination. Knowledge of the role of the oospores and other aspects of the disease cycle can directly translate into new and effective disease management strategies.

Summary Improving genetic resistance is a preferred method to manage Verticillium wilt of cotton and other hosts. Identifying host resistance is difficult because of the dearth of resistance genes against this pathogen. Previously, a novel candidate gene involved in Verticillium wilt resistance was identified by a genome‐wide association study using a panel of Gossypium hirsutum accessions. In this study, we cloned the candidate resistance gene from cotton that encodes a protein sharing homology with the TIR‐NBS‐LRR receptor‐like defence protein DSC1 in Arabidopsis thaliana (hereafter named GhDSC1). GhDSC1 expressed at higher levels in response to Verticillium wilt and jasmonic acid (JA) treatment in resistant cotton cultivars as compared to susceptible cultivars and its product was localized to nucleus. The transfer of GhDSC1 to Arabidopsis conferred Verticillium resistance in an A. thaliana dsc1 mutant. This resistance response was associated with reactive oxygen species (ROS) accumulation and increased expression of JA‐signalling‐related genes. Furthermore, the expression of GhDSC1 in response to Verticillium wilt and JA signalling in A. thaliana displayed expression patterns similar to GhCAMTA3 in cotton under identical conditions, suggesting a coordinated DSC1 and CAMTA3 response in A. thaliana to Verticillium wilt. Analyses of GhDSC1 sequence polymorphism revealed a single nucleotide polymorphism (SNP) difference between resistant and susceptible cotton accessions, within the P‐loop motif encoded by GhDSC1. This SNP difference causes ineffective activation of defence response in susceptible cultivars. These results demonstrated that GhDSC1 confers Verticillium resistance in the model plant system of A. thaliana, and therefore represents a suitable candidate for the genetic engineering of Verticillium wilt resistance in cotton.

Proteins that mediate cellular and subcellular membrane fusion are key factors in vesicular trafficking in all eukaryotic cells, including the secretion and transport of plant pathogen virulence factors. In this study, we identified vesicle-fusion components that included 22 soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), four Sec1/Munc18 (SM) family proteins, and 10 Rab GTPases encoded in the genome of the vascular wilt pathogen Verticillium dahliae Vd991. Targeted deletion of two SNARE-encoding genes in V. dahliae, VdSec22 and VdSso1, significantly reduced virulence of both mutants on cotton, relative to the wild-type Vd991 strain. Comparative analyses of the secreted protein content (exoproteome) revealed that many enzymes involved in carbohydrate hydrolysis were regulated by VdSec22 or VdSso1. Consistent with a role of these enzymes in plant cell–wall degradation, pectin, cellulose, and xylan utilization were reduced in the VdSec22 or VdSso1 mutant strains along with a loss of exoproteome cytotoxic activity on cotton leaves. Comparisons with a pathogenicity-related exoproteome revealed that several known virulence factors were not regulated by VdSec22 or VdSso1, but some of the proteins regulated by VdSec22 or VdSso1 displayed different characteristics, including the lack of a typical signal peptide, suggesting that V. dahliae employs more than one secretory route to transport proteins to extracellular sites during infection.

Summary Wilt caused by Verticillium dahliae significantly reduces cotton yields, as host resistance in commercially cultivated Gossypium species is lacking. Understanding the molecular bases of disease resistance in non-commercial Gossypium species could galvanize the development of Verticillium wilt resistance in the cultivated species. Nucleotide binding site-leucine-rich repeat (NBS-LRR) proteins play a central role in plant defense against pathogens. In this study, we focused on the relationship between a locus enriched with eight NBS-LRR genes and Verticillium wilt resistance in G. barbadense. Independent virus-induced gene silencing of each of eight NBS-LRR genes in G. barbadense cultivar Hai 7124 revealed that silencing GbaNA1 alone compromised the resistance of G. barbadense to V. dahliae isolate Vd991. In cultivar Hai 7124, GbaNA1 could be induced by V. dahliae isolate Vd991 and by ethylene, jasmonic acid, and salicylic acid. Nuclear protein localization of GbaNA1 was demonstrated by transient expression. Sequencing the GbaNA1 ortholog in nine G. hirsutum accessions revealed that all carried a non-functional allele, due to a premature peptide truncation. In addition, all ten G. barbadense and nine G. hirsutum tested carried a full length (∼1140 amino acid) homolog of the V. dahliae race 1 resistance gene Gbve1, though some sequence polymorphisms were observed. V. dahliae Vd991 is a non-race 1 isolate that lacks the Ave1 gene. Thus, the resistance imparted by GbaNA1 appears to be mediated by a mechanism distinct from recognition of the fungal effector Ave1. This article is protected by copyright. All rights reserved.

Understanding pathogen evolution over time is vital for plant breeding and deployment of host resistance. In the context of a soilborne pathogen, the potential of host-directed evolution of a Verticillium dahliae race 1 isolate and genotypic variation of V. dahliae associated with two major hosts (lettuce and tomato) were determined. In total, 427 isolates were recovered over 6 years from a resistance screening nursery infested with a single V. dahliae race 1 isolate. In a separate study, an additional 206 isolates representing 163 and 43 isolates from commercial lettuce and tomato fields, respectively, were collected. Analyses of isolates recovered from the screening nursery over 6 years revealed no changes in the race and mating type composition but did uncover seven simple sequence repeat (SSR) variant genotypes. No significant genotypic variation in V. dahliae was observed between or within fields of either lettuce or tomato but pathogen populations were significantly differentiated between these two hosts. Replicated virulence assays of variant SSR genotypes on lettuce differential cultivars suggested no significant difference in virulence from the wild-type race 1 isolate introduced into the field. This suggests that deployed race 1 host resistance will be robust against the widespread race 1 populations in lettuce-growing regions at least for 6 years unless novel pathogen genotypes or races are introduced into the system.

Seedborne pathogens and pests limit production in many agricultural systems. Quarantine programs help prevent the introduction of exotic pathogens into a country, but few regulations directly apply to reducing the reintroduction and spread of endemic pathogens. Use of phytosanitary thresholds helps limit the movement of pathogen inoculum through seed, but the costs associated with rejected seed lots can be prohibitive for voluntary implementation of phytosanitary thresholds. In this paper, we outline a framework to optimize thresholds for seedborne pathogens, balancing the cost of rejected seed lots and benefit of reduced inoculum levels. The method requires relatively small amounts of data, and the accuracy and robustness of the analysis improves over time as data accumulate from seed testing. We demonstrate the method first and illustrate it with a case study of seedborne oospores of Peronospora effusa, the causal agent of spinach downy mildew. A seed lot threshold of 0.23 oospores per seed could reduce the overall number of oospores entering the production system by 90% while removing 8% of seed lots destined for distribution. Alternative mitigation strategies may result in lower economic losses to seed producers, but have uncertain efficacy. We discuss future challenges and prospects for implementing this approach.

Fungal transcription factors (TFs) implicated in the regulation of virulence gene expression have been identified in a number of plant pathogens. In Verticillium dahliae, despite its agricultural importance, few regulators of transcription have been characterized. In this study, a T-DNA insertion mutant with significantly reduced virulence towards cotton was identified. The T-DNA was traced to VdFTF1, a gene encoding a TF containing a Fungal_trans domain. Transient expression in onion epidermal cells indicated that VdFTF1 is localized to the nucleus. The VdFTF1-deletion strains displayed normal vegetative growth, mycelial pigmentation and conidial morphology, but exhibited significantly reduced virulence on cotton, suggesting that VdFTF1 is required exclusively for pathogenesis. Comparisons of global transcription patterns of wild-type and VdFTF1-deletion strains indicated that VdFTF1 affected the expression of 802 genes, 233 of which were associated with catalytic processes. These genes encoded 69 potentially secreted proteins, 43 of which contained a carbohydrate enzyme domain known to participate in pathogenesis during infection of cotton. Targeted gene deletion of one VdFTF1-regulated gene resulted in significantly impaired vascular colonization, as measured by quantitative polymerase chain reaction, as well as aggressiveness and symptom severity in cotton. In conclusion, VdFTF1, which encodes a TF containing a Fungal_trans domain, regulates the gene expression of plant cell wall degradation enzymes in V. dahliae, which are required for full virulence on cotton.

Three growth stages of each of 20 Sclerotinia minor isolates were tested for resistance to iprodione. Sclerotia and both vegetative and mature mycelium of each isolate were plated on potato-dextrose agar (PDA) amended with 0, 1, 5, 10, 25, and 100 µg of the fungicide per ml, and radial growth was measured. All wild-type field isolates were initially sensitive and did not grow in the presence of iprodione. However, fungicide resistance arose readily in vitro. All 20 isolates produced measurable growth (≥2 mm) on iprodione at 5 µg/ml after 2 weeks when started from mature mycelium, and 18 of 20 isolates produced measurable growth after 4 weeks when started from vegetative mycelium. In general, fungicide-resistant growth arose more frequently and mean colony diameters were significantly greater (P ≤ 0.05) with mature mycelium than with vegetative mycelium at all times and concentrations. In sclerotial germination tests, at least 1% of sclerotia germinated in 18 of 20 isolates after 2 weeks on iprodione at 5 µg/ml, and in 19 of 20 isolates after 5 weeks on 100 µg/ml. Of growth produced on 79 plates containing iprodione, 73 remained viable on PDA after 5 months, and 71 retained resistance to 5 µg/ml. Seventy of these also exhibited cross-resistance to vinclozolin, another dicarboximide fungicide. Pathogenicity of five fungicide-resistant strains was tested in greenhouse, microplot, and field experiments, with and without iprodione. Two months after in vitro production, one of the five resistant strains was avirulent, but disease incidence for the other four ranged from 40 to 75%, compared with 40% for the wild-type isolates. However, the virulence of the fungicide-resistant strains declined over time. Ten months after their production, two of the isolates were avirulent and disease incidence for the others ranged from 3 to 17%, compared with 40 to 90% for the wild-type isolates. The strains that remained virulent 5 and 7 months after in vitro production were not significantly controlled by iprodione sprayed according to labeled rates, although disease was significantly controlled by the fungicide in treatments inoculated with wild-type field isolates (P > 0.05). In experiments in commercial fields to determine the efficacy of fungicide sprays on the wild-type S. minor population, there was no evidence that fungicide resistance was the cause of lack of lettuce drop control observed in many coastal California fields. Application of fungicides at a less than optimal time may account for some incidents of control failure.

Endive (Cichorium endivia) and escarole (broad-leaf type of C. endivia) are two of the many leafy vegetables produced commercially in coastal California. For several years, both crops have been affected by a disease that causes soft, watery, brown leaf decay. In the tightly appressed heads of endive and escarole plants, decay generally spreads in a concentric circle, resulting in circular whorls of brown, rotted leaves within diseased heads. Such symptoms make the heads unmarketable. In the Salinas Valley (Monterey County), the disease was much more prevalent during 1998, when weather was affected by “El Niño.” Rhizoctonia solani was isolated consistently from symptomatic leaves of both endive and escarole. Pathogenicity was tested by placing two agar plugs of representative isolates inside the leaf whorls of 15 potted plants each of endive (cv. Tres Fine Maraicchere) and escarole (cv. Full Heart). Watery, brown leaf decay, similar to symptoms observed in the field, occurred on all plants within 7 days after inoculation, and R. solani was reisolated. Control plants, treated with sterile agar plugs, did not develop disease. Tests were repeated, and results were similar. Anastomosis-group testing revealed that four endive isolates belonged to AG2-2 (1). This appears to be the first report of leaf blight of endive and escarole caused by R. solani in California. Reference: (1) Sneh et al. 1991. Identification of Rhizoctonia Species. The American Phytopathological Society, St. Paul, MN.

Isolates of Verticillium dahliae from 15 different hosts and V. albo-atrum from alfalfa were tested for their ability to cause wilt on broccoli using a root-dip inoculation method. None of the isolates caused vascular discoloration in broccoli except those from cabbage and cauliflower that were weakly pathogenic. Broccoli cultivars Baccus, Greenbelt, Parasol, Patriot, and Symphony showed resistance to Verticillium infection. Re-isolated strains from fresh samples of internally discolored broccoli and cauliflower root tissues were unable to cause disease symptoms on re-inoculation of broccoli, but caused severe disease on cauliflower. Inoculation of 5-, 7-, 9-, or 11-week-old plants did not alter the resistance in broccoli or the susceptibility in cauliflower against Verticillium. Immunity of broccoli against infection by isolates of V. dahliae from non-crucifer hosts, its resistance against crucifer isolates, and as previously described, attrition of V. dahliae microsclerotia in soil by broccoli residue, coupled with its importance as a commercial vegetable, make broccoli an attractive rotation crop for the management of Verticillium wilt in many cropping systems.

Verticillium dahliae is widely distributed in potato and olive fields in Lebanon, causing serious economic losses. However, little is known about the inoculum source, population structure, and genetic diversity of the pathogen or the mechanisms of dissemination within Lebanon. To understand the population structure, a total of 203 isolates sampled from olive (n = 78) and potato (n = 125) were characterized for species, mating type, and race, and the genetic relationships were delineated using 13 microsatellite markers. All isolates except one from potato were V. dahliae, with 55.1 and 12.1% race 1, and 43.6 and 83.1% race 2 in olive and potato, respectively. The genetic structure of the studied population was best described by two large and two small clusters. Membership in the two large clusters was determined by the presence or absence of the effector gene Ave1. Furthermore, genetic structure was moderately associated with the host of origin but was weakly associated with the geographic origin. All but four isolates represented by three multilocus haploid genotypes were MAT1-2. This study identified a clear lack of gene flow between virulence genotypes of V. dahliae despite the proximity of these cropping systems and the wide distribution of genetic diversity among hosts and geographic regions in Lebanon.

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.

Vallad, G. E., Bhat, R. G., Koike, S. T., Ryder, E. J., and Subbarao, K. V. 2005. Weedborne reservoirs and seed transmission of Verticillium dahliae in lettuce. Plant Dis. 89:317-324. The seed transmission of Verticillium dahliae was evaluated in lettuce (Lactuca sativa). Seed collected from lettuce plants infected with V. dahliae were plated with or without surface sterilization on Sorenson’s modified NP10 medium. Of the seed plated with or without surface sterilization, 90 and 66%, respectively, yielded colonies of V. dahliae. The incidence of Verticillium wilt ranged from 55 to 80% among lettuce plants grown from seed harvested from infected plants. All evaluated isolates of V. dahliae were capable of seed transmission in lettuce. A V. tricorpus isolate failed to cause significant disease in lettuce or to become seedborne. Storage of contaminated seed at seven temperatures ranging from –20 to 15°C for up to 72 weeks did not reduce the incidence of V. dahliae in seed, whereas storage at room temperature (23 ± 2°C) for 20 to 52 weeks reduced the incidence of V. dahliae without affecting seed viability. Of the 11 weed species collected from fields with a known history of Verticillium wilt of lettuce, four yielded V. dahliae. Pathogenicity tests demonstrated that isolates of V. dahliae from Sonchus oleraceus, Capsella bursa-pastoris, and Solanum sarrachoides were as virulent as or more virulent than an isolate of V. dahliae from lettuce. These results demonstrate the potential of seedborne and weedborne inoculum to disseminate V. dahliae.

Previous studies in coastal California suggested that morning leaf wetness duration and temperature immediately after the prolonged leaf wetness period affect infection of lettuce by the downy mildew pathogen, Bremia lactucae. In this study, spatial analysis tools in a geographic information system were used to interpolate disease assessment data and then relate them to weather variables measured in 1995 and 1997 at weather stations in the Salinas Valley. Among the variables monitored at these weather stations, midday temperature (10:00 A.M. to 2:00 P.M.) was related most strongly to the interpolated downy mildew incidence in a circular area (radius = 5 km) around each station (r = 0.52, P < 0.0001); the higher the midday temperature, the lower the disease incidence. High humidity and prolonged morning leaf wetness duration also were associated with high downy mildew incidence. Cluster analysis resulted in distinct regions with different midday temperatures, which overlapped well (92.2% of the total area) with regions distinguished in previous cluster analyses of downy mildew incidence. Clusters of morning relative humidity showed similar patterns, although they overlapped less well with clusters of disease incidence. These results confirmed that midday temperature is an important determining factor for lettuce downy mildew, and its effects should be incorporated into a disease warning system for coastal California. Cluster analyses based on the effects of temperature have great potential for use in regional downy mildew risk assessment.

Temporal and spatial patterns of lettuce (Lactuca sativa) drop caused by Sclerotinia minor and S. sclerotiorum were determined in lettuce fields in the Salinas, Santa Maria, and San Joaquin Valleys in California during 1995 to 1998. Of the 25 commercial fields assessed, 14 had predominantly S. minor, 9 had predominantly S. sclerotiorum, and 2 had varying levels of both species. Sclerotinia infections were classified based on symptoms: those caused directly by eruptive germination of sclerotia (type I) and those caused by the airborne ascospores (type II). The precise location of diseased and healthy plants was mapped and lettuce drop progress was determined at different crop growth stages. Spatial patterns of disease incidence were analyzed using 1-by-1-, 2-by-2-, 3-by-3-, and 4-by-4-m quadrat sizes. Regardless of the analytical method employed, disease incidence with type I infection showed an aggregated pattern in a majority of the fields evaluated and random patterns in fields where incidence was low. In all fields with type I infection, disease progress followed the monomolecular model, typical of soilborne diseases. For fields with aggregated distribution, spatial dependence was observed up to 10 m and was either isotropic or random in direction, suggesting the potential influence of tillage operations on inoculum distribution and disease incidence. Lettuce drop incidence in fields with type II infection was erratic in time and peaked within a very short time. However, disease incidence showed an aggregated pattern in all fields evaluated. Spatial dependence of quadrats generally was detected in two adjacent directions, suggesting a directional gradient perhaps caused by wind direction during ascospore dissemination. Increasing quadrat sizes usually increased the degree of aggregation of lettuce drop, but not the distribution pattern itself. These results demonstrate that the source of inoculum and the type of infections they cause are most likely to determine spatial patterns of lettuce drop in the field.

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 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.

Field experiments were conducted to determine the population dynamics of Sclerotinia minor and incidence of lettuce drop at two sites during 1995 to 1998. Rotation treatments at the Spence site, which had a low density of inoculum (3 of soil) that was distributed randomly, included: continuous lettuce (LLL), lettuce rotated with broccoli (LBL), and lettuce followed by a fallow period (LFL). Treatments at the Hartnell site, which had a high density of inoculum (>7 sclerotia per 100 cm3 of soil) that was distributed uniformly, included: continuous lettuce (LLLL), alternate crops of broccoli and lettuce (BLBL), continuous broccoli or lettuce (BBLL), and fallow-lettuce-fallow-lettuce (FLFL). Under continuous lettuce cropping (LLLL) at the Hartnell site, a progressively aggregated spatial pattern of inoculum distribution developed, despite the initial uniform distribution of high inoculum density. In the fallow treatment (FLFL), the spatial pattern tended to be aggregated following a lettuce crop and less aggregated or random when left fallow. In contrast to these two treatments, treatments involving rotations with broccoli (BLBL and BBLL) exhibited consistently random spatial patterns of inoculum regardless of the crop in the field. The marginal increases in the number of sclerotia contributed by the few diseased lettuce plants were offset by the significant reductions in the number of sclerotia by the broccoli residue. Spatial patterns of disease incidence reflected the pattern of inoculum distribution in the soil at the Hartnell site. Higher inoculum density coupled with an aggregated distribution was associated with an aggregation in disease incidence. At Spence, this correlation was poor in most seasons because of progressive decline in the lettuce drop incidence and lack of treatment differences. In greenhouse experiments, the competence volume for S. minor sclerotia was quantified, which was calculated to be 25 3 for 100% infection and 200 cm3 for 50% infection. Thus, in 100 cm3 of soil, a minimum of four to five sclerotia are needed for 100% of infection, explaining the high correlation between inoculum density and disease incidence.

Verticillium dahliae (Kleb.) is known worldwide as a destructive soilborne pathogen with a wide host range (2). Reports of V. dahliae attacking cucurbits are generally limited to ‘Casaba’ and ‘Persian’ type melons. During August and September of 2004 to 2006, fields of seedless watermelon (Citrullus lanatus [Thunb.] Matsum. & Nak.) and pollinators in Yoakum County, Texas, exhibited severe symptoms of vine decline. There was no apparent difference between diploid and triploid watermelon cultivars. Night-time temperatures during July, August, and September averaged 20°C or less. Losses were estimated in excess of one-half million dollars. Symptoms consisted of leaf yellowing, wilting, and gradual death of the leaves, but stems generally remained green. The xylem exhibited a uniform tan-to-light brown discoloration that often extended throughout the vine. Dead plants had numerous microsclerotia embedded throughout the root and crown. Crown and root sections (1 cm long) from triploid plants were surface disinfected in 0.5% NaOCl for 30 s, transferred to water agar with 100 ppm of streptomycin sulfate, and incubated at 25°C. Slow-growing colonies were transferred to potato dextrose agar after approximately 72 h. V. dahliae was identified on the basis of morphology (3). Pathogenicity of four selected isolates was determined on the watermelon cultivars used to identify races of Fusarium oxysporum f. sp. niveum (Fon). Flasks containing 100 ml of medium (1) were inoculated with a 1-ml spore suspension at 1 × 105 spores/ml for each isolate and placed on an orbital shaker for 6 days at 100 rpm with continuous near-UV/fluorescent lighting at 25°C. Roots of approximately 40 plants of each of five watermelon cultivars (1 to 2 true-leaf stage) were trimmed to 2 cm long and root dipped for 2 min in the spore suspension (1 × 106/ml) of each isolate. Each cultivar/isolate combination and controls were transplanted into 10 pots (1.5 liter) with four plants per pot. The pots were transferred to the greenhouse where soil temperatures ranged between 15 and 25°C and were fertilized (Jack's fertilizer solution) every 7 days. Plants were rated at the end of 28 days as 1 = healthy, 2 = stunting (≤50% of controls), 3 = wilting, and 4 = dead. Initial wilting was observed within 7 to 10 days postinoculation. All four isolates caused varying degrees of vascular discoloration, stunting, wilting, and plant death. The pathogen was reisolated from symptomatic plants but not the controls. Mean disease ratings for the most virulent Texas isolate (28-040215) on ‘Black Diamond’, ‘Charleston Gray’, ‘Dixie Lee’, ‘Calhoun Gray’, and ‘PI 296341 FR’ were 2.7, 3.0, 3.0, 2.9, and 2.9, respectively. All watermelon Fon differentials were equally susceptible to V. dahliae in these studies. Historically, Verticillium wilt has been a problem in this area, which has been in cotton production for approximately 100 years. In the past decade, watermelon production has increased substantially to approximately 3,600 ha in the Texas High Plains. To our knowledge, this is the first known report of Verticillium wilt on watermelon in Texas. References: (1) R. G. Esposito and A. M. Fletcher. Arch. Biochem. Biophys. 93:369, 1961. (2) G. F. Pegg and B. L. Brady. Verticillium Wilts. CABI Publishing, New York, 2002. (3) H. C. Smith. N. Z. J. Agr. Res. 8:450, 1965.

Subbarao, K. V., Kabir, Z., Martin, F. N., and Koike, S. T. 2007. Management of soilborne diseases in strawberry using vegetable rotations. Plant Dis. 91:964-972. The influence of crop rotation on soilborne diseases and yield of strawberry (Fragaria × ananassa) was determined at a site infested with Verticillium dahliae microsclerotia and at another with no known history of V. dahliae infestation during 1997 to 2000. The rotations studied at the V. dahliae-infested site were (i) broccoli-broccoli-strawberry, (ii) Brussels sproutsstrawberry, and (iii) lettuce-lettuce-strawberry; the treatments at the site with no history of V. dahliae were (i) broccoli-broccoli-strawberry, (ii) cauliflower-cauliflower-strawberry, and (iii) lettuce-lettuce-strawberry. The effects of rotation on V. dahliae and Pythium populations, strawberry vigor, Verticillium wilt severity, and strawberry fruit yield were compared with a standard methyl bromide + chloropicrin fumigated control treatment at both sites. Rotations did not alter total population levels of Pythium spp. at either study site. However, V. dahliae microsclerotia were significantly reduced with broccoli and Brussels sprouts rotations compared with lettuce rotations at the V. dahliae-infested site. Reduced propagules led to lower Verticillium wilt severity on strawberry plants in the broccoli and Brussels sprouts rotations than in lettuce-rotated plots. Strawberry vigor and fruit yield were significantly lower in lettuce-rotated plots than in broccoli- and Brussels sprouts-rotated plots. Despite no detectable microsclerotia at the other site, strawberry vigor and fruit yield were greatest in plots rotated with broccoli, intermediate with cauliflower, and lowest with lettuce. None of the rotation treatments were better than the fumigated control for all variables measured. In the absence of fumigation, rotation with broccoli and Brussels sprouts is an effective cultural practice for managing Verticillium wilt in strawberry production; whereas, in fields with no detectable V. dahliae, broccoli is also a feasible rotational crop that enhances strawberry growth and yield. According to a cost-benefit analysis, the broccoli-strawberry rotation system could be an economically viable option provided growers are able to alternate years for strawberry cultivation.

Verticillium wilt of lettuce, caused by the soilborne pathogen Verticillium dahliae, poses a serious threat to the California lettuce industry. Knowledge of disease development and its impact on postharvest marketability would facilitate better management of the affected fields. This study investigated postharvest marketability of 22 lettuce varieties harvested from two Verticillium-infested commercial lettuce fields in Salinas and Watsonville, CA, in 2005 using a randomized complete block design. Periodic sampling to monitor disease in several crisphead varieties in the field demonstrated that root symptoms developed quickly at later stages of heading, followed by the onset of foliar symptoms as the crop reached harvest maturity. Harvested marketable heads were vacuum cooled soon after harvest to about 4°C and maintained at this temperature in commercial coolers. The impact of V. dahliae on postharvest marketability was assessed based on the percentage of heads per case deemed marketable following 1, 2, and 3 weeks of refrigerated storage. Across both field experiments, the average disease incidence and postharvest marketability ranged from 4.2 to 87.5% and from 69.4 to 100.0%, respectively, among lettuce types and varieties. The Pearson correlation analysis detected no significant relationship between disease incidence and postharvest marketability across all varieties tested (r = 0.041, P = 0.727), or within lettuce types, even though V. dahliae was recovered from 34% of the plants harvested, and recovery ranged from 0 to 73.3% for V. dahliae and from 10 to 91.7% for non-V. dahliae (V. isaacii or V. klebahnii) species. These findings demonstrate that growers can harvest lettuce from an infested field before foliar symptoms develop with negligible impact by Verticillium spp. on postharvest marketability or quality.

Spatial patterns of lettuce big-vein (LBV) incidence under furrow, sprinkler, and subsurface drip irrigation systems were determined. Because LBV pathogen is a virus and is vectored by the soilborne chytrid Olpidium brassicae, different irrigation systems likely affect the movement of the vector and were hypothesized to result in different distribution patterns and levels of the disease. Lettuce plants were mapped by recording the location of each LBV-infected or healthy plant in arbitrarily selected plots of sizes 16 by 30, 20 by 30, and 18 by 50 m in Salinas, Gonzales, and Santa Maria in California. Data were arrayed into different quadrat sizes by rearrangement, and disease incidence was calculated for each quadrat. Frequency distribution analysis and spatial autocorrelation analyses were performed on this data. LBV incidence was aggregated in all furrow-irrigated fields, four of five subsurface drip-irrigated fields, and two of three sprinkler-irrigated fields. The remaining fields had a random distribution. As the quadrat size increased, index of aggregation decreased, and vice versa. In fields under sprinkler irrigation, regardless of whether the spatial pattern of LBV was random or aggregated, no directional orientation occurred. However, under furrow or subsurface drip irrigation, the aggregation mostly occurred across the rows. Although irrigation type influenced LBV distribution pattern and incidence in lettuce fields, the differential effects of irrigation type on vector O. brassicae could not be discerned in this study. The sprinkler irrigation practiced in lettuce production until thinning may influence the vector distribution and the subsequent irrigation methods adapted for the remainder of the season in individual fields may play a significant role in disease incidence.

Verticillium wilt, caused by Verticillium dahliae, is an important disease of cotton worldwide. Isolates of V. dahliae can be characterized as race 1 or race 2 based on the responses of differential cultivars of tomato and lettuce, or as defoliating or nondefoliating based on symptom expression in cotton. To investigate the frequency and distribution of races and defoliation phenotypes of cotton-associated V. dahliae, 317 isolates from China, Israel, Turkey, and the United States were tested by polymerase chain reaction (PCR) using defoliating, nondefoliating, and race 1- and race 2-specific primers DF/DR, NDF/NDR, VdAve1F/VdAve1R, and VdR2F/VdR2R, respectively. Of the total, 97.2% of isolates genotyped as defoliating were also characterized as race 2, while 90.8% of isolates genotyped as nondefoliating were also genotyped as race 1. To verify these results, three cotton cultivars—‘FM 2484B2F’ (highly resistant), ‘98M-2983’ (highly susceptible), and ‘CA4002’ (partially resistant)—used as differentials were each inoculated with 10 isolates characterized by PCR: six defoliating/race 2 strains (GH1005, GH1021, HN, XJ2008, XJ592, and reference strain Ls17) and four nondefoliating/race 1 strains (GH1015, GH1016, GH1020, and reference strain Ls16). All defoliating/race 2 isolates except for Ls17 caused defoliation on 98M-2983 and CA4002. Isolate Ls17 caused defoliation on 98M-2983 only. The nondefoliating/race 1 isolates caused Verticillium wilt symptoms devoid of defoliation on 98M-2983. The greenhouse assays confirmed the molecular identification of race and defoliation phenotype. Although the existence of races has not been previously established among V. dahliae isolates from cotton, the long-established nondefoliating and defoliating population structure corresponded with V. dahliae races 1 and 2, respectively.

Verticillium wilt caused by Verticillium dahliae is an important soilborne disease of pepper (Capsicum species) worldwide. Most commercial pepper cultivars lack resistance to this pathogen. Our objective was to identify resistance to two V. dahliae isolates in wild and cultivated Capsicum accessions from the core collection of the National Plant Germplasm System of the USDA. Screening of 397 Capsicum accessions against two V. dahliae isolates (Vdca59 and VdCf45) was performed in a greenhouse. Seventy-eight accessions selected from this screen were further evaluated in a follow-up experiment. In total, 21 (26.9%) and 13 (16.6%) Capsicum accessions tested were resistant to Verticillium wilt when inoculated with V. dahliae isolates VdCa59 and VdCf45, respectively. Eight accessions (Grif 9073, PI 281396, PI 281397, PI 438666, PI 439292, PI 439297, PI 555616, and PI 594125) were resistant to Verticillium wilt against both V. dahliae isolates. On the basis of Germplasm Resources Information Network data, two of the Capsicum annuum accessions (Grif 9073 and PI 439297) were also resistant to Phytophthora root rot disease. These sources of multiple disease resistance will be useful to pepper breeding programs.

Verticillium is a genus that includes major vascular wilt pathogens. Recently, multilocus phylogenetic analyses of the genus identified five new species, including Verticillium isaacii and V. klebahnii, both of which occur in agricultural soils in coastal California and have been isolated from asymptomatic and diseased spinach and lettuce plants. Little data are available regarding their pathogenicity and virulence on a broader range of crops important to the region. Four isolates each of V. isaacii and V. klebahnii along with two reference isolates of V. dahliae races 1 and 2 were inoculated on eight crops (artichoke, cauliflower, eggplant, lettuce, pepper, tomato, spinach, and strawberry) in a greenhouse experiment. After 8 weeks, plants were assessed for disease severity to determine the relative host ranges of Verticillium isolates. Additionally, 13 lettuce lines resistant to race 1 and partially resistant to race 2 of V. dahliae were screened against V. isaacii and V. klebahnii to evaluate their responses. Three of four V. isaacii and four of four V. klebahnii isolates tested were nonpathogenic on all crops tested except those indicated below. One V. isaacii isolate caused wilt on artichoke and ‘Salinas’ lettuce and most isolates of both species caused varying degrees of Verticillium wilt on strawberry. Lettuce lines resistant to V. dahliae race 1 and partially resistant to V. dahliae race 2 also exhibited resistance to all of the isolates of V. isaacii and V. klebahnii. Thus, at least some isolates in the populations of V. isaacii and V. klebahnii have the potential to become significant pathogens of coastal California crops. However, resistance developed against V. dahliae also offers resistance to the pathogenic isolates of both species, at least in lettuce.

Production of oospores by Peronospora effusa, the causal agent of downy mildew on spinach (Spinacia oleracea), was reported on spinach seed over three decades ago. In view of the rapid proliferation of new races of P. effusa worldwide, seedborne transmission of this pathogen has been suspected but methods to test the viability of seedborne oospores have not been available. Eighty-two seed lots of contemporary spinach cultivars were evaluated for the presence of P. effusa using a seed-wash method and the sediment was examined by microscopy. Of the analyzed seed lots, 16% were positive for oospores and an additional 6% for sporangiophores characteristic of P. effusa. Application of a P. effusa-specific quantitative polymerase chain reaction assay showed that 95% of the 59 tested seed lots were positive for P. effusa. The viability of oospores from five seed lots that were proven to carry the pathogen from the above tests was tested using two independent methods, one involving plasmolysis and the other trypan blue staining. The oospores plasmolyzed in 4 M sodium chloride and were deplasmolyzed in water, demonstrating an active and viable cell membrane. Similarly, viable oospores failed to take up the trypan blue stain. Overall, 59% of the oospores were viable in the plasmolysis test and 45% with the trypan blue test. These results indicate the presence of P. effusa oospores in contemporary spinach seed lots, and suggest that the transmission of viable oospores of P. effusa in spinach seed does occur. Elimination of the pathogen on seed, in addition to other management approaches, will be useful in reducing the extent and severity of downy mildew on spinach crops and diminishing pathogen spread through seed.

NONRACE-SPECIFIC DISEASE RESISTANCE (NDR1) is a widely characterized gene that plays a key role in defense against multiple bacterial, fungal, oomycete and nematode plant pathogens. NDR1 is required for activation of resistance by multiple NB and LRR-containing (NLR) protein immune sensors and contributes to basal defense. The role of NDR1 in positively regulating salicylic acid (SA)-mediated plant defense responses is well documented. However, ndr1-1 plants flower earlier and show accelerated development in comparison to wild type (WT) Arabidopsis plants, indicating that NDR1 is a negative regulator of flowering and growth. Exogenous application of gibberellic acid (GA) further accelerates the early flowering phenotype in ndr1-1 plants, while the GA biosynthesis inhibitor paclobutrazol attenuated the early flowering phenotype of ndr1-1, but not to WT levels, suggesting partial resistance to paclobutrazol and enhanced GA response in ndr1-1 plants. Mass spectroscopy analyses confirmed that ndr1-1 plants have 30–40% higher levels of GA3 and GA4, while expression of various GA metabolic genes and major flowering regulatory genes is also altered in the ndr1-1 mutant. Taken together this study provides evidence of crosstalk between the ndr1-1-mediated defense and GA-regulated developmental programs in plants.

Bremia lactucae is an obligate, oomycete pathogen of lettuce that causes leaf chlorosis and necrosis and adversely affects marketability. The disease has been managed with a combination of host resistance and fungicide applications with success over the years. Fungicide applications are routinely made under the assumption that inoculum is always present during favorable environmental conditions. This approach often leads to fungicide resistance in B. lactucae populations. Detection and quantification of airborne B. lactucae near lettuce crops provides an estimation of the inoculum load, enabling more judicious timing of fungicide applications. We developed a quantitative polymerase chain reaction (qPCR)-based assay using a target sequence in mitochondrial DNA for specific detection of B. lactucae. Validation using amplicon sequencing of DNA from 83 geographically diverse isolates, representing 14 Bremia spp., confirmed that the primers developed for the TaqMan assays are species specific and only amplify templates from B. lactucae. DNA from a single sporangium could be detected at a quantification cycle (Cq) value of 32, and Cq values >35 were considered to be nonspecific. The coefficient of determination (R2) for regression between sporangial density derived from flow cytometry and Cq values derived from the qPCR was 0.86. The assay was deployed using spore traps in the Salinas Valley, where nearly half of U.S. lettuce is produced. The deployment of this sensitive B. lactucae-specific assay resulted in the detection of the pathogen during the 2-week lettuce-free period as well as during the cropping season. These results demonstrate that this assay will be useful for quantifying inoculum load in and around the lettuce fields for the purpose of timing fungicide applications based on inoculum load.

Finding effective non-fumigant solutions to soil disinfestation is an international priority for sustainable strawberry production. In these studies at Ventura, California, combinations of steam, s...

Introduction: The causal agents of Verticillium wilts are globally distributed pathogens that cause significant crop losses every year. Most Verticillium wilts are caused by V. dahliae, which is pathogenic on a broad range of plant hosts, whereas other pathogenic Verticillium species have more restricted host ranges. In contrast, V. longisporum appears to prefer brassicaceous plants and poses an increasing problem to oilseed rape production. Taxonomy: Kingdom Fungi; Phylum Ascomycota; Class Sordariomycetes; Subclass Hypocreomycetida; Family Plectosphaerellaceae; genus Verticillium. Disease symptoms: Dark unilateral stripes appear on the stems of apparently healthy looking oilseed rape plants at the end of the growing season. Microsclerotia are subsequently formed in the stem cortex beneath the epidermis. Genome: Verticillium longisporum is the only non-haploid species in the Verticillium genus, as it is an amphidiploid hybrid that carries almost twice as much genetic material as the other Verticillium species as a result of interspecific hybridization. Disease management: There is no effective fungicide treatment to control Verticillium diseases, and resistance breeding is the preferred strategy for disease management. However, only a few Verticillium wilt resistance genes have been identified, and monogenic resistance against V. longisporum has not yet been found. Quantitative resistance exists mainly in the Brassica C-genome of parental cabbage lines and may be introgressed in oilseed rape breeding lines. Common name: Oilseed rape colonized by V. longisporum does not develop wilting symptoms, and therefore the common name of Verticillium wilt is unsuitable for this crop. Therefore, we propose 'Verticillium stem striping' as the common name for Verticillium infections of oilseed rape.

Biological suppression of soilborne diseases with minimal use of outside interventive actions has been difficult to achieve in high input conventional crop production systems due to the inherent risk of pest resurgence. This review examines previous approaches to the management of soilborne disease as precursors to the evolution of a systems-based approach, in which plant disease suppression through natural biological feedback mechanisms in soil is incorporated into the design and operation of cropping systems. Two case studies are provided as examples in which a systems-based approach is being developed and deployed in the production of high value crops: lettuce/strawberry production in the coastal valleys of central California (United States) and sweet basil and other herb crop production in Israel. Considerations for developing and deploying system-based approaches are discussed and operational frameworks and metrics to guide their development are presented with the goal of offering a credible alternative to conventional approaches to soilborne disease management.

Downy mildew disease of spinach, caused by Peronospora effusa, is managed in conventional fields by a combination of host resistance and scheduled fungicide applications. Fungicides are currently applied to prevent downy mildew epidemics regardless of the infection status of spinach crops. A more streamlined approach would be to develop methods to target either latent infections for fungicide application in conventional production systems or to hasten harvest in organic production. In this study, conventional polymerase chain reaction (PCR) was applied to detect P. effusa DNA in symptomless spinach leaves in three spatially and temporally separated field plots, each containing four 2-m beds, 35 m in length. Spinach leaves were sampled weekly at 3-m intervals at 48 locations throughout each plot. Initial samples were asymptomatic and yet PCR enabled detection of P. effusa DNA extracted from sampled spinach leaves. Detection of latent downy mildew infection in spinach leaves was confirmed by PCR as early as 7 days prior to symptom development. The limit of pathogen DNA detection in spinach leaves was calculated at 10 pg using the conventional PCR approach. Quantitative PCR with TaqMan methodology revealed the presence of inhibitors from spinach leaf DNA extracts and affected amplification efficiencies, but not when diluted, enabling detection of P. effusa DNA at a concentration of

Potato (Solanum tuberosum L.) is one of the most important staple foods in many parts of the world including China. In recent years, Verticillium wilt has become a severe threat to potato production in China. During 2015 to 2016, 287 samples of symptomatic potato plants were collected from 15 counties in five provinces from northern China. One hundred and eighty-seven Verticillium-like colonies were isolated from these samples and identified to species based on cultural and morphological characteristics, and multigene phylogeny based on the partial sequences of actin (ACT), elongation factor 1-alpha (EF1α), glyceraldehyde-3-phosphate dehydrogenase (GPD), and tryptophan synthase (TS) genes. A consensus-rooted most parsimonious phylogenetic tree was generated from the data. One hundred and fifteen isolates comprising 61.5% of the total were identified as Verticillium dahliae, and the remaining 38.5% of the isolates were identified as V. nonalfalfae. V. dahliae was widely distributed in Shaanxi (84.1%), Inner Mongolia (76.7%), Gansu (12.8%), and Qinghai (100%, representing a single isolate). V. dahliae was not recovered from the samples in Ningxia. V. nonalfalfae dominated the collections from Gansu (87.2%) and Ningxia (100%) but was also recovered from Shaanxi (15.9%) and Inner Mongolia (23.3%) at lower frequencies. Neither V. albo-atrum nor V. alfalfae was recovered from the sampled areas. The V. nonalfalfae isolates were predominantly isolated from the samples collected from altitudes above 1,800 m, and in contrast, V. dahliae isolates were mainly recovered from fields sampled below 1,800 m. The optimum temperature for the colony growth of V. nonalfalfae was lower (20°C) than that for V. dahliae (25°C). Pathogenicity tests demonstrated that V. dahliae and V. nonalfalfae were both pathogens of potato Verticillium wilt, with V. dahliae isolates exhibiting higher virulence than V. nonalfalfae isolates regardless of the collection area of the species. This is the first documentation of V. nonalfalfae infecting S. tuberosum in China and the higher altitudes associated with infections of V. nonalfalfae anywhere in the world.

Some volatile compounds (VC) play critical roles in intra- and interspecies interactions. To investigate roles of VC in fungal ecology, we characterized how VC produced by Verticillium spp., a group of broad-host-range soilborne fungal pathogens, affect plant growth and development. VC produced by 19 strains corresponding to 10 species significantly enhanced the growth of Arabidopsis thaliana and Nicotiana benthamiana. Analysis of VC produced by four species revealed the presence of diverse compounds, including those previously shown to affect plant growth. Using A. thaliana, we investigated the mechanism underpinning plant growth enhancement by Verticillium dahliae VC. Allometric analysis indicated that VC caused preferential resource allocation for root growth over shoot growth. Growth responses of A. thaliana mutants defective in auxin or ethylene signaling suggested the involvement of several components of auxin signaling, with TIR3 playing a key role. AUX1, TIR1, and AXR1 were also implicated but appeared to play lesser roles. Inhibition of auxin efflux using 1-naphthylphthalamic acid blocked VC-mediated growth enhancement. Spatial and temporal expression patterns of the auxin-responsive reporter DR5::GUS indicated that the activation of auxin signaling occurred before enhanced plant growth became visible. Results from this study suggest critical yet overlooked roles of VC in Verticillium ecology and pathology.

© 2018 The American Phytopathological Society. Plant pathogens migrate to new regions through human activities such as trade, where they may establish themselves and cause disease on agriculturally important crops. Verticillium wilt of lettuce, caused by Verticillium dahliae, is a soilborne fungus that was introduced to coastal California via infested spinach seeds. It has caused significant losses for lettuce growers. Once introduced, Verticillium wilt could be managed by fumigating with methyl bromide and chloropicrin, but this option is no longer available. Growers can also manage the disease by planting broccoli or not planting spinach. These control options require long-term investments for future gain. Verticillium wilt can also be prevented or controlled by testing and providing spinach seeds with little or no V. dahliae infestation. However, seed companies have been reluctant to test or clean spinach seeds, as spinach crops are not affected by Verticillium wilt. Thus, available control options are affected by externalities. Renters and other producers with short time horizons will not undertake long-term investments and seed companies do not take into account the effect of their decision not to test on lettuce producers. We review the literature on the economics of managing crop disease; discuss the economics of managing Verticillium wilt; and review the recent research on the externalities that arise with short-term growers, and between seed companies and growers due to Verticillium wilt. An externality arises whenever the actions of one individual or firm affects the payoffs to another individual or firm not involved in a specific transaction. These externalities have important implications for the management of Verticillium wilt and, more broadly, for the management of migratory pathogens and the diseases they cause in agriculture in general. This review is of interest to policy-makers, the producers, marketers, seed companies, and researchers.

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.

Cutinases have been implicated as important enzymes during the process of fungal infection of aerial plant organs. The function of cutinases in the disease cycle of fungal pathogens that invade plants through the roots has been less studied. Here, functional analysis of 13 cutinase (carbohydrate esterase family 5 domain–containing) genes (VdCUTs) in the highly virulent vascular wilt pathogen Verticillium dahliae Vd991 was performed. Significant sequence divergence in cutinase family members was observed in the genome of V. dahliae Vd991. Functional analyses demonstrated that only VdCUT11, as purified protein, induced cell death and triggered defense responses in Nicotiana benthamiana, cotton, and tomato plants. Virus-induced gene silencing showed that VdCUT11 induces plant defense responses in Nicotiana benthamania in a BAK1 and SOBIR-dependent manner. Furthermore, coinfiltration assays revealed that the carbohydrate-binding module family 1 protein (VdCBM1) suppressed VdCUT11-induced cell death and other defense responses in N. benthamiana. Targeted deletion of VdCUT11 in V. dahliae significantly compromised virulence on cotton plants. The cutinase VdCUT11 is an important secreted enzyme and virulence factor that elicits plant defense responses in the absence of VdCBM1.