Short-Term Host Selection Pressure Has Little Effect on the Evolution of a Monoclonal Population of Verticillium dahliae Race 1.

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.

Identification of fungal metabolites from inside Gallus gallus domesticus eggshells by non-invasively detecting volatile organic compounds (VOCs).

The natural porosity of eggshells allows hen eggs to become contaminated with microbes from the nesting material and environment. Those microorganisms can later proliferate due to the humid ambient conditions while stored in refrigerators, causing a potential health hazard to the consumer. The microbes' volatile organic compounds (mVOCs) are released by both fungi and bacteria. We studied mVOCs produced by aging eggs likely contaminated by fungi and fresh eggs using the non-invasive detection method of gas-phase sampling of volatiles followed by gas chromatography/mass spectrometry (GC/MS) analysis. Two different fungal species (Cladosporium macrocarpum and Botrytis cinerea) and two different bacteria species (Stenotrophomas rhizophila and Pseudomonas argentinensis) were identified inside the studied eggs. Two compounds believed to originate from the fungi themselves were identified. One fungus-specific compound was found in both egg and the fungi: trichloromethane. Graphical abstract Trichloromethane is a potential biomarker of fungal contamination of eggs.

Nondefoliating and Defoliating Strains from Cotton Correlate with Races 1 and 2 of Verticillium dahliae.

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.

Mapping Fusarium wilt race 1 resistance genes in cotton by inheritance, QTL and sequencing composition.

Knowledge of the inheritance of disease resistance and genomic regions housing resistance (R) genes is essential to prevent expanding pathogen threats such as Fusarium wilt [Fusarium oxysporum f.sp. vasinfectum (FOV) Atk. Sny & Hans] in cotton (Gossypium spp.). We conducted a comprehensive study combining conventional inheritance, genetic and quantitative trait loci (QTL) mapping, QTL marker-sequence composition, and genome sequencing to examine the distribution, structure and organization of disease R genes to race 1 of FOV in the cotton genome. Molecular markers were applied to F(2) and recombinant inbred line (RIL) interspecific mapping populations from the crosses Pima-S7 (G. barbadense L.) × 'Acala NemX' (G. hirsutum L.) and Upland TM-1 (G. hirsutum) × Pima 3-79 (G. barbadense), respectively. Three greenhouse tests and one field test were used to obtain sequential estimates of severity index (DSI) of leaves, and vascular stem and root staining (VRS). A single resistance gene model was observed for the F(2) population based on inheritance of phenotypes. However, additional inheritance analyses and QTL mapping indicated gene interactions and inheritance from nine cotton chromosomes, with major QTLs detected on five chromosomes [Fov1-C06, Fov1-C08, (Fov1-C11 ( 1 ) and Fov1-C11 ( 2)) , Fov1-C16 and Fov1-C19 loci], explaining 8-31% of the DSI or VRS variation. The Fov1-C16 QTL locus identified in the F(2) and in the RIL populations had a significant role in conferring FOV race 1 resistance in different cotton backgrounds. Identified molecular markers may have important potential for breeding effective FOV race 1 resistance into elite cultivars by marker-assisted selection. Reconciliation between genetic and physical mapping of gene annotations from marker-DNA and new DNA sequences of BAC clones tagged with the resistance-associated QTLs revealed defenses genes induced upon pathogen infection and gene regions rich in disease-response elements, respectively. These offer candidate gene targets for Fusarium wilt resistance response in cotton and other host plants.

Analysis of Verticillium dahliae Suggests a Lack of Correlation Between Genotypic Diversity and Virulence Phenotypes.

Verticillium dahliae causes severe wilt and recurring losses in numerous agricultural and ornamental hosts worldwide. Two virulence phenotypes (races) have been identified based on the Ve resistance gene and its homologs but their distribution and evolutionary history are unknown. Sequence analyses of the intergenic spacer of the ribosomal DNA and amplified fragment length polymorphism markers suggested an absence of correlation between genotypic diversity and virulence phenotypes. Additionally, both race 1 and 2 phenotypes were isolated in various geographic regions and hosts. Sustained levels of migration of both virulence phenotypes among various geographic regions were evident, and the study also suggested that both virulence phenotypes infect a variety of hosts, regardless of the availability of resistant cultivars. Given the high genotypic diversity observed in V. dahliae, more than the two known virulence phenotypes may be present in nature but not yet identified because of the current lack of sources of resistance other than the Ve gene and its homologs. The inclusion of various genotypes exhibiting the same virulence phenotype may greatly improve the long-term effectiveness of resistance to race 2 of V. dahliae regardless of the host. This study also confirms the transcontinental gene flow and high genotypic diversity of V. dahliae affecting lettuce in coastal California regardless of the molecular markers employed.

Population structure of Rhizoctonia oryzae-sativae in California rice fields.

Six pairs of single-locus microsatellite primers were developed to study the population structure of Rhizoctonia oryzae-sativae, the cause of aggregate sheath spot disease of rice, among and within three rice-growing areas in California over a 3-year period. A high level of gene flow among growing areas was indicated by low population subdivision according to analysis of molecular variance and moderate to no population differentiation between pairs of populations based on the fixation index (F(ST)). Gametic equilibrium of most pairs of microsatellite loci, high numbers of unique multilocus genotypes, and high genotypic diversity indicated extensive sexual recombination within growing areas. Because there was little differentiation among populations in all hierarchical levels, including among growing areas within sampling years, fields within growing areas, and corners within individual fields, a high level of gene flow was revealed in all levels. Basidiospores were likely the main vehicle of gene flow among populations, including short and long distances. Asexual inocula (sclerotia and mycelia) probably overwinter because a few clones were detected over a 2-year period within the same field. A few clones were shared among fields but were not commonly shared among growing areas.

Efficacy of Natural Plant Products on the Control of Aggregate Sheath Spot of Rice.

Aqueous extracts from ginger, pepper, basil, and garlic plants and essential oils from neem, garlic, lemongrass, and cinnamon were evaluated for their antagonistic effects against Rhizoctonia oryzae-sativae, the cause of aggregate sheath spot of rice. The compounds in 5% concentrations in water or agar were tested on several R. oryzae-sativae isolates. Cinnamon oil, the most efficacious plant product in vitro, was further tested in the greenhouse for the control of the disease on two rice cultivars inoculated with R. oryzae-sativae. One milliliter of each of four cinnamon oil concentrations (12.5, 37.5, 62.5, or 87.5%) diluted in vegetable oil was applied to the surface of the water in constantly flooded pots. Cinnamon oil failed to reduce the disease caused by one of the isolates at any concentration. Cinnamon oil suppressed the disease caused by the other isolate on one of the cultivars at a concentration of 37.5%, and on both cultivars at a concentration of 62.5 and 87.5%. However, cinnamon oil at 87.5% was phytotoxic. Cinnamon oil has potential to control aggregate sheath spot but relatively high concentrations were required for disease suppression.

Effect of Soil Inoculum Density of Fusarium oxysporum f. sp. vasinfectum Race 4 on Disease Development in Cotton.

The effect of soil inoculum density (0 to 106 conidia/g of potting mix) of Fusarium oxysporum f. sp. vasinfectum race 4 on plant growth (weight, height, and number of nodes), Fusarium wilt symptoms, vascular discoloration, and number of CFU per gram of stem tissue was determined on five cotton cultivars grown in a greenhouse. In the susceptible cv. DP744, symptoms of wilt and reductions in plant growth occurred at inoculum levels of 103 conidia/g of potting mix and higher, whereas plant growth of the resistant Pima cv. Ph800 was not affected by any soil inoculum densities. Cvs. DP340, Ph72, and UltEF all responded similarly, with a relatively moderate negative growth response to soil inoculum densities generally beginning at 104 conidia/g of potting mix. F. oxysporum f. sp. vasinfectum race 4 was recovered from the stems of all cultivars by plating macerated stem tissue on a semiselective medium. There was a significant positive correlation between CFU and inoculum density above 104 conidia/g of potting mix for DP744 and UltEF. The fungus was recovered relatively infrequently from stems of Ph 800 plants inoculated with any inoculum density of F. oxysporum f. sp. vasinfectum. Fusarium wilt caused by F. oxysporum f. sp. vasinfectum race 4 is an inoculum density- dependent disease; the disease was generally mild and the fungus was rarely recovered from stems at inoculum levels less than 104 conidia/g of potting mix.

Pythiogeton manoomin, a new species causing root and basal stalk rot of wild rice in the United States.

A new species of Pythiogeton isolated from wild rice exhibiting rot and dieback of roots and stalks in California is described. Pythiogeton manoomin sp. nov. is characterized by coenocytic hyphae, club-like appressorium, and terminal or intercalary sporangia, which are often a short distance from the end of supporting hyphae. The protoplasm is discharged through a discharge tube into an elongate transient vesicle, which soon disappears, leaving the naked protoplasm to differentiate into reniform zoospores. Pythiogeton manoomin also produces thick-walled pigmented chlamydospores, not found in other Pythiogeton species. In greenhouse trials, Pg. manoomin did not infect economically important crops such as rice, bean, chard, corn, carrot, lettuce, oat, radish, sweet pepper, tomato, or wheat. Phylogenetic analysis based on ITS data supports the conclusion that this organism is a new species that is most closely related to Pg. ramosum. In this paper, we describe morphological characteristics, temperature-growth relationships, pathogenicity, and phylogenetic relationships that support the description of this taxon as a new species, Pythiogeton manoomin sp. nov. urn:lsid:zoobank.org:pub:4C63AAA4-4D4A-4679-A344-79B75121A5C6.

Genome Sequence of a California Isolate of Fusarium oxysporum f. sp. lycopersici Race 3, a Fungus Causing Wilt Disease on Tomato.

Fusarium wilt of tomato, caused by the soilborne fungus Fusarium oxysporum f. sp. lycopersici, is an increasingly important disease of tomato. This paper reports the high-quality draft genome assembly of F. oxysporum f. sp. lycopersici isolate D11 (race 3), which consists of 39 scaffolds with 57,281,978 bp (GC content, 47.5%), an N 50 of 4,408,267 bp, a mean read coverage of 99.8×, and 17,682 predicted genes.

Susceptibility of Selected Cotton Cultivars to Seedling Disease Pathogens and Benefits of Chemical Seed Treatments.

The susceptibility of 12 Upland cotton cultivars to three soilborne fungi, Pythium ultimum, Rhizoctonia solani, and Thielaviopsis basicola, was evaluated in greenhouse experiments. Based on symptom development and seedling survival, cultivars highly resistant to P. ultimum included Delta Pine (DP) 6166, Prema, DP 6100, and Maxxa. A relatively low incidence of pre-emergence damping-off caused by R. solani occurred in ChemBred 7, DP 6100, and Royale, although all cultivars subsequently suffered significant post-emergence damping-off. All cultivars were equally susceptible to T. basicola. In both greenhouse and field evaluations, there were no benefits of metalaxyl seed treatment in Pythium-resistant cultivars. Seed treatment with carboxin-pentachloronitrobenzene for the control of Rhizoctonia-induced damping-off resulted in stand increases in all 12 cultivars in greenhouse tests and in 3 of 6 cultivars in field trials.

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.

Characterization and Control of Garlic Rust in California.

In 1998, a devastating outbreak of rust disease severely damaged the garlic crop in California, resulting in yield losses of 51% and an economic loss of 27% to the industry. The disease also occurred in 1999 and 2000, indicating that rust may have become an annual problem in some parts of the state. The presence of urediniospores, two-celled teliospores, and telial paraphyses indicated that the pathogen was Puccinia allii. Isolates from garlic infected onion and chives, but not leek, elephant garlic, or shallot in inoculation experiments. Garlic cloves obtained from diseased plants were planted under controlled conditions, but the resulting plants did not develop rust. Fungicide trials were conducted for 3 years and showed that none of the currently registered materials gave satisfactory control. However, tebuconazole and azoxystrobin provided good protection against rust if sprayed at 10-day intervals. A variety trial of 34 garlic cultivars and selections was planted, inoculated, and evaluated for resistance to rust. Although there was variability in rust severity among the selections, acceptable levels of resistance were not observed in any cultivar.

Identification and Differentiation of Verticillium Species and V. longisporum Lineages by Simplex and Multiplex PCR Assays.

Accurate species identification is essential for effective plant disease management, but is challenging in fungi including Verticillium sensu stricto (Ascomycota, Sordariomycetes, Plectosphaerellaceae), a small genus of ten species that includes important plant pathogens. Here we present fifteen PCR assays for the identification of all recognized Verticillium species and the three lineages of the diploid hybrid V. longisporum. The assays were based on DNA sequence data from the ribosomal internal transcribed spacer region, and coding and non-coding regions of actin, elongation factor 1-alpha, glyceraldehyde-3-phosphate dehydrogenase and tryptophan synthase genes. The eleven single target (simplex) PCR assays resulted in amplicons of diagnostic size for V. alfalfae, V. albo-atrum, V. dahliae including V. longisporum lineage A1/D3, V. isaacii, V. klebahnii, V. nonalfalfae, V. nubilum, V. tricorpus, V. zaregamsianum, and Species A1 and Species D1, the two undescribed ancestors of V. longisporum. The four multiple target (multiplex) PCR assays simultaneously differentiated the species or lineages within the following four groups: Verticillium albo-atrum, V. alfalfae and V. nonalfalfae; Verticillium dahliae and V. longisporum lineages A1/D1, A1/D2 and A1/D3; Verticillium dahliae including V. longisporum lineage A1/D3, V. isaacii, V. klebahnii and V. tricorpus; Verticillium isaacii, V. klebahnii and V. tricorpus. Since V. dahliae is a parent of two of the three lineages of the diploid hybrid V. longisporum, no simplex PCR assay is able to differentiate V. dahliae from all V. longisporum lineages. PCR assays were tested with fungal DNA extracts from pure cultures, and were not evaluated for detection and quantification of Verticillium species from plant or soil samples. The DNA sequence alignments are provided and can be used for the design of additional primers.

Development of a DNA-based macroarray for the detection and identification of Amanita species.

A DNA-based macroarray was designed to quickly and accurately identify certain Amanita mushroom specimens at the species level. The macroarray included probes for Amanita phalloides and Amanita ocreata, toxic species responsible for most mushroom poisonings, and Amanita lanei and Amanita velosa, edible species sometimes confused with toxic species, based on sequences of the highly variable internal transcribed spacer (ITS) region of rDNA. A cryptic species related to A. ocreata and one related to A. lanei, identifiable by ITS sequences, were also included. Specific multiple oligonucleotide probes were spotted onto nylon membranes and the optimal hybridization temperatures were determined. The Amanita DNA array was highly specific, sensitive (0.5 ng DNA/µL and higher were detected), and reproducible. In two case studies, the method proved useful when only small amounts of mushroom tissue remained after a suspected poisoning. An identification could be completed in 12 h.

The ascomycete Verticillium longisporum is a hybrid and a plant pathogen with an expanded host range.

Hybridization plays a central role in plant evolution, but its overall importance in fungi is unknown. New plant pathogens are thought to arise by hybridization between formerly separated fungal species. Evolution of hybrid plant pathogens from non-pathogenic ancestors in the fungal-like protist Phytophthora has been demonstrated, but in fungi, the most important group of plant pathogens, there are few well-characterized examples of hybrids. We focused our attention on the hybrid and plant pathogen Verticillium longisporum, the causal agent of the Verticillium wilt disease in crucifer crops. In order to address questions related to the evolutionary origin of V. longisporum, we used phylogenetic analyses of seven nuclear loci and a dataset of 203 isolates of V. longisporum, V. dahliae and related species. We confirmed that V. longisporum was diploid, and originated three different times, involving four different lineages and three different parental species. All hybrids shared a common parent, species A1, that hybridized respectively with species D1, V. dahliae lineage D2 and V. dahliae lineage D3, to give rise to three different lineages of V. longisporum. Species A1 and species D1 constituted as yet unknown taxa. Verticillium longisporum likely originated recently, as each V. longisporum lineage was genetically homogenous, and comprised species A1 alleles that were identical across lineages.

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.

Detection of the sour-rot pathogen Geotrichum candidum in tomato fruit and juice by using a highly specific monoclonal antibody-based ELISA.

Geotrichum candidum is a common soil-borne fungus that causes sour-rot of tomatoes, citrus fruits and vegetables, and is a major contaminant on tomato processing equipment. The aim of this work was to produce a monoclonal antibody and diagnostic assay for its detection in tomato fruit and juice. Using hybridoma technology, a cell line (FE10) was generated that produced a monoclonal antibody belonging to the immunoglobulin class M (IgM) that was specific to G. candidum and the closely related teleomorphic species Galactomyces geotrichum and anamorphic species Geotrichum europaeum and Geotrichum pseudocandidum in the G. geotrichum/G. candidum complex. The MAb did not cross-react with a wide range of unrelated fungi, including some likely to be encountered during crop production and processing. The MAb binds to an immunodominant high molecular mass (> 200 kDa) extracellular polysaccharide antigen that is present on the surface of arthroconidia and hyphae of G. candidum. The MAb was used in a highly specific enzyme-linked immunosorbent assay (ELISA) to accurately detect the fungus in infected tomato fruit and juice. Specificity of the ELISA was confirmed by sequencing of the internally transcribed spacer (ITS) 1-5.8S-ITS2 rRNA-encoding regions of fungi isolated from naturally-infected tomatoes.