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Botrytis is an important genus of plant pathogens causing pre- and postharvest disease on diverse crops worldwide. This study evaluated Botrytis isolates collected from strawberry, blueberry, and table grape berries in California. Isolates were evaluated for resistance to eight different fungicides, and 60 amplicon markers were sequenced (neutral, species identification, and fungicide resistance associated) distributed across 15 of the 18 B . cinerea chromosomes. Fungicide resistance was common among the populations, with resistance to pyraclostrobin and boscalid being most frequent. Isolates from blueberry had resistance to the least number of fungicides, whereas isolates from strawberry had resistance to the highest number. Host and fungicide resistance-specific population structure explained 12 and 7 to 26%, respectively, of the population variability observed. Fungicide resistance was the major driver for population structure, with select fungicides explaining up to 26% and multiple fungicide resistance explaining 17% of the variability observed. Shared and unique significant single-nucleotide polymorphisms (SNPs) associated with host and fungicide (fluopyram, thiabendazole, pyraclostrobin, and fenhexamid) resistance-associated population structures were identified. Although overlap between host and fungicide resistance SNPs were detected, unique SNPs suggest that both host and fungicide resistance play an important role in Botrytis population structure.

This perspective presents a synopsis of the topics contained in the Phytopathology Pathogen Spotlight on Botrytis spp. causing gray mold, including pathogen biology and systematics, genomic characterization of new species, perspectives on genome editing, and fungicide resistance. A timely breakthrough to engineer host plant resistance against the gray mold fungus has been demonstrated in planta and may augment chemical controls in the near future. While B. cinerea has garnered much of the research attention, other economically important Botrytis spp. have been identified and characterized via morphological and genome-based approaches. Gray mold control is achieved primarily through fungicide applications but resistance to various chemical classes is a major concern that threatens global plant health and food security. In this issue, new information on molecular mechanism(s) of fungicide resistance and ways to manage control failures are presented. Finally, a significant leap in fundamental pathogen biology has been achieved via development of CRISPR/Cas9 to assess gene function in the fungus which likely will spawn new control mechanisms and facilitate gene discovery studies.

Botrytis cinerea , the causal agent of gray mold, has high genetic diversity and a broad host range. In Vitis sp. and Prunus spp., B . cinerea causes pre- and postharvest diseases, and fungicides are routinely applied to prevent yield loss. In total, 535 isolates of B. cinerea collected from Vitis sp. and Prunus spp. in 2012, 2016, and 2017 were genotyped using 18 microsatellite markers and the transposable elements (TEs) Boty and Flipper . Only nine of the polymorphic markers and the two TEs were considered informative and retained for the final analyses. Of the 532 isolates, 297 were tested for resistance to seven fungicides representing six Fungicide Resistance Action Committee classes. After clone correction, 295 multilocus genotype groups were retained across the 3 years in 326 individuals, and four genetic subpopulations were detected. High levels of clonality were observed across the dataset. Significant pairwise differentiation was detected among years, locations, and TE composition. However, most of the diversity observed was within a subpopulation and not among subpopulations. No genetic differentiation was detected among resistant and sensitive isolates for individual fungicide classes. When resistance to the total number of fungicides was compared, regardless of the fungicide class, significant differentiation was detected among isolates that are resistant to two fungicide classes and those resistant to three or four fungicide groups. Fungicide resistance frequencies were stable for most chemistries evaluated with the exception of fluopyram, which increased from 2012 to 2016/2017.

Phacidiopycnis washingtonensis is the cause of speck rot of apple and leaf blight of Pacific madrone in Washington State. In total, 314 isolates were collected from apple production areas in eastern Washington and Pacific madrone in western Washington. Using eight microsatellite markers designed in this study, 58 unique multilocus haplotypes were identified. Only one of the haplotypes was shared between the apple and Pacific madrone populations. Analysis of molecular variance showed no genetic differentiation between the apple and Pacific madrone populations. Genetic variation was present within each subpopulation of apple from different geographic locations. The apple population possessed higher genotypic diversity than the Pacific madrone population, suggesting that isolates from apple may represent an older population and could have been introduced into the native habitat of Pacific madrone. P. washingtonensis likely reproduces asexually because populations examined in this study were not in linkage equilibrium. In pathogenicity tests, representative isolates from apple and Pacific madrone all incited leaf blight on Pacific madrone and speck rot on apple fruit regardless of their host of origin. Overall, our findings indicate that the P. washingtonensis population in Washington State is largely asexual, with high genotypic flow and that apple, crabapple, and Pacific madrone could serve as sources of P. washingtonensis inoculum for these hosts.

Fruit rot caused by Alternaria spp. is one of the most important factors affecting the postharvest quality and shelf life of blueberry fruit. The aims of this study were to characterize Alternaria isolates using morphological and molecular approaches and test their pathogenicity to blueberry fruit. Alternaria spp. isolates were collected from decayed blueberry fruit in the Central Valley of California during 2012 and 2013. In total, 283 isolates were obtained and five species of Alternaria, including Alternaria alternata, A. tenuissima, A. arborescens, A. infectoria, and A. rosae, were identified based on DNA sequences of the plasma membrane ATPase, Alt a1 and Calmodulin gene regions in combination with morphological characters of the culture and sporulation. Of the 283 isolates, 61.5% were identified as A. alternata, 32.9% were A. arborescens, 5.0% were A. tenuissima, and only one isolate of A. infectoria and one isolate of A. rosae were found. These fungi were able to grow at temperatures from 0 to 35°C, and mycelial growth was arrested at 40°C. Optimal radial growth occurred between 20 to 30°C. Pathogenicity tests showed that all five Alternaria spp. were pathogenic on blueberry fruit at 0, 4, and 20°C, with A. alternata, A. arborescens, and A. tenuissima being the most virulent species, followed by A. infectoria and A. rosae. Previously A. tenuissima has been reported to be the primary cause of Alternaria fruit rot of blueberry worldwide. Our results indicated that the species composition of Alternaria responsible for Alternaria fruit rot in blueberry can be dependent on geographical region. A. alternata, A. arborescens, A. infectoria, and A. rosae are reported for the first time on blueberry in California. This is also the first report of A. infectoria and A. rosae infecting blueberry fruit.

Verticillium dahliae is a soilborne, economically significant fungal plant pathogen that persists in the soil for up to 14 years as melanized microsclerotia (ms). Similarly, V. longisporum is a very significant production constraint on members of the family Brassicaceae. Management of Verticillium wilt has relied on methods that reduce ms below crop-specific thresholds at which little or no disease develops. Methyl bromide, a broad-spectrum biocide, has been used as a preplant soil fumigant for over 50 years to reduce V. dahliae ms. However, reductions in the number of ms in the vertical and horizontal soil profiles and the rate at which soil recolonization occurs has not been studied. The dynamics of ms in soil before and after methyl bromide+chloropicrin fumigation were followed over 3 years in six 8-by-8-m sites in two fields. In separate fields, the dynamics of ms in the 60-cm-deep vertical soil profile pre- and postfumigation with methyl bromide+chloropicrin followed by various cropping patterns were studied over 4 years. Finally, ms densities were assessed in six 8-by-8-m sites in a separate field prior to and following a natural 6-week flood. Methyl bromide+chloripicrin significantly reduced but did not eliminate V. dahliae ms in either the vertical or horizontal soil profiles. In field studies, increases in ms were highly dependent upon the crop rotation pattern followed postfumigation. In the vertical soil profile, densities of ms were highest in the top 5 to 20 cm of soil but were consistently detected at 60-cm depths. Six weeks of natural flooding significantly reduced (on average, approximately 65% in the total viable counts of ms) but did not eliminate viable ms of V. longisporum.

Botrytis cinerea isolates obtained from apple orchards were screened for resistance to the quinone outside inhibitor (QoI) pyraclostrobin. Of the 220 isolates tested, 43 (19.5%) were resistant to pyraclostrobin. Analysis of partial sequences of the cytochrome b gene (cyt b) in five pyraclostrobin-resistant (PR) and five pyraclostrobin-sensitive (PS) isolates showed that PR isolates harbored the point mutation leading to the substitution of glycine by alanine at codon position 143 in cyt b (G143A). Two pairs of allele-specific primers were designed based on this point mutation, and allele-specific polymerase chain reaction analysis with these primers showed that all 73 PR isolates (including 30 collected from decayed apple fruit) harbored the G143A mutation but PS isolates did not. Six pairs of primers were designed to analyze the presence of various introns in cyt b. There were six types (I to VI) of cyt b present in 247 isolates of B. cinerea collected from various apple-production areas in Washington State. Of the 247 isolates, 23 had type I cyt b containing all four introns (Bcbi-67/68, Bcbi-131/132, Bcbi-143/144, and Bcbi-164), 176 had type II cyt b containing three introns (Bcbi-67/68, Bcbi-131/132, and Bcbi-164), six had type III cyt b containing two introns (Bcbi-67/68 and Bcbi-131/132), one had type IV cyt b containing two introns (Bcbi-131/132 and Bcbi-164), one had type V cyt b containing only the Bcbi-131/132 intron, and 40 had type VI cyt b containing no introns. This is the first report of types III to VI cyt b present in B. cinerea. All 73 PR isolates did not carry the Bcbi-143/144 intron in cyt b. Of the 247 isolates tested, >90% did not carry the Bcbi-143/144 intron in cyt b, suggesting that B. cinerea populations from apple pose a high inherent risk for the development of resistance to QoIs because the presence of this intron in cyt b prevents the occurrence of G143A-mediated resistance. Analysis of genetic background based on three microsatellite primers showed that PR isolates originated from different lineages, and there was no correlation between cyt b types (I, II, and III) and the genetic background of the isolates; however, isolates carrying type VI cyt b might originate from the same lineage.

Phenotype stability, fitness, and competitive ability of pyraclostrobin- and boscalid-resistant isolates of Botrytis cinerea from apple were investigated. Stability of resistance was determined after consecutive transfers on potato dextrose agar (PDA) or being cycled on apple fruit. In vitro fitness components mycelial growth, osmotic sensitivity, conidial germination, and sporulation were evaluated on agar media. Pathogenicity, virulence and sporulation on apple fruit were evaluated at both 20 and 0°C. Competition between fungicide-resistant and -sensitive isolates on apple fruit also was evaluated. Resistance to the two fungicides was retained at levels similar to that of the initial generation after 20 and 10 transfers on PDA and five and three disease cycles on apple fruit at 20 and 0°C, respectively. Great variability in individual fitness components tested was observed among isolates within the same phenotype groups either sensitive or resistant to the fungicides but, when compared as phenotype groups, there were no significant differences in the mean values of these fitness components between resistant and sensitive phenotypes except that the phenotype resistant only to boscalid produced fewer conidia in vitro than sensitive isolates. Resistant isolates were as pathogenic and virulent on apple fruit as sensitive isolates. There was no significant correlation between the values of individual fitness components tested and the level of resistance to pyraclostrobin or boscalid, except that virulence at 20°C positively correlated with the level of resistance to the two fungicides. The final frequency of pyraclostrobin-resistant individuals in the populations was significantly decreased compared with the initial generation and no boscalid-resistant individuals were detected after four disease cycles on apple fruit inoculated with a pair mixture of a dual-sensitive isolate and one isolate each of the three phenotypes resistant to pyraclostrobin, boscalid, or both. The results suggest that resistance of B. cinerea to pyraclostrobin and boscalid was stable in the absence of the fungicides and that resistance to the two fungicides did not significantly impair individual fitness components tested. However, both pyraclostrobin- and boscalid-resistant isolates exhibited competitive disadvantage over the dual-sensitive isolate on apple fruit.

Botrytis cinerea isolates obtained from apple orchards were screened for resistance to boscalid. Boscalid-resistant (BosR) isolates were classified into four phenotypes based on the levels of the concentration that inhibited fungal growth by 50% relative to control. Of the 220 isolates tested, 42 were resistant to boscalid, with resistant phenotypes ranging from low to very high resistance. There was cross resistance between boscalid and carboxin. Analysis of partial sequences of the iron-sulfur subunit of succinate dehydrogenase gene in B. cinerea (BcSdhB) from 13 BosR and 9 boscalid-sensitive (BosS) isolates showed that point mutations in BcSdhB leading to amino acid substitutions at the codon position 272 from histidine to either tyrosine (H272Y) or arginine (H272R) were correlated with boscalid resistance. Allele-specific polymerase chain reaction (PCR) analysis of 66 BosR isolates (including 24 additional isolates obtained from decayed apple fruit) showed that 19 carried the point mutation H272Y and 46 had the point mutation H272R, but 1 BosR isolate gave no amplification product. Analysis of the BcSdhB sequence of this isolate revealed a different point mutation at codon 225, resulting in a substitution of proline (P) by phenylalanine (F) (P225F). The results indicated that H272R/Y in BcSdhB were the dominant genotypes of mutants in field BosR isolates from apple. A multiplex allele-specific PCR assay was developed to detect point mutations H272R/Y in a single PCR amplification. Levels of boscalid resistance ranged from low to very high within isolates carrying either the H272R or H272Y mutation, indicating that, among BosR isolates, different BosR phenotypes (levels of resistance) were not associated with particular types of point mutations (H272R versus H272Y) in BcSdhB. Analysis of genetic relationships between 39 BosR and 56 BosS isolates based on three microsatellite markers showed that 39 BosR isolates and 30 BosS isolates were clustered into two groups, and the third group consisted of only BosS isolates, suggesting that the development of resistance to boscalid in B. cinerea likely is not totally random, and resistant populations may come from specific genetic groups.

Potebniamyces pyri is the cause of Phacidiopycnis rot of d'Anjou pear, which is grown primarily in Washington and Oregon. To estimate the population structure of P. pyri, 146 single-spore isolates were sampled from five major pear-production areas and scored for variation at eight sequence-characterized amplified region (SCAR) loci. Significant genetic differentiation was detected among five subpopulations and a total of 54 multilocus genotypes were identified, with significant genotypic diversity in each subpopulation. No genotype was shared among more than three subpopulations. To estimate the relationship between phenotype and multilocus SCAR genotype, four to five representative isolates of each dominant SCAR genotype in each subpopulation were assayed for growth rate on oatmeal agar and virulence on d'Anjou pear fruit. Significant differences in daily growth rates and virulence were detected among genotypes; however, genotype was not predictive of virulence. To assess the mating system of the pathogen, 10 ascospores were sampled from each of 20 apothecia from a commercial orchard and scored for five SCAR markers. Segregation of alleles at one or more SCAR loci was detected among 18 of 20 ascospore progeny sets, indicating that P. pyri is likely a heterothallic fungus with a predominantly outcrossing mating system.

Penicillium expansum is the primary cause of blue mold, a major postharvest disease of apple. Fludioxonil and pyrimethanil are two newly registered postharvest fungicides for pome fruit in the United States. To evaluate the potential risk of resistance development in P. expansum to the new postharvest fungicides, one isolate of each of thiabendazole-resistant (TBZ-R) and -sensitive (TBZ-S) P. expansum was exposed to UV radiation to generate fungicide-resistant mutants. Four fludioxonil highly-resistant mutants (EC(50) > 1,000 microg/ml) and four pyrimethanil-resistant mutants (EC(50) > 10 microg/ml) were tested for sensitivities to thiabendazole, fludioxonil, and pyrimethanil, and fitness parameters including mycelial growth, sporulation on potato dextrose agar (PDA), sensitivity to osmotic stress, and pathogenicity and sporulation on apple fruit. The stability of resistance of the mutants was tested on PDA and apple fruit. Efficacy of the three fungicides to control blue mold incited by the mutants was evaluated on apple fruit. Six fungicide-resistant phenotypes were identified among the parental wild-type isolates and their mutants based upon their resistance levels. All four fludioxonil highly-resistant mutants were sensitive to pyrimethanil and retained the same phenotypes of resistance to TBZ as the parental isolates. All four pyrimethanil-resistant mutants had a low level of resistance to fludioxonil with a resistance factor >15. The two pyrimethanil-resistant mutants derived from a TBZ-S isolate became resistant to TBZ at 5 microg/ml. After 20 successive generations on PDA and four generations on apple fruit, the mutants retained the same phenotypes as the original generations. All mutants were pathogenic on apple fruit at both 0 and 20 degrees C, but fludioxonil highly-resistant mutants were less virulent and produced fewer conidia on apple fruit than pyrimethanil-resistant mutants and their parental wild-type isolates. Compared with the parental isolates, all four fludioxonil highly-resistant mutants had an increased sensitivity to osmotic stress on PDA amended with NaCl, while the pyrimethanil-resistant mutants did not. Pyrimethanil was effective against blue mold caused by fludioxonil-resistant mutants at both 0 and 20 degrees C. Pyrimethanil and fludioxonil reduced blue mold incited by pyrimethanil-resistant mutants during 12-week storage at 0 degrees C but were not effective at 20 degrees C. TBZ was not effective against pyrimethanil-resistant mutants derived from TBZ-S wild-type isolates at room temperature but provided some control at 0 degrees C. The results indicate that: (i) a fitness cost was associated with fludioxonil highly resistant mutants of P. expansum in both saprophytic and pathogenic phases of the pathogen but not pyrimethanil-resistant mutants; (ii) pyrimethanil possessed a higher risk than fludioxonil in the development of resistance in P. expansum; and (iii) triple resistance to the three apple-postharvest fungicides could emerge and become a practical problem if resistance to pyrimethanil develops in P. expansum populations.

ABSTRACT Potebniamyces pyri is the causal agent of Phacidiopycnis rot, a postharvest disease of pears. Infection of fruit occurs in the orchard, and symptoms develop during storage. Conidial germination of P. pyri in response to nutrient, temperature, wetness duration, relative humidity (RH), and pH was determined in vitro. Conidia germinated by either budding or developing germ tubes in various concentrations of pear juice solutions. The mode of conidial germination was nutrient-dependent. Low nutrient levels favored budding, whereas high nutrient levels favored germ tube development. Conidia germinated at 0 to 30 degrees C but not at 35 degrees C, with optimum temperature between 20 and 25 degrees C. Wetness durations of 4 to 5 h and 6 to 8 h at optimum temperature were required for budding and developing germ tubes, respectively, and 20 to 24 h of wetness was required to reach germination peaks. Regardless of temperature, conidia germinated primarily by budding in 10% pear juice. Secondary conidia, produced by budding of conidia, initially increased their dimensions and later germinated at 0 to 25 degrees C in the same manner as mother conidia. No germination of secondary conidia occurred at 30 degrees C. Germ tubes from conidia elongated at 0 to 25 degrees C but not at 30 degrees C. No germination occurred at

ABSTRACT Colletotrichum crown rot of strawberry in Florida is caused primarily by Colletotrichum gloeosporioides. To determine potential inoculum sources, isolates of Colletotrichum spp. from strawberry and various noncultivated plants growing in the areas adjacent to strawberry fields were collected from different sites. Species-specific internal transcribed spacer primers for C. gloeosporioides and C. acutatum were used to identify isolates to species. Random amplified polymorphic DNA (RAPD) markers were used to determine genetic relationships among isolates recovered from noncultivated hosts and diseased strawberry plants. Selected isolates also were tested for pathogenicity on strawberry plants in the greenhouse. In all, 39 C. gloeosporioides and 3 C. acutatum isolates were recovered from diseased strawberry crowns, and 52 C. gloeosporioides and 1 C. acutatum isolate were recovered from noncultivated hosts. In crown inoculation tests, 18 of the 52 C. gloeosporioides isolates recovered from noncultivated hosts were pathogenic to strawberry. Phylogenetic analysis using RAPD marker data divided isolates of C. gloeosporioides from noncultivated hosts into two separate clusters. One cluster contained 50 of the 52 isolates and a second cluster contained 2 isolates that were homothallic in culture. Isolates from strawberry were interspersed within the cluster containing the 50 isolates that were recovered from noncultivated hosts. The results are not inconsistent with the hypothesis that C. gloeosporioides isolates obtained from strawberry and noncultivated hosts adjacent to strawberry fields are from the same population and that noncultivated hosts can serve as potential inoculum sources for Colletotrichum crown rot of strawberry.

ABSTRACT Cauliflower root and plant growth and Verticillium wilt development were evaluated under different moisture regimes in the presence or absence of V. dahliae. Treatments included two main plots (V. dahliae-infested and fumigated), two subplots (furrow and subsurface drip irrigation), and three sub-subplots (deficit, moderate, and excessive regimes) that were arranged in a split-split-plot design in the field. Soil cores with roots were periodically sampled at 5 and 25 cm distance from plants. Total roots in each soil core were extracted with a hydropneumatic root elutriator, and root length from each sample was determined with a digital image analysis system. Incidence and severity of Verticillium wilt, plant height, number of leaves, and dry weights of leaves and roots were determined on 10 plants sampled at 7- to 10-day intervals 1 month after cauliflower transplanting and continued until harvest. To evaluate the effects of Verticillium wilt-induced stress on cauliflower plants, stomatal resistance was measured in upper healthy and lower (or diseased) leaves. Root length density at 5 and 25 cm from plant was significantly (P < 0.05) higher in subsurface drip than in furrow irrigation. Root length density was significantly higher in excessive irrigation regime than in the other regimes. Concomitantly, there was higher wilt incidence and severity in excessive and moderate regimes than deficit regime regardless of the irrigation method. Plant height was affected by irrigation methods and deficit regime. Neither the method of irrigation nor the quantity of water affected the other variables. Stomatal resistance in lower diseased leaves was significantly higher in infested than in fumigated plots but it was not in the upper healthy leaves. In this study, cauliflower yield was not affected by V. dahliae and irrigation method, but the deficit irrigation regime resulted in reduced yield even though it suppressed wilt in cauliflower. Thus, higher moisture levels resulted in higher root length density in V. dahliae-infested plots that in turn lead to greater incidence of Verticillium wilt and severity. The pathogen also affected physiological processes such as hydraulic conductance of cauliflower leaves, but not shoot growth or yield under these experimental conditions.

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

ABSTRACT Microplot and field experiments were conducted to evaluate the effects of inoculum density on Verticillium wilt and cauliflower growth. Soil containing Verticillium dahliae microsclerotia was mixed with various proportions of fumigated soil to establish different inoculum densities (fumigated soil was used as the noninfested control). Seven inoculum density treatments replicated four times were established, and the treatments were arranged in a randomized complete block design. Soil was collected from each microplot immediately after soil infestation for V. dahliae assay by plating onto sodium polypectate agar (NP-10) selective medium using the Anderson sampler technique. Five-week-old cauliflower was transplanted into two beds within each 1.2- by 1.2-m microplot. At the same time, several extra plants were also transplanted at the edge of each bed for destructive sampling to examine the disease onset (vascular discoloration) after planting. Cauliflower plants were monitored for Verticillium wilt development. Stomatal resistance in two visually healthy upper and two lower, diseased leaves in each microplot was measured three times at weekly intervals after initial wilt symptoms occurred. At maturity, all plants were uprooted, washed free of soil, and wilt incidence and severity, plant height, number of leaves, and dry weights of leaves and roots were determined. The higher the inoculum density, the earlier was disease onset. A density of 4 microsclerotia per g of dry soil caused 16% wilt incidence, but about 10 microsclerotia per g of soil caused 50% wilt incidence. Both wilt incidence and severity increased with increasing inoculum density up to about 20 microsclerotia per g of soil, and additional inoculum did not result in significantly higher disease incidence and severity. A negative exponential model described the disease relationships to inoculum levels under both microplot and field conditions. Stomatal resistance of diseased leaves was significantly higher at higher inoculum densities; in healthy leaves, however, no treatment differences occurred. The height, number of leaves, and dry weights of leaves and roots of plants in the fumigated control were significantly higher than in infested treatments, but the effects of inoculum density treatments were variable between years. Timing of cauliflower infection, crop physiological processes related to hydraulic conductance, and wilt intensity (incidence and severity) were thus affected by the inoculum density. Verticillium wilt management methods used in cauliflower should reduce inoculum density to less than four micro-sclerotia per g of soil to produce crops with the fewest number of infected plants.

ABSTRACT The spatial patterns of microsclerotia of Verticillium dahliae in soil and wilt symptoms on cauliflower were determined at three sites in each of two fields in 1994 and 1995. Each site was an 8 x 8 grid divided into 64 contiguous quadrats (2 by 2 m each). Soil samples were collected to a depth of 15 cm with a probe (2.5 cm in diameter), and samples from four sites in each quadrat were bulked. Plants in each quadrat were cut transversely, and the number of plants with vascular discoloration and the number without discoloration were recorded. The soil was assayed for microsclerotia by the modified Anderson sampler technique. Lloyd's index of patchiness (LIP) was used as an indicator to evaluate the aggregation of microsclerotia in the field. Spatial autocorrelation and geostatistical analyses were also used to assess the autocorrelation of microsclerotia among quadrats. The LIP for microsclerotia was greater than 1, indicating aggregation of propagules; however, the degree of aggregation at most sites was not high. Significant autocorrelation within or across rows was detected in some spatial autocorrelograms of propagules, and anisotropic patterns were also detected in some oriented semivariograms from geostatistical analyses for microsclerotia, indicating the influence of bed preparation in the fields on pathogen distribution. The parameter estimates p and theta in the beta-binomial distribution and the index of dispersion (D) associated with the distribution were used to assess the aggregation of diseased plants at each site. A random pattern of wilt incidence was detected at 7 of 12 sites, and an aggregated pattern was detected at 5 of 12 sites. The degree of aggregation was not high. A regular pattern of wilt severity was detected at all sites. The high disease incidence (77 to 98%) observed at 11 of the 12 sites could be explained by high inoculum density.