Phytophthora capsici: Recent Progress on Fundamental Biology and Disease Management 100 Years After Its Description.

Phytophthora capsici is a destructive oomycete pathogen of vegetable, ornamental, and tropical crops. First described by L.H. Leonian in 1922 as a pathogen of pepper in New Mexico, USA, P. capsici is now widespread in temperate and tropical countries alike. Phytophthora capsici is notorious for its capability to evade disease management strategies. High genetic diversity allows P. capsici populations to overcome fungicides and host resistance, the formation of oospores results in long-term persistence in soils, zoospore differentiation in the presence of water increases epidemic potential, and a broad host range maximizes economic losses and limits the effectiveness of crop rotation. The severity of disease caused by P. capsici and management challenges have led to numerous research efforts in the past 100 years. Here, we discuss recent findings regarding the biology, genetic diversity, disease management, fungicide resistance, host resistance, genomics, and effector biology of P. capsici.

Field Response of Cucurbit Hosts to Pseudoperonospora cubensis in Michigan.

Downy mildew, caused by Pseudoperonospora cubensis, is a severe foliar disease of many cucurbit crops worldwide. Forty-one cucurbit cultigens (commercial cultivars and plant introductions) from five genera (Cucumis, Citrullus, Cucurbita, Lagenaria, and Luffa) were assessed for susceptibility to P. cubensis in a research field exposed to natural inoculum in Michigan. Eight cultigens from a differential set for pathotype determination were included within the 41 cultigens to detect changes in dominant P. cubensis pathotypes present. No pathotype differences were found between 2010 and 2011. Cucumis melo cultigen MR-1 was less susceptible to Michigan P. cubensis populations than other C. melo cultigens. No symptoms or signs of infection were detected on cultigens of Cucurbita moschata and C. pepo. Disease onset was later in 2011 than 2010; greater than 90% disease severity in pickling cucumber 'Vlaspik' was observed in both years. This study confirmed that Cucumis is the most susceptible cucurbit genus, while Citrullus and Cucurbita cultigens were the least susceptible genera to populations of P. cubensis in Michigan. Area under the disease progress curve values indicated that disease progress was limited on all Citrullus cultigens compared with Cucumis cultigens, and pathogen sporulation was not detected under field conditions. Future studies should evaluate the ability of a reduced fungicide program to control downy mildew on less susceptible Cucumis melo 'Edisto 47', 'Primo', 'Athena', 'Strike', 'Ananas', 'Banana', and 'Tam-Dew'. Many of the melon cultivars evaluated were selected on the basis of reported resistance to downy mildew, yet they showed significant disease symptoms. It is important to evaluate candidate cultigens for resistance to local P. cubensis populations.

Development and Field Evaluation of Multiple Virus-Resistant Bottle Gourd (Lagenaria siceraria).

In an effort to develop bottle gourd (Lagenaria siceraria) as a widely adapted rootstock for watermelon grafting, we sought to identify lines with broad resistance to several cucurbit viruses that are economically important in the United States. Preliminary analysis under greenhouse conditions indicated that the currently available commercial watermelon rootstocks were either highly susceptible or somewhat tolerant to one or more viruses. However, in greenhouse screening, several breeding lines of bottle gourd displayed broad-spectrum resistance to four viruses tested, including Zucchini yellow mosaic virus, Watermelon mosaic virus (WMV), Papaya ringspot virus watermelon strain (PRSV-W), and Squash vein yellowing virus. Resistance to PRSV-W and WMV was confirmed through field trials in two consecutive years at two different locations in South Carolina. Two breeding lines (USVL#1-8 and USVL#5-5) with broad-spectrum virus resistance could be useful materials for watermelon rootstock development.

Outbreak of Cucurbit Powdery Mildew on Watermelon Fruit Caused by Podosphaera xanthii in Southwest Florida.

Cucurbit powdery mildew caused by the obligate parasite Podosphaera xanthii occurs commonly on foliage, petioles, and stems of most cucurbit crops grown in the United States. (3). However, in the field, fruit infection on cucurbits including watermelon (Citrullus lanatus), is rarely, if ever, observed (2). Consequently, it was atypical when severe powdery mildew-like symptoms were observed on seedless and seeded watermelon fruit on several commercial farms in southwestern Florida during November and December 2010. Severe powdery mildew was also observed on 'Tri-X 313' and 'Mickey Lee' fruit grown at SWFREC, Immokalee, FL. Infected fruit developed poorly and were not marketable. Powdery mildew symptoms were mainly observed on young immature fruit, but not on mature older fruit. Abundant powdery mildew conidia occurred on fruit surface, but not on the leaves. Conidia were produced in chains and averaged 35 × 21 µm. Observation of conidia in 3% KOH indicated the presence of fibrosin bodies commonly found in the cucurbit powdery mildew genus Podosphaera (3). Orange-to-dark brown chasmothecia (formerly cleisthothecia) containing a single ascus were detected on the surface of some fruit samples. Conidial DNA was subjected to PCR using specific primers designed to amplify the internal transcribed spacer (ITS) region of Podosphaera (4). The resulting amplicons were sequenced and found to be 100% identical to the ITS sequences of P. xanthii in the NCBI database (D84387, EU367960, AY450961, AB040322, AB040315). Sequences from the watermelon fruit isolate were also identical to several P. fusca (synonym P. xanthii), P. phaseoli (GQ927253), and P. balsaminae (AB462803) sequences. On the basis of morphological characteristics and ITS sequence analysis, the pathogen infecting watermelon fruit can be considered as P. xanthii (1,3,4). The powdery mildew isolate from watermelon fruit was maintained on cotyledons of squash (Cucurbita pepo, 'Early Prolific Straight Neck'). Cotyledons and leaves of five plants each of various cucurbits and beans were inoculated with 10 µl of a conidial suspension (105conidia/ml) in water (0.02% Tween 20). Two weeks after inoculation, abundant conidia were observed on cucumber (Cucumis sativus, 'SMR-58') and melon (Cucumis melo) powdery mildew race differentials 'Iran H' and 'Vedrantais'. However, no growth was observed on melon differentials 'PI 414723', 'Edisto 47', 'PMR 5', 'PMR 45', 'MR 1', and 'WMR 29' (2,3). The powdery mildew isolate from watermelon fruit behaved as melon race 1 (3). Mycelium and conidia were also observed on fruit surface of watermelon 'Sugar Baby' and a susceptible U.S. plant introduction (PI 538888) 3 weeks after inoculation. However, the disease was not as severe as what was observed in the fields in fall 2010. The pathogen did not grow on plants of Impatiens balsamina or on select bean (Phaseolus vulgaris) cultivars ('Red Kidney', 'Kentucky Blue', and 'Derby Bush'), but did grow and produce abundant conidia on 'Pinto bush bean'. Powdery mildew on watermelon fruit in production fields can be considered as a potentially new and serious threat requiring further studies to develop management strategies. References: (1) U. Braun and S. Takamatsu. Schlechtendalia 4:1, 2000. (2) A. R. Davis et al. J. Am. Soc. Hortic. Sci. 132:790, 2007. (3) M. T. McGrath and C. E. Thomas. In: Compendium of Cucurbit Diseases. American Phytopathological Society, St. Paul, MN, 1996. (4) S. Takamatsu and Y. Kano. Mycoscience 42:135, 2001.

Cucurbit yellow stunting disorder virus Detected in Pigweed in Florida.

Pigweeds (genus Amaranthus) are problematic weeds in crop production throughout the world and are responsible for significant yield losses in many crops (2). Members of this genus can produce hundreds of thousands of seeds per plant and are also capable of supporting populations of important crop pathogens including viruses, nematodes, fungi, and oomycetes. Thirty-one pigweed samples (tentatively identified as Amaranthus lividus L. based on leaf notch and growth habit) were collected in November and December of 2009 from a watermelon field near Immokalee, FL, previously found to contain watermelon plants infected with three whitefly-transmitted viruses: Cucurbit yellow stunting disorder virus (CYSDV), Cucurbit leaf crumple virus (CuLCrV), and Squash vein yellowing virus (SqVYV). Although no obvious virus symptoms were observed on any of the pigweed plants, whiteflies (Bemisia tabaci), a known vector of CYSDV, CuLCrV, and SqVYV, were observed on leaves. Consequently, replica tissue blots were made from all pigweed samples and tested independently by tissue blot nucleic acid hybridization assay for CYSDV, CuLCrV, or SqVYV (3). Tissue blots indicated CYSDV infection in six pigweed samples. Neither CuLCrV nor SqVYV was detected. Three of the tissue blot-positive pigweed samples were further tested by reverse transcription (RT)-PCR amplification from total RNA (extracted from leaf tissue with TRIzol Reagent [Invitrogen, Carlsbad, CA]) with HSP70 and coat protein (CP) gene primers (1). HSP70 and CP gene RT-PCR products of the expected sizes (175 and 707 nt, respectively) were amplified, sequenced, and found to be 100% identical for all three pigweed samples. The partial HSP70 gene sequence from pigweed shared 98.3 to 100% nucleotide identity with CYSDV isolates from Arizona, California, and Spain (GenBank Accession Nos. FJ492808, EU596530, and NC_004810, respectively). The partial CP gene sequence from pigweed shared 88.8 to 100% nucleotide identity with CYSDV isolates from Arizona, Saudi Arabia, Texas, and Spain (GenBank Accession Nos. EF210558, AF312811, AF312806, and AF312808, respectively). To our knowledge, this is the first report of CYSDV infection of pigweed in Florida. Infection of redroot pigweed (A. retroflexus) was recently reported in California (4). These results collectively indicate that control of noncucurbit weeds may be important for effective management of CYSDV in cucurbit crops. References: (1) S. Adkins et al. Online publication. doi:10.1094/PHP-2009-1118-01-BR. Plant Health Progress, 2009. (2) L. Holm et al. World's Weeds: Natural Histories and Distributions. John Wiley and Sons, Inc. New York, NY, 1997. (3) W. W. Turechek et al. Phytopathology 100:1194, 2010. (4) W. M. Wintermantel et al. Plant Dis. 93:685, 2009.

First Report of Southern Blight on Bottle Gourd (Lagenaria siceraria) Caused by Sclerotium rolfsii in South Carolina.

Bottle gourd (Lagenaria siceraria (Mol.) Standl.) is an important rootstock in watermelon production in several countries such as Japan, China, and Israel where 60 to 70% of watermelons are grafted (2). We are evaluating bottle gourds for their ability to improve disease resistance when used as rootstock for watermelon (3). In the summer of 2007, symptoms of wilting and crown necrosis appeared on bottle gourd seedlings 1 month after transplanting in a field in Charleston, SC. Infection was observed on commercial cv. Emphasis and four advanced breeding lines. In October of 2007, 35 of 85 plants examined (41%) had stem rot at the crown area just above the soil line where coarse, white mycelia and abundant sclerotia were observed. The fungus tentatively identified as Sclerotium rolfsii produced sclerotia that were white or light to dark brown and measured 0.6 to 2.5 mm in diameter (mean = 1.1 mm). Diseased tissues with sclerotia from four plants were disinfested for 1 min in 0.5% sodium hypochlorite and plated on acidified potato dextrose agar (APDA). Fungal colonies that produced white mycelia and tan-to-brown sclerotia were isolated from four wilted plants. A single PCR product of approximately 680 bp was amplified from DNA extracted from two isolates using the primers ITS1 and ITS4 (4). One PCR product was cloned into the TOPO TA cloning vector (Invitrogen, Carlsbad, CA) and sequenced (GenBank Accession No. EU338381). BLASTN analysis of the sequence in the NCBI databases revealed 99% similarity to the internal transcribed spacer (ITS) sequences of S. rolfsii and Athelia rolfsii (perfect stage of S. rolfsii), confirming that the pathogen was indeed S. rolfsii. Two S. rolfsii isolates were used to test pathogenicity. Each isolate was used to inoculate five young seedlings and five adult (10-week-old) bottle gourd plants. For inoculation, 10 sclerotia obtained from the APDA plates were placed on the surface of the potting soil 0.5 to 1 cm from the collar region of each bottle gourd plant growing in 10-cm pots. Inoculations were done carefully to ensure that the plants were not injured. After inoculation, the plants were maintained at high humidity and 25°C for 3 days and then transferred to laboratory benches. Four young seedlings and three adult noninoculated plants kept under the same conditions served as controls. The pathogenicity test was repeated once with similar results. All inoculated plants developed symptoms of southern blight. The inoculated plants developed symptoms of wilting 4 to 5 days after inoculation and completely wilted within 7 to 10 days. Symptoms of wilting were soon followed by the appearance of white-to-light brown sclerotia on the collar region. No symptoms were observed on the noninoculated plants. S. rolfsii was reisolated from the inoculated plants on APDA. Although southern blight caused by S. rolfsii has been reported on many crop plants in the southern United States, to our knowledge, this disease has not been reported previously on bottle gourd in North America. However, the disease has been reported on bottle gourd in India (1). Identifying sources of resistance to southern blight in bottle gourds may be necessary to make them suitable as rootstocks in areas where S. rolfsii is present. References: (1) K. S. Amin. Indian Phytopathol. 34:253, 1981. (2) R. Cohen et al. Plant Dis. 91:916, 2007. (3) K. S. Ling and A. Levi. HortScience 42:1124, 2007. (4) T. J. White et al. PCR Protocols: A Guide to Methods and Amplifications. Academic Press, San Diego, 1990.

First Report of Insensitivity to Cyazofamid Among Isolates of Phytophthora capsici from the Southeastern United States.

Phytophthora capsici is rapidly becoming an important limiting factor in vegetable production in the southeastern United States, particularly on cucurbits as fruit rots. One of the strategies used to manage diseases caused by P. capsici is the regular application of fungicides. Recently the new fungicide cyazofamid (trade name Ranman, FRAC Group 21, FMC Corporation, EPA Reg. No. 71512-3-279) was registered for management of P. capsici on cucurbits. Cyazofamid has been reported to be very effective against P. capsici on peppers (1). In a recent evaluation, we observed that cyazofamid was not very effective on fruit rot of watermelon in a field artificially infested with P. capsici (3). Hence, we evaluated our collection of isolates for sensitivity to cyazofamid. We confirmed our isolates as P. capsici based on morphology of colonies and sporangia and amplification of internal transcribed spacer regions using specific PCR primers (4). Mycelial growth of 28 isolates from the southeastern United States including North (NC) and South Carolina (SC), Georgia (GA), and Florida (FL) was evaluated on Ranman amended (0, 25, 100, 310, 518, and 1,000 mg/liter of the active ingredient cyazofamid) V8 juice agar using similar techniques as described before (2). The EC50 (50% effective concentration) values ranged from 3.8 to 535 mg/liter. Thirteen isolates (8 GA, 3 SC, 1 NC, and 1 FL) had EC50 >100 mg/liter. Similar results were obtained when technical grade cyazofamid was used. The same 28 isolates were evaluated on media amended with technical grade cyazofamid (0, 1, 10, and 100 mg/liter) and 100 mg/liter of salicylhydroxaymic acid, which was added to inhibit the alternative oxidase enzyme. The EC50 values ranged from <1 to >100 mg/liter. Six isolates (5 GA and 1 NC) had EC50 >100 mg/liter. Three isolates, one sensitive and two insensitive, were used to inoculate cucumber (Cucumis sativus) fruits treated with commercial Ranman at 0, 10, 100, 300, and 1,000 mg/liter of cyazofamid plus the surfactant Silwett L-77 (0.52 ml/liter). Mycelial plugs (7-mm diameter) were placed on nonwounded fruits. Fruits were kept under high humidity at 25 ± 1°C in an incubator for 3 days. Two measurements of each lesion at right angles were averaged to get the lesion diameter. The EC50 value for lesion diameter on fruits varied from 13 mg/liter for the sensitive isolate to >233 mg/liter for the insensitive isolates. EC50 values for diameter of the lesion with sporulation ranged from 3 to 107 mg/liter. Relative lesion diameters of the insensitive isolates at 100 mg/liter treatment compared with nonsprayed check were 70 to 93%, and at 300 mg/liter, it was 38 to 80%. Similarly in another experiment, watermelon (Citrullus lanatus var. lanatus) fruits were sprayed with a recommended field rate of Ranman (284 mg of cyazofamid/liter) plus Silwett L-77 (0.52 ml/liter) till runoff and inoculated with four isolates. The relative lesion diameter for insensitive isolates on Ranman treated watermelon fruits were 76 to 100% of nonsprayed fruits. To our knowledge, these insensitive isolates were collected from fields that were never sprayed with Ranman. Because of the existence of cyazofamid insensitive P. capsici isolates, it should be rotated with fungicides from other chemical classes to prevent extensive selection of insensitive isolates. References: (1) K. L. Ivors et al. Plant Dis. Manage. Rep. 1:V088, 2007. (2) A. P. Keinath. Plant Dis. 91:743, 2007. (3) C. S. Kousik and R. Hassell. Plant Dis. Manage. Rep. 1:V010, 2007. (4) J. B. Ristaino et al. Appl. Environ. Microbiol. 64:948, 1998.

Reduced genetic variation occurs among genes of the highly clonal plant pathogen Xanthomonas axonopodis pv. vesicatoria, including the effector gene avrBs2.

The bacterial plant pathogen Xanthomonas axonopodis pv. vesicatoria, also known as Xanthomonas campestris pv. vesicatoria group A, is the causal agent of bacterial spot in pepper and tomato. In order to test different models that may explain the coevolution of avrBs2 with its host plants, we sequenced avrBs2 and six chromosomal loci (total of 5.5 kb per strain) from a global sample of 55 X. axonopodis pv. vesicatoria strains collected from diseased peppers. We found an extreme lack of genetic variation among all X. axonopodis pv. vesicatoria genomic loci (average nucleotide diversity, pi = 9.1 x 10(-5)), including avrBs2. This lack of diversity is consistent with X. axonopodis pv. vesicatoria having undergone a recent population bottleneck and/or selective sweep followed by population expansion. Coalescent analysis determined that approximately 1.4 x 10(4) to 7.16 x 10(4) bacterial generations have passed since the most recent common ancestor (MRCA) of the current X. axonopodis pv. vesicatoria population. Assuming a range of 50 to 500 bacterial generations per year, only 28 to 1,432 years have passed since the MRCA. This time frame coincides with human intervention with the pathogen's host plants, from domestication to modern agricultural practices. Examination of 19 mutated (loss-of-function) avrBs2 alleles detected nine classes of mutations. All mutations affected protein coding, while no synonymous changes were found. The nature of at least one of the avrBs2 mutations suggests that it may be possible to observe one stage of an evolutionary arms race as X. axonopodis pv. vesicatoria responds to selection pressure to alter avrBs2 to escape host plant resistance.

Temperature Sensitivity of the Hypersensitive Response of Bell Pepper to Xanthomonas axonopodis pv. vesicatoria.

ABSTRACT When bacterial spot-resistant pepper plants carrying resistance gene Bs2 and infiltrated with incompatible strains of Xanthomonas axonopodis pv. vesicatoria carrying a functional avrBs2 gene (races P1 and P3) were incubated at 32 degrees C, they exhibited an electrolyte leakage and bacterial multiplication pattern in planta similar to that obtained with a compatible strain (race P4) carrying a nonfunctional avrBs2 gene. They also developed disease-like symptoms. Pretreatment of incompatible bacteria at 32 degrees C before infiltration caused a delay in electrolyte leakage less pronounced than that caused by exposing plants to 32 degrees C. Also, plants had to be exposed to 32 degrees C for an hour prior to inoculation to increase symptom expression. These data suggest that the Bs2 gene is temperature sensitive. In other experiments, the avrBs1-Bs1 interaction appeared to be the most heat tolerant and thus the least likely to revert to compatible, whereas the avrBs3-Bs3 interaction had an intermediate sensitivity to elevated temperatures.

A non-hypersensitive resistance in pepper to the bacterial spot pathogen is associated with two recessive genes.

ABSTRACT The pepper genotype, ECW-12346, was developed with bacterial spot resistance derived from Pep13, PI 271322, and ECW123 (Early Calwonder containing Bs1, Bs2, and Bs3 genes). For genetic analysis of this resistance, ECW12346, ECW123, F(1), F(2), and backcrosses were inoculated with a pepper race 6 (P6) strain. Two recessive genes were identified that determined resistance. The genes are designated bs5 and bs6 for the resistance derived from PI 271322 and Pep13, respectively. In greenhouse and field studies, ECW12346 was highly resistant, whereas ECW123 had significant defoliation. In growth-room studies, electrolyte leakage and population dynamics were determined. Following infiltration of both genotypes with 10(8) CFU/ml of a P6 strain, there was no rapid increase in electrolyte leakage within 72 h, whereas a rapid increase in electrolyte leakage occurred within 24 h when a similar concentration of a P3 strain (containing the avrBs2 gene) was infiltrated into the intercellular spaces of the leaf. When 10(5) CFU/ml of a P6 strain was infiltrated into leaves, complete tissue collapse was evident in ECW123 10 days later as determined by visual assessment and electrolyte leakage data, but no confluent necrosis was detected in ECW12346. Internal populations were at least two logarithmic units higher in ECW123 than in ECW12346. Therefore, ECW12346 inhibits population build-up without inducing the typical hypersensitive reaction characterized by an increase in electrolyte leakage.

Resistance to Bacterial Spot in Bell Pepper Induced by Acibenzolar-S-Methyl.

Bell pepper plants sprayed with the chemical acibenzolar-S-methyl (ABM, Actigard 50 WG) showed resistance to subsequent infections with the bacterial spot agent Xanthomonas axonopodis pv. vesicatoria. Induction of resistance was independent of the cultivar used, and was expressed as early as 3 days after treatment and continued for at least 2 weeks. In the field, applications of ABM every 2 weeks, alone or in combination with copper, resulted in disease control similar to the standard treatment of copper plus maneb. Yield response was variable, with certain combinations of chemical treatments and cultivars producing yields as large as the copper plus maneb treatment. In contrast, weekly applications during the entire crop season had a negative impact on yield. In plots maintained free of bacterial spot, applications of ABM every 2 weeks caused a reduction in yield for one cultivar of six tested. The use of chemical inducers for the control of bacterial spot on bell pepper, while generally promising, may result in an unpredictable loss in fruit yield.

Development of bacterial spot on near-isogenic lines of bell pepper carrying gene pyramids composed of defeated major resistance genes.

ABSTRACT Disease severity caused by races 1 through 6 of Xanthomonas campestris pv. vesicatoria on eight near-isogenic lines (isolines) of Early Calwonder (ECW) with three major resistance genes (Bs1, Bs2, and Bs3) in different combinations was evaluated in the greenhouse and field. Strains representing races 1, 3, 4, and 6 caused similar high levels of disease severity, followed by races 2 and 5 on susceptible ECW. Race 3 caused severe disease on all isolines lacking resistance gene Bs2. Race 4, which defeats Bs1 and Bs2, caused less disease on isoline ECW-12R (carries Bs1 + Bs2), than on isolines ECW, ECW-10R (carries Bs1), and ECW-20R (carries Bs2). Similar results were obtained with race 4 strains in field studies conducted during 1997 and 1998. In greenhouse studies, race 6, which defeats all three major genes, caused less disease on isoline ECW-13R (carries Bs1 + Bs3) and ECW-123R (carries Bs1 + Bs2 + Bs3) than on isolines ECW, ECW-10R, ECW-20R, and ECW-30R (carries Bs3), but not on ECW-23R (carries Bs2 + Bs3). In greenhouse studies with commercial hybrids, strains of races 4 and 6 caused less disease on Boynton Bell (carries Bs1 + Bs2) than on Camelot (carries no known resistance genes), King Arthur (carries Bs1), and X3R Camelot (carries Bs2). Race 6 caused less disease on hybrid R6015 (carries Bs1 + Bs2 + Bs3) and Sentinel (carries Bs1 + Bs3) than on Camelot. Residual effects were not as evident in field studies with race 6 strains. Defeated major resistance genes deployed in specific gene combinations (i.e., gene pyramids) were associated with less area under the disease progress curve than when genes were deployed individually in isolines of ECW or commercial hybrids. Successful management of bacterial spot of pepper is achieved incrementally by integrating multiple tactics. Although there is evidence of residual effects from defeated genes, these effects alone likely will not provide acceptable bacterial spot control in commercial production fields. However, when combined with sanitation practices and a judicious spray program, pyramids of defeated resistance genes may aid in reducing the risk of major losses due to bacterial spot.

Response of Bell Pepper Cultivars to Bacterial Spot Pathogen Races that Individually Overcome Major Resistance Genes.

The effect of major resistance genes (Bs1, Bs2, and Bs3) or gene combinations for resistance to bacterial spot of bell peppers (Xanthomonas campestris pv. vesicatoria) in 15 commercial cultivars on disease reduction and yield were studied during 1995 and 1996. Reaction of cultivars to specific races (races 1, 2, or 3) of the pathogen corresponded with seed company claims for resistance against these races. Races 1 to 4 were used as initial inoculum in 1995, and races 1 to 6 in 1996 field experiments. Cultivars with no known resistance genes to bacterial spot (e.g., Camelot, Jupiter, and Valiant), a single resistance gene (X3R Camelot, King Arthur), or a combination of Bs1 and Bs3 genes (Guardian, Sentinel, and Admiral) were severely diseased. Yields were reduced in all inoculated cultivars compared to non-inoculated cultivars used as controls. Although races 4 and 6 caused significant disease in cultivars with only Bs1 (King Arthur) or Bs2 (X3R Camelot) genes, cultivars with a combination of Bs1 and Bs2 (Boynton Bell, PR9300-8) had much lower levels of bacterial spot. Roger 4178, a hybrid with a combination of Bs1, Bs2, and Bs3 genes, had the lowest disease ratings. Overall, race 3 was predominant during 1995, while races 3 and 6 were recovered most frequently in 1996.