Loop-mediated isothermal amplification for detection of the tomato and potato late blight pathogen, Phytophthora infestans.

AIMS: To design and validate a colorimetric loop-mediated isothermal amplification assay for rapid detection of Phytophthora infestans DNA. METHODS AND RESULTS: Two sets of loop-mediated isothermal amplification (LAMP) primers were designed and evaluated for their sensitivity and specificity for P. infestans. ITSII primers targeted a portion of the internal transcribed spacer region of ribosomal DNA. These primers had a limit of detection of 2 pg P. infestans DNA and cross-reacted with the closely related species Phytophthora nicotianae. Rgn86_2 primers, designed to improve assay specificity, targeted a portion of a conserved hypothetical protein. These primers had a limit of detection of 200 pg P. infestans DNA and did not cross-react with P. nicotianae. The specificity of the Rgn86_2 assay was tested further using the closely related species P. andina, P. ipomoeae, P. mirabilis and P. phaseoli. Cross-reactions occurred with P. andina and P. mirabilis, but neither species occurs on tomato or potato. Both primer sets were able to detect P. infestans DNA extracted from tomato late blight leaf lesions. CONCLUSIONS: Two colorimetric LAMP assays detected P. infestans DNA from pure cultures as well as infected leaf tissue. The ITSII primers had higher sensitivity, and the Rgn86_2 primers had higher specificity. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of a LAMP assay for the detection of P. infestans, the causal organism of potato and tomato late blight. These assays have potential for immediate utility in plant disease research and diagnostic laboratories.

Diversity of Xanthomonas campestris Isolates from Symptomatic Crucifers in New York State.

To assess the diversity of Xanthomonas campestris spp. infecting crucifers in New York, 154 isolates were collected over 10 years across the state. The goal was to determine if isolates of the pathogen were overwintering in New York and serving as primary inoculum in subsequent years, or if novel isolates were entering the state each year. Pure cultures of isolates were characterized using multilocus sequence analysis (MLSA), a greenhouse pathogenicity assay, repetitive element-polymerase chain reaction (Rep-PCR) using the BOX-A1R primer, and enzyme-linked immunosorbent assay. The MLSA scheme proved to be more efficient than Rep-PCR for a large sample population and for comparison with global isolates. X. campestris isolated from crucifers in New York comprised of X. campestris pv. campestris and X. campestris pv. raphani, with X. campestris pv. raphani being predominately isolated from transplants. Evidence for unique haplotypes persisting on the same farm for several years due to improper seedbed rotations was documented in addition to novel haplotypes being spread throughout states through infected transplants and seed. Rep-PCR confirmed the high diversity of X. campestris and was used to generate 15 unique fingerprint patterns from isolates collected in the first 5 years. A worldwide comparison of isolates suggests that the X. campestris pv. campestris population appears to be very homogenous with dominant haplotypes persisting for extended periods and being globally disseminated.

Five Reasons to Consider Phytophthora infestans a Reemerging Pathogen.

Phytophthora infestans has been a named pathogen for well over 150 years and yet it continues to "emerge", with thousands of articles published each year on it and the late blight disease that it causes. This review explores five attributes of this oomycete pathogen that maintain this constant attention. First, the historical tragedy associated with this disease (Irish potato famine) causes many people to be fascinated with the pathogen. Current technology now enables investigators to answer some questions of historical significance. Second, the devastation caused by the pathogen continues to appear in surprising new locations or with surprising new intensity. Third, populations of P. infestans worldwide are in flux, with changes that have major implications to disease management. Fourth, the genomics revolution has enabled investigators to make tremendous progress in terms of understanding the molecular biology (especially the pathogenicity) of P. infestans. Fifth, there remain many compelling unanswered questions.

Differential Susceptibility of 39 Tomato Varieties to Phytophthora infestans Clonal Lineage US-23.

During the summers of 2012 and 2013, 39 tomato (Solanum lycopersicum) lines or varieties were evaluated for resistance to late blight in three separate field trials. In each trial, late blight was caused by field isolates of Phytophthora infestans clonal lineage US-23. Varieties with the late blight resistance genes Ph-1, Ph-2, Ph-3, and Ph-2 + Ph-3 were included, along with several heirloom varieties with grower-reported resistance and varieties with no known resistance. All six varieties with Ph-2 + Ph-3, along with NC25P, which is homozygous for Ph-3 only, showed a high level of resistance. Plum Regal F1, which is heterozygous for Ph-3 only, showed moderate resistance. Legend, the only variety with Ph-2 alone, also showed moderate resistance. Three heirloom varieties, Matt's Wild Cherry, Lemon Drop, and Mr. Stripey, showed a high level of resistance comparable with that of varieties with Ph-2 + Ph-3. New Yorker, possessing Ph-1 only, showed no resistance. Indeterminate varieties had significantly less disease than determinate varieties in two of the three trials. Overall, this study suggests that tomato varieties with both Ph-2 and Ph-3 can be used to effectively manage late blight caused by P. infestans clonal lineage US-23. Varieties possessing only Ph-2, or heterozygous for Ph-3, were better protected than those without any late blight resistance but might still require supplemental fungicide applications, while the variety that was homozygous for Ph-3 was highly resistant. Several heirloom varieties were also highly resistant, and the unknown mechanism of their resistance warrants further research. Finally, the plasticity observed in United States P. infestans populations over the past several decades necessitates continued monitoring for genetic changes within P. infestans that could lead to the breakdown of resistance reported here.

The 2009 Late Blight Pandemic in the Eastern United States - Causes and Results.

The tomato late blight pandemic of 2009 made late blight into a household term in much of the eastern United States. Many home gardeners and many organic producers lost most if not all of their tomato crop, and their experiences were reported in the mainstream press. Some CSAs (Community Supported Agriculture) could not provide tomatoes to their members. In response, many questions emerged: How did it happen? What was unusual about this event compared to previous late blight epidemics? What is the current situation in 2012 and what can be done? It's easiest to answer these questions, and to understand the recent epidemics of late blight, if one knows a bit of the history of the disease and the biology of the causal agent, Phytophthora infestans.

Population Structure and Resistance to Mefenoxam of Phytophthora capsici in New York State.

In 2006, 2007, and 2008, we sampled 257 isolates of Phytophthora capsici from vegetables at 22 sites in four regions of New York, to determine variation in mefenoxam resistance and population genetic structure. Isolates were assayed for mefenoxam resistance and genotyped for mating type and five microsatellite loci. We found mefenoxam-resistant isolates at a high frequency in the Capital District and Long Island, but none were found in western New York or central New York. Both A1 and A2 mating types were found at 12 of the 22 sites, and we detected 126 distinct multilocus genotypes, only nine of which were found at more than one site. Significant differentiation (FST) was found in more than 98% of the pairwise comparisons between sites; approximately 24 and 16% of the variation in the population was attributed to differences among regions and sites, respectively. These results indicate that P. capsici in New York is highly diverse, but gene flow among regions and fields is restricted. Therefore, each field needs to be considered an independent population, and efforts to prevent movement of inoculum among fields need to be further emphasized to prevent the spread of this pathogen.

Which animals are at risk? Predicting species susceptibility to Covid-19.

In only a few months, the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic, leaving physicians, scientists, and public health officials racing to understand, treat, and contain this zoonotic disease. SARS-CoV-2 has made the leap from animals to humans, but little is known about variations in species susceptibility that could identify potential reservoir species, animal models, and the risk to pets, wildlife, and livestock. While there is evidence that certain species, such as cats, are susceptible, the vast majority of animal species, including those in close contact with humans, have unknown susceptibility. Hence, methods to predict their infection risk are urgently needed. SARS-CoV-2 spike protein binding to angiotensin converting enzyme 2 (ACE2) is critical for viral cell entry and infection. Here we identified key ACE2 residues that distinguish susceptible from resistant species using in-depth sequence and structural analyses of ACE2 and its binding to SARS-CoV-2. Our findings have important implications for identification of ACE2 and SARS-CoV-2 residues for therapeutic targeting and identification of animal species with increased susceptibility for infection on which to focus research and protection measures for environmental and public health.

Dispersal and movement mechanisms of Phytophthora capsici sporangia.

Understanding the mechanisms of Phytophthora capsici sporangial dissemination is paramount to understanding epidemic initiation and development. Direct laboratory observations showed P. capsici sporangial dispersal occurred in water with capillary force, but did not occur in response to wind or a reduction in relative humidity. Atmospheric sporangial concentrations were monitored under field conditions using a volumetric spore sampler in a commercial cucurbit field and in an experimental setting where copious sporangia were continuously available in close proximity to the spore trap. Dispersal was infrequent (0.7% of total hours monitored) during sampling in a commercial field; 14 sporangia were detected during a 7.5-week sampling period. In the experimental field situation, dispersal occurred in 4.6% of the hours sampled and 438 sporangia were impacted onto tapes during a 7-week sampling period. Airborne sporangial concentrations were positively associated with rainfall at both sites, but not vapor pressure deficit. Furthermore, in the experimental field situation, wind speed was not significant in regression analysis. Wind speed was not measured in the commercial field. Hence, both direct laboratory observations and volumetric spore sampling indicate that dispersal of sporangia via wind currents is infrequent, and sporangia are unlikely to be naturally dispersed among fields by wind alone.

Induction of plant defense gene expression by plant activators and Pseudomonas syringae pv. tomato in greenhouse-grown tomatoes.

Plant activators provide an appealing management option for bacterial diseases of greenhouse-grown tomatoes. Two types of plant activators, one that induces systemic acquired resistance (SAR) and a second that activates induced systemic resistance (ISR), were evaluated for control of Pseudomonas syringae pv. tomato and effect on plant defense gene activation. Benzothiadiazole (BTH, SAR-inducing compound) effectively reduced bacterial speck incidence and severity, both alone and in combination with the ISR-inducing product. Application of BTH also led to elevated activation of salicylic acid and ethylene-mediated responses, based on real-time polymerase chain reaction analysis of marker gene expression levels. In contrast, the ISR-inducing product (made up of plant growth-promoting rhizobacteria) inconsistently modified defense gene expression and did not provide disease control to the same level as did BTH. No antagonism was observed by combining the two activators as control of bacterial speck was similar to or better than BTH alone.

Efficacy of Muscodor albus for the Control of Phytophthora Blight of Sweet Pepper and Butternut Squash.

The efficacy of Muscodor albus, a potential soil biofumigant, to control root and stem rot by Phytophthora capsici, was examined in a greenhouse study. P. capsici-infested potting mix was treated with three rates of M. albus, mefenoxam (Ridomil Gold EC, Syngenta Crop Protection, Inc.), or nothing. Seedlings of five sweet pepper cultivars and one butternut squash cultivar were transplanted into the treated potting mix. After 7 days, the plants were rated on a scale of 0 (healthy) to 5 (dead). The experiment was conducted three times and there was a significant interaction between pepper cultivar and soil treatment. Treatment with the highest rate of M. albus resulted in a slight but significant reduction in disease severity on Alliance, Aristotle, Paladin, and Revolution pepper compared with the pathogen-only control, while no significant decreases in disease severity were observed with butternut squash or the highly susceptible pepper cv. Red Knight. Of the four less-susceptible pepper cultivars, Paladin (the most tolerant cultivar) was the only one on which M. albus, as applied in this study, reduced disease severity to commercially acceptable levels.

Host-Pathogen Interactions Between Phytophthora infestans and the Solanaceous Hosts Calibrachoa × hybridus, Petunia × hybrida, and Nicotiana benthamiana.

Late blight, caused by the pathogen Phytophthora infestans, is a devastating disease of potato and tomato, but can also damage other solanaceous hosts. To gain a better understanding of the interaction between P. infestans and these other hosts, the susceptibility of species in three solanaceous genera was investigated. Of the 10 Calibrachoa × hybridus cultivars tested, four were susceptible and six were resistant to the pathogen; susceptible cultivars supported only very limited growth of P. infestans. The majority of the Petunia × hybrida (petunia) cultivars were susceptible, although less so than susceptible potatoes or tomatoes. Two petunia cultivars displayed differential resistance, suggesting the presence of R genes against P. infestans. The hypersensitive response was present in susceptible, partially resistant, and resistant petunia-P. infestans interactions, but was predominant in the resistant interaction. Young petunias (3 weeks) were more susceptible than older petunias (7 weeks). Nicotiana benthamiana was susceptible to all four P. infestans isolates tested in the lab and became infected during a field epidemic. Several of these isolates were tested for the presence of the inf1 gene, and were found to have and express the gene in vitro. In addition, culture filtrate from these isolates contained 10-kDa proteins and also elicited the hypersensitive response in Nicotiana tabacum and N. benthamiana.

Gene profiling of a compatible interaction between Phytophthora infestans and Solanum tuberosum suggests a role for carbonic anhydrase.

Late blight of potato, caused by the oomycete pathogen Phytophthora infestans, is a devastating disease that can cause the rapid death of plants. To investigate the molecular basis of this compatible interaction, potato cDNA microarrays were utilized to identify genes that were differentially expressed in the host during a compatible interaction with P. infestans. Of the 7,680 cDNA clones represented on the array, 643 (12.9%) were differentially expressed in infected plants as compared with mock-inoculated control plants. These genes were classified into eight groups using a nonhierarchical clustering method with two clusters (358 genes) generally down-regulated, three clusters (241 genes) generally up-regulated, and three clusters (44 genes) with a significant change in expression at only one timepoint. Three genes derived from two down-regulated clusters were evaluated further, using reverse transcription real-time polymerase chain reaction analysis. For these analyses, both incompatible and compatible interactions were included to determine if suppression of these genes was specific to compatibility. One gene, plastidic carbonic anhydrase (CA), was found to have a very different expression pattern in compatible vs. incompatible interactions. Virus-induced gene silencing was used to suppress expression of this gene in Nicotiana benthamiana. In CA-silenced plants, the pathogen grew more quickly, indicating that suppression of CA increases susceptibility to P. infestans.

Partial resistance of tomato to Phytophthora infestans is not dependent upon ethylene, jasmonic acid, or salicylic acid signaling pathways.

We compared tomato defense responses to Phytophthora infestans in highly compatible and partially compatible interactions. The highly compatible phenotype was achieved with a tomato-specialized isolate of P. infestans, whereas the partially compatible phenotype was achieved with a nonspecialized isolate. As expected, there was induction of the hypersensitive response (HR) earlier during the partially compatible interaction. However, contrary to our expectation, pathogenesis-related (PR) gene expression was not stimulated sooner in the partially compatible interaction. While the level of PR gene expression was quite similar in the two interactions, the LeDES gene (which encodes an enzyme necessary for the production of divinyl ethers) was expressed at a much higher level in the partially compatible interaction at 48 h after inoculation. Host reaction to the different pathogen genotypes was not altered (compared with wild type) in mutant tomatoes that were ethylene-insensitive (Never-ripe) or those with reduced ability to accumulate jasmonic acid (def-1). Similarly, host reaction was not altered in NahG transgenic tomatoes unable to accumulate salicylic acid. These combined data indicate that partial resistance in tomato to P. infestans is independent of ethylene, jasmonic acid, and salicylic acid signaling pathways.

Environmental and Genetic Factors Influencing Self-Fertility in Phytophthora infestans.

ABSTRACT Phytophthora infestans is generally regarded as heterothallic-requiring physical proximity of two individuals of different mating type (A1 and A2) for oosporogenesis. Recent reports of limited selfing in young cultures of this oomycete stimulated us to investigate factors contributing to the phenomenon. The ability to produce oospores rapidly (within 2 weeks) in pure, single individual cultures (self-fertility) was tested in 116 individual isolates. The 116 isolates were from geographically diverse locations (16 countries) and were genetically diverse. Mating type and growth medium were the most prominent factors in determining if an isolate would be self-fertile. The majority of A2 isolates (45 of 47 tested) produced oospores when grown on a 50:50 mixture of V8 and rye B medium. In contrast, the majority of A1 isolates (65 of 69 tested) did not produce oospores on this medium. None of the 116 isolates produced oospores when grown on rye B medium (with no V8 juice). Further tests on representative A1 and A2 isolates revealed that oatmeal agar, tomato juice agar, and V8-juice agar all induced the A2 mating type isolate to produce oospores but did not induce the A1 mating type isolate to produce oospores. Calcium carbonate and pH did not alter the self-fertile oospore production in either A1 or A2 mating type isolates. For in vivo tests, the application of fungicide to potato or tomato leaf tissue either before or after inoculation did not stimulate any individual isolate (one A2 and one A1 isolate) to produce oospores in infected tissue. However, in all of the controls for all experiments (in vivo and in vitro), many oospores were produced rapidly if both strains grew in physical proximity.

Genetic Change Within Populations of Phytophthora infestans in the United States and Canada During 1994 to 1996: Role of Migration and Recombination.

ABSTRACT Dramatic changes occurred within populations of Phytophthora infestans in the United States and Canada from 1994 through 1996. Occurrence of the US-8 genotype, detected rarely during 1992 and 1993, increased rapidly and predominated in most regions during 1994 through 1996. US-7, which infected both potato and tomato and made up almost 50% of the sample during 1993, was detected only rarely among 330 isolates from the United States analyzed during 1994. It was not detected at all in more limited samples from 1996. Thus, ability to infect both potato and tomato apparently did not increase the fitness of this genotype relative to US-8, as predicted previously. US-1, the previously dominant genotype throughout the United States and Canada, made up 8% or less of the samples analyzed during 1994 through 1996. A few additional genotypes were detected, which could indicate the beginnings of sexual reproduction of P. infestans within the United States and Canada. However, clonal reproduction still predominated in all locations sampled; opportunities for sexual reproduction probably were limited, because the A1 and A2 mating types usually were separated geographically. The high sensitivity of the US-1 genotype to the fungicide metalaxyl also could have reduced opportunities for contact between the mating types in fields where this compound was applied. The previous correlation between metalaxyl sensitivity and genotype was confirmed and extended to a new genotype, US-17: all US-1 isolates tested were sensitive; all isolates of the US-7, US-8, and US-17 genotypes tested to date have been resistant. Isolates of P. capsici and P. erythroseptica, two other species often found on tomato and potato, could be easily distinguished from each other and from P. infestans using a simple allozyme assay for the enzyme glucose-6-phosphate isomerase. This technique could be useful for rapid identification of species, in addition to genotype of P. infestans. It generally was not possible to predict which genotypes would be present in a location from 1 year to the next. Long-distance movement of US-8 in seed tubers was documented, and this was probably the primary means for the rapid spread of this genotype from 1993 through 1996.

Population Structure of Phytophthora infestans in the Toluca Valley Region of Central Mexico.

ABSTRACT We tested the hypothesis that the population of Phytophthora infestans in the Toluca valley region is genetically differentiated according to habitat. Isolates were sampled in three habitats from (i) wild Solanum spp. (WILD), (ii) land-race varieties in low-input production systems (RURAL), and (iii) modern cultivars in high-input agriculture (VALLEY). Isolates were sampled in 1988-89 (n= 179) and in 1997-98 (n= 389). In both sampling periods, the greatest genetic diversity was observed in RURAL and VALLEY habitats. Based on the Glucose-6-phosphate isomerase and Peptidase allozymes, the subpopulations from the three habitats were significantly differentiated in both sampling periods. In contrast to allozyme data for 1997-98, no differences were found among the three subpopulations for sensitivity to metalaxyl. Two groups of isolates identical for allozyme and mating type were further investigated by restriction fragment length polymorphism fingerprinting; 65% of one group and 85% of another group were demonstrated to be unique. The genetic diversity data and the chronology of disease occurrence during the season are consistent with the hypothesis that populations of P. infestans on wild Solanum populations are derived from populations on cultivated potatoes in the central highlands of Mexico near Toluca.

A Novel Population of Phytophthora, Similar to P. infestans, Attacks Wild Solanum Species in Ecuador.

ABSTRACT Twenty-six isolates of a Phytophthora population from two wild solanaceous species, Solanum tetrapetalum (n 11) and S. brevifolium (n = 15), were characterized morphologically, with genetic and phenotypic markers, and for pathogenicity on potato and tomato. Based on morphology, ribosomal internal transcribed spacer region 2 (ITS2) sequence, and pathogenicity, all isolates closely resembled P. infestans and were tentatively placed in that species. Nonetheless, this population of Phytophthora is novel. Its primary host is neither potato nor tomato, and all isolates had three restriction fragment length polymorphism (RFLP) bands (probe RG57) and a mitochondrial DNA haplotype that have not been reported for P. infestans. All the isolates were the A2 mating type when tested with a P. infestans A1 isolate. The A2 mating type has not been found among isolates of P. infestans from potato or tomato in Ecuador. Geographical substructing of the Ecuadorian A2 population was detected. The three isolates from the village of Nono, identical to the others in all other aspects, differed by three RFLP bands; those from Nono lacked bands 10 and 16, but possessed band 19. Most of the Ecuadorian A2 isolates were nonpathogenic on potato and tomato, but a few caused very small lesions with sparse sporulation on necrotic tissue. Cluster analysis of multilocus genotypes (RFLP, mating type, and two allozymes) dissociated this A2 population from genotypes representing clonally propagated populations of P. infestans worldwide. The current hypotheses for the historical global movements of P. infestans do not satisfactorily explain the origin or possible time of introduction into Ecuador of this A2 population. Assuming the population is P. infestans, its presence in Ecuador suggests either a hitherto unreported migration of the pathogen or an indigenous population that had not previously been detected.

rep-PCR-Mediated Genomic Fingerprinting: A Rapid and Effective Method to Identify Clavibacter michiganensis.

ABSTRACT The genomic DNA fingerprinting technique known as repetitive-sequence-based polymerase chain reaction (rep-PCR) was evaluated as a tool to differentiate subspecies of Clavibacter michiganensis, with special emphasis on C. michiganensis subsp. michiganensis, the pathogen responsible for bacterial canker of tomato. DNA primers (REP, ERIC, and BOX), corresponding to conserved repetitive element motifs in the genomes of diverse bacterial species, were used to generate genomic fingerprints of C. michiganensis subsp. michiganensis, C. michiganensis subsp. sepedonicus, C. michiganensis subsp. nebraskensis, C. michiganensis subsp. tessellarius, and C. michiganensis subsp. insidiosum. The rep-PCR-generated patterns of DNA fragments observed after agarose gel electrophoresis support the current division of C. michiganensis into five subspecies. In addition, the rep-PCR fingerprints identified at least four types (A, B, C, and D) within C. michiganensis subsp. michiganensis based on limited DNA polymorphisms; the ability to differentiate individual strains may be of potential use in studies on the epidemiology and host-pathogen interactions of this organism. In addition, we have recovered from diseased tomato plants a relatively large number of naturally occurring avirulent C. michiganensis subsp. michiganensis strains with rep-PCR fingerprints identical to those of virulent C. michiganensis subsp. michiganensis strains.

Implications of Sexual Reproduction for Phytophthora infestans in the United States: Generation of an Aggressive Lineage.

Phytophthora infestans isolates (n = 26) collected in the Columbia Basin of Oregon and Washington in 1993, which had been characterized previously for mating type, metalaxyl sensitivity, and alleles at the glucose-6-phosphate isomerase locus, were analyzed for nuclear restriction fragment length polymorphism (RFLP) bands detected by probe RG57 and mitochondrial haplotype. Analyses involving the larger set of markers indicated that this group of isolates satisfied expectations of a sexual progeny: they contained much greater genetic diversity than has been reported for most other epidemic populations of P. infestans in the United States and Canada (16 unique multilocus genotypes); both mating types were present in proximity; all possible combinations of alleles occurred at many pairs of polymorphic loci; and two distinct mitochondrial haplotypes were distributed among the isolates. An in vitro laboratory cross involving the putative parents (US-6 and US-7) as parental strains produced progeny with the same general characteristics as the field isolates. Among the field progeny were two genotypes, US-11 and US-16, that had been described previously but from subsequent and largely clonal collections. Isolates obtained from tomatoes (n = 40) and potatoes (n = 7) in 24 counties in California in 1998 were analyzed as described above, and all except one US-8 isolate from potatoes were of the US-11 clonal lineage, consistent with the hypothesis that the US-11 lineage is an especially fit clonal lineage that has survived over time and can dominate pathogen populations over a large area. We conclude that the 1993 Columbia Basin collection represents a sexual progeny that generated the US-11 lineage, and that this lineage is particularly fit when tomatoes are part of the agroecosystem.

Oospore Survival and Pathogenicity of Single Oospore Recombinant Progeny from a Cross Involving US-17 and US-8 Genotypes of Phytophthora infestans.

Oospores of Phytophthora infestans produced in vitro and in planta, from a cross between US-17 and US-8 genotypes, were exposed to a variety of environments and their survival was assessed. Additionally, the pathogenic characteristics of some resultant progeny isolates were assessed. Viability of oospores as measured by plasmolysis declined slightly over a period of 18 months whether they were stored in water at 4°C, in soil at 18°C, or in soil under natural field conditions. In comparison, viability as measured by germination was lower overall but appeared to increase after storage in soil. Oospores produced in planta were buried in the field in the fall of 1998, and were capable of infecting both tomato and potato leaflets when recovered in May 1999. Single oospore progeny (n = 53) from the in vitro cross were analyzed individually for genetic and pathogenicity characteristics. All 53 progeny tested for restriction fragment length polymorphisms with probe RG57 were hybrids. All but one progeny produced sporulating lesions on detached potato or tomato leaflets in growth chamber tests, but most lesions were smaller and developed more slowly than those produced by either parental isolate. In a further test of pathogenicity, under field conditions, none of a subset of 10 A2 progeny was capable of initiating a detectable epidemic in small plots of either potatoes or tomatoes.