Genome-Wide Association Study Identifies Single Nucleotide Polymorphism Markers Associated with Mycelial Growth (at 15, 20, and 25°C), Mefenoxam Resistance, and Mating Type in Phytophthora infestans.

Phenotypic diversity among individuals defines the potential for evolutionary selection in a species. Phytophthora infestans epidemics are generally thought to be favored by moderate to low temperatures, but temperatures in many locations worldwide are expected to rise as a result of global climate change. Thus, we investigated variation among individuals of P. infestans for relative growth at different temperatures. Isolates of P. infestans came from three collections: (i) individual genotypes recently dominant in the United States, (ii) recently collected individuals from Central Mexico, and (iii) progeny of a recent sexual recombination event in the northeastern United States. In general, these isolates had optimal mycelial growth rates at 15 or 20°C. However, two individuals from Central Mexico grew better at higher temperatures than did most others and two individuals grew relatively less at higher temperatures than did most others. The isolates were also assessed for mefenoxam sensitivity and mating type. Each collection contained individuals of diverse sensitivities to mefenoxam and individuals of the A1 and A2 mating type. We then searched for genomic regions associated with phenotypic diversity using genotyping-by-sequencing. We found one single nucleotide polymorphism (SNP) associated with variability in mycelial growth at 20°C, two associated with variability in mycelial growth at 25°C, two associated with sensitivity to mefenoxam, and one associated with mating type. Interestingly, the SNPs associated with mefenoxam sensitivity were found in a gene-sparse region, whereas the SNPs associated with growth at the two temperatures and mating type were found both at more gene-dense regions.

Phytophthora betacei, a new species within Phytophthora clade 1c causing late blight on Solanum betaceum in Colombia.

Over the past few years, symptoms akin to late blight disease have been reported on a variety of crop plants in South America. Despite the economic importance of these crops, the causal agents of the diseases belonging to the genus Phytophthora have not been completely characterized. In this study, a new Phytophthora species was described in Colombia from tree tomato (Solanum betaceum), a semi-domesticated fruit grown in northern South America. Comprehensive phylogenetic, morphological, population genetic analyses, and infection assays to characterize this new species, were conducted. All data support the description of the new species, Phytophthora betacei sp. nov. Phylogenetic analyses suggest that this new species belongs to clade 1c of the genus Phytophthora and is a close relative of the potato late blight pathogen, P. infestans. Furthermore, it appeared as the sister group of the P. andina strains collected from wild Solanaceae (clonal lineage EC-2). Analyses of morphological and physiological characters as well as host specificity showed high support for the differentiation of these species. Based on these results, a complete description of the new species is provided and the species boundaries within Phytophthora clade 1c in northern South America are discussed.

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.

Phenotypic Characterization of Recent Clonal Lineages of Phytophthora infestans in the United States.

Phytophthora infestans, the causal agent of late blight disease, has been reported in the United States and Canada since the mid-nineteenth century. Due to the lack of or very limited sexual reproduction, the populations of P. infestans in the United States are primarily reproducing asexually and, thus, show a simple genetic structure. The emergence of new clonal lineages of P. infestans (US-22, US-23, and US-24) responsible for the late blight epidemics in the northeastern region of the United States in the summers of 2009 and 2010 stimulated an investigation into phenotypic traits associated with these genotypes. Mating type, differences in sensitivity to mefenoxam, differences in pathogenicity on potato and tomato, and differences in rate of germination were studied for clonal lineages US-8, US-22, US-23, and US-24. Both A1 and A2 mating types were detected. Lineages US-22, US-23, and US-24 were generally sensitive to mefenoxam while US-8 was resistant. US-8 and US-24 were primarily pathogenic on potato while US-22 and US-23 were pathogenic on both potato and tomato. Indirect germination was favored at lower temperatures (5 and 10°C) whereas direct germination, though uncommon, was favored at higher temperatures (20 and 25°C). Sporangia of US-24 released zoospores more rapidly than did sporangia of US-22 and US-23. The association of characteristic phenotypic traits with genotype enables the prediction of phenotypic traits from rapid genotypic analyses for improved disease management.

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.

Characterization and mapping of RPi-ber, a novel potato late blight resistance gene from Solanum berthaultii.

Phytophthora infestans, the causal agent of late blight, threatens potato production worldwide. An important tool in the management of the disease is the use of resistant varieties. Eleven major resistance genes have been identified and introgressed from Solanum demissum. However, new sources of resistance are continually sought. Here, we report the characterization and refined genetic localization of a resistance gene previously identified as Rber in a backcross progeny of Solanum tuberosum and Solanum berthaultii. In order to further characterize Rber, we developed a set of P. infestans isolates capable of identifying each of the 11 R-genes known to confer resistance to late blight in potato. Our results indicate that Rber is a new resistance gene, different from those recognized in S. demissum, and therefore, it has been named RPi-ber according to the current system of nomenclature. In order to add new molecular markers around RPi-ber, we used a PCR-based mapping technique, named MASP-map, which located RPi-ber in a 3.9 cM interval between markers CT240 and TG63 on potato chromosome X. The location of RPi-ber coincides with an area involved in resistance to different pathogens of potato and tomato.

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.

Temperature and Leaf Wetness Requirements for Pathogen Establishment, Incubation Period, and Sporulation of Phytophthora infestans on Petunia × hybrida.

The temperature and leaf wetness requirements for pathogen establishment (germination, infection, and colonization) and the temperature effects on incubation period and sporulation of Phytophthora infestans on petunia were compared with those on tomato. The responses to environmental parameters were found to be similar on petunia and tomato and agreed with those previously reported for late blight development on tomato and potato. In the current study, temperatures ranging from 13 to 23°C generally were conducive to establishment. Very little establishment occurred at 28°C. The minimum leaf wetness period that enabled pathogen establishment was 2 h, whereas most establishment occurred within 6 h of inoculation. The incubation period (time period from inoculation to lesion development) and the time required for development of sporangia after lesions were formed were shortest at 23 and 28°C, respectively. Production of sporangia was greatest (per square centimeter) at 18°C and was nearly absent at 28°C on both petunia and tomato. The sporulation density at 18°C was only slightly less on petunia compared with tomato (20,000 and 24,000 sporangia/cm2, respectively); however, the total lesion area on petunia was only 20% of that on tomato.

Epidemiology and Management of Petunia and Tomato Late Blight in the Greenhouse.

Factors affecting the management of petunia and tomato late blight, caused by Phytophthora infestans, under greenhouse conditions were investigated. Late blight-infected petunias (Petunia × hybrida) and tomatoes (Lycopersicon esculentum) each produced sporangia that were dispersed throughout the greenhouse via air currents. Infected petunias produced and released fewer sporangia than infected tomatoes, but infected petunias released sporangia two times longer. Surface-directed irrigation reduced disease incidence compared with overhead irrigation that wetted the foliage. The fungicides dimethomorph-mancozeb, fosetyl-Al, azoxystrobin, and dipo-tassium phosphonate/phosphate suppressed late blight development, as did the plant defense activator acibenzolar-S-methyl. All products were applied twice at 7-day intervals. The other plant defense activator (harpin protein) and the bioantagonists (Trichoderma harzianum, Glio-cladium virens, and Bacillus subtilis) were ineffective at the rates tested.

Soilborne Oospores of Phytophthora infestans in Central Mexico Survive Winter Fallow and Infect Potato Plants in the Field.

Survival and infectivity of oospores in soils naturally infested with P. infestans oospores were studied in central Mexico. Sporangia were selectively eliminated from soil samples to determine infectivity attributable to the presence of oospores. Selective elimination of sporangia was achieved by two cycles of wetting and drying the soil. Oospore concentration, viability, and infectivity varied among soils collected during the winter fallow in different locations of central Mexico. In some soils, oospores were infective regardless of the time at which they were collected during the winter fallow. However, oospore viability and infectivity decreased following 2 years of intercropping. The number of stem lesions and initial disease severity were significantly higher in soils with moderate (20 to 39 oospores g-1 soil) oospore infestation compared with soils with low (0 to 19 oospores g-1 soil) infestation. Our study confirms that oospores can survive winter fallow and serve as a source of primary inoculum in the central highlands of Mexico. Oospore survival appeared lower in the Toluca Valley soil, which may be an indication of soil suppressiveness.

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.

Formation of Phytophthora infestans Oospores in Nature on Tubers in Central Mexico.

Oospore formation by Phytophthora infestans in nature has been detected on potato leaflets in central Mexico (1), but there are no reports of oospore formation on tubers. A severe late blight epidemic occurred in Calimaya, Mexico, in fields where potato cv. Alpha was planted during the summer of 2000. Yield was reduced despite numerous applications of fungicide. Four hundred potato tubers left in the field were collected from the upper 10 cm of soil and examined for late blight symptoms. Tubers with soft and dry rot symptoms were observed, but symptoms of pink rot (Phytophthora erythroseptica) were not found. Four percent of the tubers showed late blight symptoms. Sections of 10 tubers with late blight symptoms were air-dried for 2 weeks in the laboratory and homogenized with a mortar and pestle. Glycerol was added to the homogenized tissue and observed microscopically. Aplerotic oospores (10 to 15 oospores per tuber) with amphyginous antheridia typical of P. infestans were observed. P. mirabilis morphologically similar to P. infestans is present in the area but it does not infect potato tubers. The number of oospores observed in our tuber sample was much lower than the number reported on leaflets (>1,000 oospores per leaflet) in the Toluca Valley. Low numbers of oospores have been reported on tubers artificially inoculated with P. infestans under field conditions (2). Infected tubers left in the field may act as a source of primary inoculum. To our knowledge, this is the first report of oospores of P. infestans found on tubers in Mexico under natural field conditions. References: (1) M. E. Gallegly and J. Galindo. Phytopathology 48:274, 1958. (2) A. Levin et al. Phytopathology 91:579, 2001.

Quantifying the Rate of Release and Escape of Phytophthora infestans Sporangia from a Potato Canopy.

ABSTRACT A means for determining the rate of release, Q (spores per square meter per second), of spores from a source of inoculum is paramount for quantifying their further dispersal and the potential spread of disease. Values of Q were obtained for Phytophthora infestans sporangia released from an area source of diseased plants in a potato canopy by comparing the concentrations of airborne sporangia measured at several heights above the source, with the concentrations predicted by a Lagrangian Stochastic simulation model. An independent estimate of Q was obtained by quantifying the number of sporangia per unit area of source at the beginning of each sampling day by harvesting diseased plant tissue and enumerating sporangia from these samples. This standing spore crop was the potential number of sporangia released per area of source during the day. The standing spore crop was apportioned into time segments corresponding to sporangia concentration measurement periods using the time trace of sporangia sampled above the source by a Burkard continuous suction spore sampler. This apportionment of the standing spore crop yielded potential release rates that were compared with modeled release rates. The two independent estimates of Q were highly correlated (P = 0.003), indicating that the model has utility for predicting release rates for P. infestans sporangia and the spread of disease between fields.

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.

The Roles of Three Fungicides in the Epidemiology of Potato Late Blight.

Three fungicides were tested in the field for efficacy on late blight caused by Phytophthora infestans. The effects of these fungicides on epidemic development, lesion growth rate and sporulation were measured. No fungicide completely arrested epidemic development under the environmental conditions of these experiments. However, the fungicide mixture, propamocarb hydrochloride plus chlorothalonil, had the most suppressive effect of the fungicides tested. The mechanism of effect included suppression of disease progress and lesion expansion. Growth chamber studies demonstrated that 24°C compared to 10 or 16°C limited cymoxanil efficacy.

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.