Phenotypic and Genotypic Characterization of Phytophthora infestans Isolates Associated with Tomato and Potato Crops in Colombia.

Late blight disease, caused by the plant pathogen Phytophthora infestans, is one of the major threats for tomato and potato crops. Monitoring the populations of P. infestans is important to determine if there are changes in the sensitivity to fungicides and host preference. In this study, microsatellite markers and mitochondrial haplotypes were used to assess the genotype of isolates of P. infestans collected from tomato and potato plants in Colombia. Furthermore, sensitivity to the three fungicides cymoxanil (penetrant fungicide), mefenoxam, and fluopicolide (systemic fungicides), and tomato-potato host preference, were evaluated. Mitochondrial haplotyping showed that isolates collected on tomato were from the genetic groups Ia and Ib, while isolates collected on potatoes belonged to group IIa. Microsatellite analyses showed that isolates from tomato form two groups, including the Ib mitochondrial haplotype (which is genetically close to the US-1 clonal lineage) and the Ia haplotype (related to the EC-3 lineage), whereas Colombian isolates from potato formed a separate group. Furthermore, differences in sensitivity to fungicides were observed. Eighty-one percent of the isolates tested were resistant to mefenoxam with an EC50 >10 µg ml-1. Forty-two percent of the isolates showed an intermediate resistance to cymoxanil. The EC50 values ranged between 1 and 10 µg ml-1. For fluopicolide, 90% of the isolates were sensitive, with EC50 <1 µg ml-1. Host preference assays showed that potato isolates infected both host species. Thus, isolates that infect potatoes may pose a risk for tomato crops nearby.

Differential Susceptibility of Tree Tomato (Solanum betaceum) Cultivars to Late Blight Caused by Phytophthora betacei.

Host-pathogen interactions of a new species of Phytophthora, causal agent of late blight of tree tomato (Solanum betaceum Cav.), identified as Phytophthora betacei, were investigated with four different cultivars. Thirty-six P. betacei isolates, collected from southern Colombia between 2008 and 2009, were used to inoculate common tree tomato cultivars, Común, Híbrido, Injerto, and Holandés. Data on incubation and latent periods as well as infection efficiency, lesion development, and total sporulation were collected via detached leaf assays. Significant differences in susceptibility, based on the parameters measured, were observed. Común was the most susceptible cultivar, followed by Injerto, Híbrido, and Holandés. The mean incubation period was lowest for Común at 125.6 h post-inoculation (hpi) and highest for Híbrido at 139.4 hpi. No significant differences in latent period were observed. All 36 isolates produced necrotic lesions on Común, and 33, 24, and 21 caused infection on Injerto, Híbrido, and Holandés, respectively. Two isolates were able to cause infection only on Común, and 13 isolates were able to infect all four cultivars. Infection efficiency was significantly higher for the cultivar Común, followed by Injerto, Híbrido, and Holandés. Average lesion size was larger on Común than on any other cultivar. An inverse relationship of lesion size and total sporulation was observed. Común had significantly lower total sporulation than Híbrido and Holandés, which had the smallest average lesion sizes. These data show variation in pathogenicity of P. betacei isolates, under controlled conditions, and differential susceptibility of four distinct S. betaceum cultivars.

Is the Phenomenon of Mefenoxam-Acquired Resistance in Phytophthora infestans Universal?

Phytophthora infestans is the causal agent of late blight disease of potatoes and tomatoes. This disease causes devastating economic losses each year, and control is mainly achieved by the use of fungicides. Unfortunately, populations of P. infestans resistant to fungicides have been documented. Furthermore, studies have reported that sensitive isolates to the phenylamide fungicide, mefenoxam, become less sensitive in vitro after a single passage through sublethal concentrations of fungicide-amended medium. The first objective of this study was to investigate if isolates of P. infestans are capable of acquiring resistance to two additional systemic fungicides, fluopicolide (benzamide) and cymoxanil (cyanoacetamide-oxime). In contrast to the situation with mefenoxam, exposure of isolates to sublethal concentrations of fluopicolide and cymoxanil did not induce reduced sensitivity to these two fungicides. The second objective was to assess if reduced sensitivity to mefenoxam could occur in naturally sensitive isolates of other Phytophthora species and of Phytopythium sp., another oomycete plant pathogen. All Phytophthora spp. assessed (P. infestans, P. betacei, and P. pseudocryptogea) as well as Phytopythium sp. acquired resistance to mefenoxam after previous exposure through medium containing 1 µg ml-1 of mefenoxam. Interestingly, isolate 66 of Phytopythium sp. and the isolate of P. pseudocryptogea tested do not seem to be acquiring resistance to mefenoxam after exposure to medium containing 5 µg ml-1 of this fungicide. The tested isolates of P. palmivora and P. cinnamomi were extremely sensitive to mefenoxam, and thus it was not possible to perform a second transfer to access acquisition of resistance to this fungicide.

Phytophthora infestans: New Tools (and Old Ones) Lead to New Understanding and Precision Management.

New tools have revealed that migrations of Phytophthora infestans have been a dominant feature of the population biology of this pathogen for the past 50 years, and maybe for the past 170 years. We now have accurate information on the composition of many P. infestans populations. However, migration followed by selection can lead and has led to dramatically rapid changes in populations over large regions. Except for the highlands of central Mexico, many populations of P. infestans have probably been in flux over the past several decades. There is some evidence that this pathogen has different characteristics in the field than it does in the lab, and early field phenotypic analyses of hypotheses concerning fitness and pathogenicity would be beneficial. The newly available capacity to acquire and process vast amounts of weather and weather forecast data in combination with advancements in molecular diagnostics enables much greater precision in late blight management to produce recommendations that are site, host, and pathogen specific.

The Irish potato famine pathogen Phytophthora infestans originated in central Mexico rather than the Andes.

Phytophthora infestans is a destructive plant pathogen best known for causing the disease that triggered the Irish potato famine and remains the most costly potato pathogen to manage worldwide. Identification of P. infestan's elusive center of origin is critical to understanding the mechanisms of repeated global emergence of this pathogen. There are two competing theories, placing the origin in either South America or in central Mexico, both of which are centers of diversity of Solanum host plants. To test these competing hypotheses, we conducted detailed phylogeographic and approximate Bayesian computation analyses, which are suitable approaches to unraveling complex demographic histories. Our analyses used microsatellite markers and sequences of four nuclear genes sampled from populations in the Andes, Mexico, and elsewhere. To infer the ancestral state, we included the closest known relatives Phytophthora phaseoli, Phytophthora mirabilis, and Phytophthora ipomoeae, as well as the interspecific hybrid Phytophthora andina. We did not find support for an Andean origin of P. infestans; rather, the sequence data suggest a Mexican origin. Our findings support the hypothesis that populations found in the Andes are descendants of the Mexican populations and reconcile previous findings of ancestral variation in the Andes. Although centers of origin are well documented as centers of evolution and diversity for numerous crop plants, the number of plant pathogens with a known geographic origin are limited. This work has important implications for our understanding of the coevolution of hosts and pathogens, as well as the harnessing of plant disease resistance to manage late blight.

De novo pyrimidine biosynthesis in the oomycete plant pathogen Phytophthora infestans.

The oomycete Phytophthora infestans, causal agent of the tomato and potato late blight, generates important economic and environmental losses worldwide. As current control strategies are becoming less effective, there is a need for studies on oomycete metabolism to help identify promising and more effective targets for chemical control. The pyrimidine pathways are attractive metabolic targets to combat tumors, virus and parasitic diseases but have not yet been studied in Phytophthora. Pyrimidines are involved in several critical cellular processes and play structural, metabolic and regulatory functions. Here, we used genomic and transcriptomic information to survey the pyrimidine metabolism during the P. infestans life cycle. After assessing the putative gene machinery for pyrimidine salvage and de novo synthesis, we inferred genealogies for each enzymatic domain in the latter pathway, which displayed a mosaic origin. The last two enzymes of the pathway, orotate phosphoribosyltransferase and orotidine-5-monophosphate decarboxylase, are fused in a multi-domain enzyme and are duplicated in some P. infestans strains. Two splice variants of the third gene (dihydroorotase) were identified, one of them encoding a premature stop codon generating a non-functional truncated protein. Relative expression profiles of pyrimidine biosynthesis genes were evaluated by qRT-PCR during infection in Solanum phureja. The third and fifth genes involved in this pathway showed high up-regulation during biotrophic stages and down-regulation during necrotrophy, whereas the uracil phosphoribosyl transferase gene involved in pyrimidine salvage showed the inverse behavior. These findings suggest the importance of de novo pyrimidine biosynthesis during the fast replicative early infection stages and highlight the dynamics of the metabolism associated with the hemibiotrophic life style of pathogen.

IDENTIFICATION OF KEY MOLECULAR COMPONENTS OF THE RESISTANCE OF CHERRY TOMATO AGAINSTPhytophthora infestans / Identificación de los principales componentes moleculares de la resistencia de tomate cherry contra Phytophthora infestans

Cherry tomato Solanum lycopersicum var cerasiforme cv Matt's wild cherry is a very resistant cultivar to most Phytophthora infestans isolates. Two isolates were identified, US940480 and US970001 that cause an incompatible and a compatible interaction respectively. US970001 is one of the few isolates producing a compatible interaction with this cultivar. To identify genes with a differential gene expression between compatible and incompatible interactions, gene expression patterns were analyzed with tomato cDNA microarrays including 12,899 independent tomato cDNA clones at different time points after inoculation. A diverse set of statistical tools were used to identify key components of the plant response to the pathogen. Forty-three genes were up-regulated during the incompatible reaction at time point 36 hours, 15 globally at all time points and twelve were found both in globally and at 36 hours. Northern blots analysis was performed to confirm differential expression showed by microarray analysis and to study the differential expression of more plant resistance genes (PR) genes between compatible and incompatible interactions for this interaction.

El tomate cherry Solanum lycopersicum var cerasiforme cv Matt's es bastante resistente a la gran parte de aislamientos de Phytophthora infestans. Se han identificado dos aislamientos, US940480 y US970001 que causan interacción incompatible y compatible respectivamente. US970001 es uno de los pocos aislamientos causantes de interacción compatible con este cultivo. Con el fin de identificar genes con expresión diferencial en interacciones compatible e incompatible, analizamos DNA copia de 12899 clones independientes en tres tiempos posteriores a la inoculación del patógeno. Se aplicaron diversas herramientas estadísticas para identificar componentes moleculares claves de la respuesta de la planta al patógeno. Cuarenta y tres genes fueron detectados como activados durante la interacción incompatible a las 36 horas posinoculación, 15 genes se detectaron como activados globalmente tomando en conjunto los 3 tiempos analizados y 12 genes tanto globalmente como a las 36 horas. Análisis de Northern blot permitieron confirmar la expresión diferencial detectada con los análisis de microarreglos y estudiar la expresión diferencial de otros genes de resistencia en plantas (PR) en in teracciones compatible e incompatible en esta interacción.

Characterization of Phytophthora infestans populations in Colombia: first report of the A2 mating type.

Phytophthora infestans, the causal agent of late blight in crops of the Solanaceae family, is one of the most important plant pathogens in Colombia. Not only are Solanum lycopersicum, and S. tuberosum at risk, but also several other solanaceous hosts (Physalis peruviana, S. betaceum, S. phureja, and S. quitoense) that have recently gained importance as new crops in Colombia may be at risk. Because little is known about the population structure of Phytophthora infestans in Colombia, we report here the phenotypic and molecular characterization of 97 isolates collected from these six different solanaceous plants in Colombia. All the isolates were analyzed for mating type, mitochondrial haplotypes, genotype for several microsatellites, and sequence of the internal transcribed spacer (ITS) region. This characterization identified a single individual of A2 mating type (from Physalis peruviana) for the first time in Colombia. All isolates had an ITS sequence that was at least 97% identical to the consensus sequence. Of the 97 isolates, 96 were mitochondrial haplotype IIa, with the single A2 isolate being Ia. All isolates were invariant for the microsatellites. Additionally, isolates collected from S. tuberosum and P. peruviana (64 isolates) were tested for: aggressiveness on both hosts, genotype for the isozymes (glucose-6-phosphate isomerase and peptidase), and restriction fragment length polymorphism fingerprint pattern as detected by RG57. Isolates from S. tuberosum were preferentially pathogenic on S. tuberosum, and isolates from P. peruviana were preferentially pathogenic on P. peruviana. The population from these two hosts was dominated by a single clonal lineage (59 of 64 individuals assayed), previously identified from Ecuador and Peru as EC-1. This lineage was mating type A1, IIa for mitochondrial DNA, invariant for two microsatellites, and invariant for both isozymes. The remaining four A1 isolates were in lineages very closely related to EC-1 (named EC-1.1, CO-1, and CO-2). The remaining lineage (the A2 mating type) had characteristics of the US-8 lineage (previously identified in Mexico, the United States, and Canada). These results have important epidemiological implications for the production of these two crops in Colombia.

The Population Structure of Phytophthora infestans from the Toluca Valley of Central Mexico Suggests Genetic Differentiation Between Populations from Cultivated Potato and Wild Solanum spp.

ABSTRACT The population structure of Phytophthora infestans in the Toluca Valley of central Mexico was assessed using 170 isolates collected from cultivated potatoes and the native wild Solanum spp., S. demissum and S. xendinense. All isolates were analyzed for mitochondrial DNA (mtDNA) haplotype and amplified fragment length polymorphism (AFLP) multi-locus fingerprint genotype. Isolate samples were monomorphic for mtDNA haplotype because all isolates tested were of the Ia haplotype. A total of 158 multilocus AFLP genotypes were identified among the 170 P. infestans isolates included in this study. P. infestans populations sampled in the Toluca Valley in 1997 were highly variable and almost every single isolate represented a unique genotype based on the analysis of 165 AFLP marker loci. Populations of P. infestans collected from the commercial potato-growing region in the valley, the subsistence potato production area along the slopes of the Nevado de Toluca, and the native Solanum spp. on the forested slopes of the volcano showed a high degree of genetic diversity. The number of polymorphic loci varied from 20.0 to 62.4% for isolates collected from the field station and wild Solanum spp. On average, 81.8% (135) of the AFLP loci were polymorphic. Hetero-zygosity varied between 7.7 and 19.4%. Significant differentiation was found at the population level between strains originating from cultivated potatoes and wild Solanum spp. (P = 0.001 to 0.022). Private alleles were observed in individual isolates collected from all three populations, with numbers of unique dominant alleles varying from 9 to 16 for isolates collected from commercial potato crops and native Solanum spp., respectively. Four AFLP markers were exclusively found present in isolates collected from S. demissum. Indirect estimation of gene flow between populations indicated restricted gene flow between both P. infestans populations from cultivated potatoes and wild Solanum hosts. There was no evidence found for the presence of substructuring at the subpopulation (field) level. We hypothesize that population differentiation and genetic isolation of P. infestans in the Toluca Valley is driven by host-specific factors (i.e., R-genes) widely distributed in wild Solanum spp. and random genetic drift.

Characterization of Isolates of Phytophthora infestans from Southern and Southeastern Brazil from 1998 to 2000.

The population of Phytophthora infestans in Brazil was first characterized 12 years ago. In this research, isolates of P. infestans from potato (n = 184) and tomato (n = 267) collected in southern and southeastern Brazil were characterized to provide more detailed analysis of the current structure of the population. All 451 isolates were analyzed for mating type, and subsets of the isolates were analyzed for allozymes, restriction fragment length polymorphism fingerprint, mtDNA haplotypes, and metalaxyl resistance. Tomato isolates were all of A1 mating type, mtDNA Ib, and US-1 genotype or some variant within this clonal lineage. Of the potato isolates, 82% were A2 mating type, mtDNA IIa, BR-1 genotype, which is a new lineage of P. infestans. All A2 isolates were found on potato, whereas 91% of the A1 isolates were from tomato. A1 and A2 isolates were never found in the same field. The frequency of resistance to metalaxyl was higher in isolates from tomato (55%) than in isolates from potato (38%). After more than a decade of coexistence of isolates of the A1 and A2 mating types, the population was highly clonal, dominated by the BR-1 and US-1 clonal lineages.

Potato cultivars from the mexican national program: sources and durability of resistance against late blight.

ABSTRACT The Mexican National Potato Program has produced several cultivars with high levels of field resistance. We evaluated the durability of resistance to potato late blight of a selection of 12 such cultivars using data from 1960 to the present. Data were extracted from the field notebooks located in the archives of the Mexican National Potato Program in the John S. Niederhauser Library in Toluca, Mexico. There was a trend indicating that field resistances to potato late blight of Mexican cultivars released between 1965 to 1999 were durable. At least two of the cultivars, namely 'Sangema' and 'Tollocan', have been grown on at least 4 to 5% of the potato acreage and over long periods of time without decay in levels of field resistance. Pedigrees of the 12 cultivars indicate that most of the field resistance was introgressed from Solanum demissum. Field resistance might also be derived from commonly grown land-race cultivars such as 'Amarilla de Puebla' and 'Leona'. These have been grown in Mexico since about the 1780s. They have the appearance of S. andigena-derived material but their genetic background is unknown.