Gregory A. Forbes

Population Genetic Structure of Phytophthora infestans in Ecuador

ABSTRACT The population genetic structure of Phytophthora infestans in Ecuador was assessed from 101 isolates collected from 1990 to 1992 and 111 isolates collected in 1993. All isolates were analyzed for mating type and allozyme genotype. Both samples were dominated (>95%) by a clonal lineage (EC-1) defined from neutral markers: 90/100 genotype for glucose-6-phosphate isomerase, 96/100 genotype for peptidase, A1 mating type, and a previously unreported nuclear DNA fingerprint. The remaining isolates belonged to the US-1 clonal lineage, which has a worldwide distribution. Isolates in the 1993 sample were analyzed for virulence and metalaxyl sensitivity. All representatives of EC-1 had complex patho-types, with three pathotypes representing >60% of the collection. There was variation for metalaxyl sensitivity. There was no evidence for geographical substructuring on the basis of neutral markers, but there was evidence for limited substructuring based on metalaxyl sensitivity and specific virulence. We hypothesize that EC-1 has been recently introduced to Ecuador.

Host Specificity of Phytophthora infestans on Tomato and Potato in Ecuador

ABSTRACT Sixty Ecuadorian isolates of Phytophthora infestans from potato and 60 isolates from tomato were compared for dilocus allozyme genotype, mitochondrial DNA haplotype, mating type, and specific virulence on 11 potato R-gene differential plants and four tomato cultivars, two of which contained different Ph genes. Restriction fragment length polymorphism (RFLP) fingerprints of subsamples of isolates from each host were compared by using RG57 as the probe. All potato isolates had the allozyme genotype, haplotype, and mating type of the clonal lineage EC-1, which had been previously described in Ecuador. With the same markers, only one isolate from tomato was classified as EC-1; all others belonged to the globally distributed US-1 clonal lineage. RFLP fingerprints of isolate subsets corroborated this clonal lineage classification. Specific virulence on potato differentials was broadest among potato isolates, while specific virulence on tomato cultivars was broadest among tomato isolates. Some tomato isolates infected all tomato differentials but no potato differentials, indicating that specific virulence for the two hosts is probably controlled by different avirulence genes in P. infestans. In two separate experiments, the diameters of lesions caused by nine isolates from potato and 10 from tomato were compared on three tomato and three potato cultivars. All isolates produced larger lesions on the host from which they were isolated. No isolates were found that were highly aggressive on both tomato and potato. We conclude that there are two different populations of P. infestans in Ecuador and that they are separated by host.

Genetic Diversity of Phytophthora infestans sensu lato in Ecuador Provides New Insight Into the Origin of This Important Plant Pathogen

ABSTRACT The metapopulation structure of Phytophthora infestans sensu lato is genetically diverse in the highlands of Ecuador. Previous reports documented the diversity associated with four putative clonal lineages of the pathogen collected from various hosts in the genus Solanum. This paper simultaneously analyzes diversity of the complete collection of isolates, including a large number that had not yet been reported. This analysis confirmed the existence of three pathogen populations, which all appear to be clonal lineages, and that correspond to those previously reported as US-1, EC-1, and EC-3. No evidence was found from the analyses of recently collected isolates that would contradict earlier reports about these three lineages. In contrast, new data from a group of isolates from several similar hosts caused us to modify the previous description of clonal lineage EC-2 and its previously proposed hosts, S. brevifolium and S. tetrapetalum. Given the uncertainty associated with the identification of these hosts, which all belong to the section Anarrhichomenum, we refer to them as the Anarrhichomenum complex, pending further taxonomic clarification. New pathogen genotypes associated with the Anarrhichomenum complex were isolated recently that are A1 mating type and Ia mitochondrial DNA (mtDNA) haplotype, and therefore differ from the previously described EC-2 lineage, which is A2 and Ic, respectively. Because of uncertainty on host identification, we do not know if the new genotypes are limited to one host species and therefore represent yet another host-adapted clonal lineage. For now, we refer to the new genotypes and previously described EC-2 genotypes, together, as the pathogen group attacking the Anarrhichomenum complex. Two A2 isolates identical to the previously described EC-2 archetype were collected from severely infected plants of pear melon (S. muricatum). Pear melon is generally attacked by US-1, and this is the first clear case we have documented in which two distinct pathogen genotypes have caused severe epidemics on the same host. Based on presence of unique marker alleles (restriction fragment length polymorphism [RFLP] and mtDNA) and genetic similarity analysis using RFLP and amplified fragment length polymorphism data, EC-3 and isolates from the Anarrhichomenum complex are genetically distinct from all genotypes of P. infestans that have been reported previously. No current theory of historical migrations for this pathogen can adequately support a Mexican origin for EC-3 and genotypes of the Anarrhichomenum complex and they may, therefore, be palaeoendemic to the Andean highlands. To date, we have identified 15 hosts in the genus Solanum, in addition to the Anarrhichomenum complex, and some unidentified species of P. infestans sensu lato in Ecuador. Five of the Solanum hosts are cultivated. One isolate was collected from Brugmansia sanguinea, which represents the first report from Ecuador of a host of this pathogen that is not in the genus Solanum. However, P. infestans sensu lato was only found on flower petals of B. sanguinea. This study provides new insights into the population structure of highly specialized genotypes of P. infestans sensu lato in the Andean highlands. The results are discussed in light of previous hypotheses regarding the geographic origin of the pathogen.

The Plant Pathogen Phytophthora andina Emerged via Hybridization of an Unknown Phytophthora Species and the Irish Potato Famine Pathogen, P. infestans

Emerging plant pathogens have largely been a consequence of the movement of pathogens to new geographic regions. Another documented mechanism for the emergence of plant pathogens is hybridization between individuals of different species or subspecies, which may allow rapid evolution and adaptation to new hosts or environments. Hybrid plant pathogens have traditionally been difficult to detect or confirm, but the increasing ease of cloning and sequencing PCR products now makes the identification of species that consistently have genes or alleles with phylogenetically divergent origins relatively straightforward. We investigated the genetic origin of Phytophthora andina, an increasingly common pathogen of Andean crops Solanum betaceum, S. muricatum, S. quitoense, and several wild Solanum spp. It has been hypothesized that P. andina is a hybrid between the potato late blight pathogen P. infestans and another Phytophthora species. We tested this hypothesis by cloning four nuclear loci to obtain haplotypes and using these loci to infer the phylogenetic relationships of P. andina to P. infestans and other related species. Sequencing of cloned PCR products in every case revealed two distinct haplotypes for each locus in P. andina, such that each isolate had one allele derived from a P. infestans parent and a second divergent allele derived from an unknown species that is closely related but distinct from P. infestans, P. mirabilis, and P. ipomoeae. To the best of our knowledge, the unknown parent has not yet been collected. We also observed sequence polymorphism among P. andina isolates at three of the four loci, many of which segregate between previously described P. andina clonal lineages. These results provide strong support that P. andina emerged via hybridization between P. infestans and another unknown Phytophthora species also belonging to Phytophthora clade 1c.

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

Significance The potato late blight pathogen was introduced to Europe in the 1840s and caused the devastating loss of a staple crop, resulting in the Irish potato famine and subsequent diaspora. Research on this disease has engendered much debate, which in recent years has focused on whether the geographic origin of the pathogen is South America or central Mexico. Different lines of evidence support each hypothesis. We sequenced four nuclear genes in representative samples from Mexico and the South American Andes. An Andean origin of P. infestans does not receive support from detailed analyses of Andean and Mexican populations. This is one of a few examples of a pathogen with a known origin that is secondary to its current major host. 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.

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.

Simulation of Potato Late Blight in the Andes. I: Modification and Parameterization of the LATEBLIGHT Model

ABSTRACT LATEBLIGHT, a mathematical model that simulates the effects of weather, host growth and resistance, and fungicide use on asexual development and growth of Phytophthora infestans on potato foliage, was modified so that it can be used in the Andes and, eventually, worldwide. The modifications included (i) the incorporation of improved equations for the effect of temperature on lesion growth rate (LGR) and sporulation rate (SR); (ii) the incorporation of temperature-dependent latent period (LP); and (iii) the use of experimentally measured parameters of LGR, SR, and LP for specific potato cultivars and pathogen lineages. The model was parameterized for three Peruvian potato cultivars (Tomasa, Yungay, and Amarilis) infected with isolates of a new clonal lineage of P. infestans that is currently predominant in Ecuador and Peru (EC-1).

Estimating the level of susceptibility to Phytophthora infestans in potato genotypes.

Resistance and susceptibility are closely related terms but differ in their underlying assumptions and measurement. Standardized methods for determining the level of resistance and susceptibility in potato to Phytophthora infestans, the causal agent of late blight, have traditionally been semiquantitative and are not based on a true interval scale, thus making their use in most mathematical and statistical operations inappropriate. Recently, researchers have attempted to develop interval scales using regression analysis of the direct or transformed area under the disease progress curve (AUDPC). In this article, a similar approach is described based on the relative AUDPC (RAUDPC) of one or two reference cultivars and tested using a data set of field trials involving cultivars with varying levels of susceptibility evaluated in different environments in several countries. The coefficient of variation (CV) among trials of the AUDPC was reduced when the RAUDPC was used and even more so when the RAUDPC was made relative to the RAUDPC of cv. Bintje (RaRAUDPC), which was present in all trials. The RaRAUDPC was used in regression models to estimate scale values for eight potato cultivars in 13 to 15 locations (depending on cultivar). The CVs of scale values measuring variation among sites were similar to those of the RaRAUDPC. Using two cultivars gave a slight improvement in CV, which was statistically significant. The scale developed here has ascending numbers for increasing susceptibility, is simple, and can be constructed as a ratio measure, which permitted the calculation of mean, variance, and CV.

Phylogenetic relationships of Phytophthora andina, a new species from the highlands of Ecuador that is closely related to the Irish potato famine pathogen Phytophthora infestans

Phylogenetic relationships of Phytophthora infestans sensu lato in the Andean highlands of South America were examined. Three clonal lineages (US-1, EC-1, EC-3) and one heterogeneous lineage (EC-2) were found in association with different host species in genus Solanum. The EC-2 lineage includes two mitochondrial (mtDNA) haplotypes, Ia and Ic. Isolates of P. infestans sensu lato EC-2 fit the morphological description of P. infestans but are different from any genotypes of P. infestans described to date. All isolates of P. infestans sensu lato from Ecuador were amplified by a P. infestans specific primer (PINF), and restriction fragment length patterns were identical in isolates amplified with ITS primers 4 and 5. The EC-1 clonal lineage of P. infestans sensu lato from S. andreanum, S. columbianum, S. paucijugum, S. phureja, S. regularifolium, S. tuberosum and S. tuquerense was confirmed to be P. infestans based on sequences of the cytochrome oxidase I (cox I) gene and intron 1 of ras gene. The EC-2 isolates with the Ic haplotype formed a distinct branch in the same clade with P. infestans and P. mirabilis, P. phaseoli and P. ipomoeae for both cox I and ras intron 1 phylogenies and were identified as the newly described species P. andina. Ras intron 1 sequence data suggests that P. andina might have arisen via hybridization between P. infestans and P. mirabilis.

Qualification of a Plant Disease Simulation Model: Performance of the LATEBLIGHT Model Across a Broad Range of Environments.

ABSTRACT The concept of model qualification, i.e., discovering the domain over which a validated model may be properly used, was illustrated with LATEBLIGHT, a mathematical model that simulates the effect of weather, host growth and resistance, and fungicide use on asexual development and growth of Phytophthora infestans on potato foliage. Late blight epidemics from Ecuador, Mexico, Israel, and the United States involving 13 potato cultivars (32 epidemics in total) were compared with model predictions using graphical and statistical tests. Fungicides were not applied in any of the epidemics. For the simulations, a host resistance level was assigned to each cultivar based on general categories reported by local investigators. For eight cultivars, the model predictions fit the observed data. For four cultivars, the model predictions overestimated disease, likely due to inaccurate estimates of host resistance. Model predictions were inconsistent for one cultivar and for one location. It was concluded that the domain of applicability of LATEBLIGHT can be extended from the range of conditions in Peru for which it has been previously validated to those observed in this study. A sensitivity analysis showed that, within the range of values observed empirically, LATEBLIGHT is more sensitive to changes in variables related to initial inoculum and to weather than to changes in variables relating to host resistance.