Christine D. Smart

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.

Comparative Analyses of Potato Expressed Sequence Tag Libraries

The cultivated potato (Solanum tuberosum) shares similar biology with other members of the Solanaceae, yet has features unique within the family, such as modified stems (stolons) that develop into edible tubers. To better understand potato biology, we have undertaken a survey of the potato transcriptome using expressed sequence tags (ESTs) from diverse tissues. A total of 61,940 ESTs were generated from aerial tissues, below-ground tissues, and tissues challenged with the late-blight pathogen (Phytophthora infestans). Clustering and assembly of these ESTs resulted in a total of 19,892 unique sequences with 8,741 tentative consensus sequences and 11,151 singleton ESTs. We were able to identify a putative function for 43.7% of these sequences. A number of sequences (48) were expressed throughout the libraries sampled, representing constitutively expressed sequences. Other sequences (13,068, 21%) were uniquely expressed and were detected only in a single library. Using hierarchal and k means clustering of the EST sequences, we were able to correlate changes in gene expression with major physiological events in potato biology. Using pair-wise comparisons of tuber-related tissues, we were able to associate genes with tuber initiation, dormancy, and sprouting. We also were able to identify a number of characterized as well as novel sequences that were unique to the incompatible interaction of late-blight pathogen, thereby providing a foundation for further understanding the mechanism of resistance.

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.

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.

Invasions by the Late Blight Pathogen: Renewed Sex and Enhanced Fitness

Introductions of Phytophthora infestans, the oomycete plant pathogen responsible for late blight of potato and tomato, have had devastating human impacts wherever they have occurred. This pathogen was apparently sequestered in central Mexico until the mid-19th century, when introductions into the USA and Europe led to a series of crop failures – the most notable of which resulted in the Irish potato famine. A second series was documented in the late 20th century. There were terrible effects also from the second set of introductions, but no population was as vulnerable as the Irish peasants in the mid-19th century. Examination and comparison of these introductions has taught us that we must be prepared for the invasion of species that are more fit than the current population.

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.

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? Its 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.

Late Blight of Potato and Tomato in the Genomics Era

Tremendous progress in the availability of genomic data has been made in the past decade. With the advent of high throughput and affordable DNA sequencing, the entire hereditary blueprint of an organism can be determined. A widespread transition into research enabled by the tools and resources of genomics is consequently occurring, and it has already affected countless disciplines within the biological sciences, including plant pathology. This new era of research allows a comprehensive study of gene structure and function and has already delivered tremendous insights into the basic biology of living organisms. Genomics offers new opportunities for applied biological sciences and is poised to impact plant health and plant disease management. More than 150 years have elapsed since Phytophthora infestans caused the Irish potato famine, but strategies for managing potato and tomato late blight often remain unsustainable and costly. In effect, P. infestans continues to cost billions of dollars annually through losses in potato and tomato production and increased fungicide costs (15,45,47,48). In the United States and other developed countries, chronic use of chemicals to manage late blight reduces the profit margins of farmers and is not always successful. Most populations of the pathogen are resistant to metalaxyl (and the active enantiomer mefenoxam), which had previously been very successful for controlling P. infestans (19,47). In developing countries, late blight also affects subsistence potato production. Recent epidemics in the 1990s, notably in eastern Europe, have caused significant losses in yield (20,46). In 2003, potato production was nearly eliminated in Papua New Guinea, one of the few countries in the world that was previously free of the disease (4). Remarkably, the disease spread through the entire country within 2 months of first incidence. Disturbing reports predict that potato late blight will continue to cause food shortages and hunger in several parts of the world (20,46).

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.

Over-expression of snakin-2 and extensin-like protein genes restricts pathogen invasiveness and enhances tolerance to Clavibacter michiganensis subsp. michiganensis in transgenic tomato (Solanum lycopersicum)

Two tomato proteins were evaluated by over-expression in transgenic tomato for their ability to confer resistance to Clavibacter michiganensis subsp. michiganensis (Cmm). Snakin-2 (SN2) is a cysteine-rich peptide with broad-spectrum antimicrobial activity in vitro while extensin-like protein (ELP) is a major cell-wall hydroxyproline-rich glycoprotein linked with plant response to pathogen attack and wounding. Tomato plants, cultivar Mountain Fresh, were transformed via Agrobacterium tumefaciens harboring a binary vector for expression of the full-length SN2 gene or ELP cDNA under the regulation of the CaMV 35S promoter. Molecular characterization of PCR-positive putative T0 transgenic plants by Northern analysis revealed constitutive over-expression of SN2 and ELP mRNA. Junction fragment analysis by Southern blot showed that three of the four SN2 over-expressing T0 lines had single copies of complete T-DNAs while the other line had two complete T-DNA copies. All four ELP over-expressing T0 lines had a single copy T-DNA insertion. Semi-quantitative RT-PCR analysis of T1 plants revealed constitutive over-expression of SN2 and ELP. Transgenic lines that accumulated high levels of SN2 or ELP mRNA showed enhanced tolerance to Cmm resulting in a significant delay in the development of wilt symptoms and a reduction in the size of canker lesions compared to non-transformed control plants. Furthermore, in transgenic lines over-expressing SN2 or ELP bacterial populations were significantly lower (100–10,000-fold) than in non-transformed control plants. These results demonstrate that SN2 and ELP over-expression limits Cmm invasiveness suggesting potential in vivo antibacterial activity and possible biotechnological application for these two defense proteins.