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.

Searching for the Mechanism that Mediates Mefenoxam-Acquired Resistance in Phytophthora infestans and How It Is Regulated.

Phytophthora infestans, the causal agent of late blight disease of potatoes, is mainly controlled by the use of fungicides. Isolates that are resistant to commonly used fungicides have been reported. Also, several studies show that originally mefenoxam-sensitive isolates acquire resistance to this fungicide when exposed to sublethal concentrations. This phenomenon, termed "mefenoxam-acquired resistance," has been observed in different Phytophthora species and seems to be unique to mefenoxam. In this study, we aimed to elucidate the molecular mechanism mediating this type of resistance as well as a possible regulatory process behind it. A combination of computational analyses and experimental approaches was used to identify differentially expressed genes with a potential association to the phenomenon. These genes were classified into seven functional groups. Most of them seem to be associated with a pleiotropic drug resistance (PDR) phenotype, typically involved in the expulsion of diverse metabolites, drugs, or other substances out of the cell. Despite the importance of RNA Polymerase I for the constitutive resistance of P. infestans to mefenoxam, our results indicate no clear interaction between this protein and the acquisition of mefenoxam resistance. Several small non-coding RNAs were found to be differentially expressed and specifically related to genes mediating the PDR phenotype, thus suggesting a possible regulatory process. We propose a model of the molecular mechanisms acting within the cell when P. infestans acquires resistance to mefenoxam after exposed to sublethal concentrations of the fungicide. This study provides important insights into P. infestans' cellular and regulatory functionalities.

Phytophthora infestans Sporangia Produced in Culture and on Tomato Leaflet Lesions Show Marked Differences in Indirect Germination Rates, Aggressiveness, and Global Transcription Profiles.

Sporangia of Phytophthora infestans from pure cultures on agar plates are typically used in lab studies, whereas sporangia from leaflet lesions drive natural infections and epidemics. Multiple assays were performed to determine if sporangia from these two sources are equivalent. Sporangia from plate cultures showed much lower rates of indirect germination and produced much less disease in field and moist-chamber tests. This difference in aggressiveness was observed whether the sporangia had been previously incubated at 4°C (to induce indirect germination) or at 21°C (to prevent indirect germination). Furthermore, lesions caused by sporangia from plates produced much less sporulation. RNA-Seq analysis revealed that thousands of the >17,000 P. infestans genes with a RPKM (reads per kilobase of exon model per million mapped reads) >1 were differentially expressed in sporangia obtained from plate cultures of two independent field isolates compared with sporangia of those isolates from leaflet lesions. Among the significant differentially expressed genes (DEGs), putative RxLR effectors were overrepresented, with almost half of the 355 effectors with RPKM >1 being up- or downregulated. DEGs of both isolates include nine flagellar-associated genes, and all were down-regulated in plate sporangia. Ten elicitin genes were also detected as DEGs in both isolates, and nine (including INF1) were up-regulated in plate sporangia. These results corroborate previous observations that sporangia produced from plates and leaflets sometimes yield different experimental results and suggest hypotheses for potential mechanisms. We caution that use of plate sporangia in assays may not always produce results reflective of natural infections and epidemics.

Genetic Variation within Clonal Lineages of Phytophthora infestans Revealed through Genotyping-By-Sequencing, and Implications for Late Blight Epidemiology.

Genotyping-by-sequencing (GBS) was performed on 257 Phytophthora infestans isolates belonging to four clonal lineages to study within-lineage diversity. The four lineages used in the study were US-8 (n = 28), US-11 (n = 27), US-23 (n = 166), and US-24 (n = 36), with isolates originating from 23 of the United States and Ontario, Canada. The majority of isolates were collected between 2010 and 2014 (94%), with the remaining isolates collected from 1994 to 2009, and 2015. Between 3,774 and 5,070 single-nucleotide polymorphisms (SNPs) were identified within each lineage and were used to investigate relationships among individuals. K-means hierarchical clustering revealed three clusters within lineage US-23, with US-23 isolates clustering more by collection year than by geographic origin. K-means hierarchical clustering did not reveal significant clustering within the smaller US-8, US-11, and US-24 data sets. Neighbor-joining (NJ) trees were also constructed for each lineage. All four NJ trees revealed evidence for pathogen dispersal and overwintering within regions, as well as long-distance pathogen transport across regions. In the US-23 NJ tree, grouping by year was more prominent than grouping by region, which indicates the importance of long-distance pathogen transport as a source of initial late blight inoculum. Our results support previous studies that found significant genetic diversity within clonal lineages of P. infestans and show that GBS offers sufficiently high resolution to detect sub-structuring within clonal populations.

Toward improvements of oomycete transformation protocols.

Some of the most important plant pathogens worldwide are oomycetes, and billions of dollars are expended annually to suppress diseases they cause. More efficient disease suppression technologies will be derived from a better understanding of the basic biology of these organisms, but inefficient transformation currently limits basic molecular investigations. Of the various approaches, transformation of protoplasts using polyethylene glycol/calcium chloride remains most successful, but the frequency of stable transformation remains low and inconsistent. Here we report that modifications of a protocol, previously used for Arabidopsis mesophyll cells, successfully releases protoplasts from four different oomycetes (Phytophthora citricola, Phytophthora infestans, Phytophthora sojae, and Pythium aphanidermatum). The protoplasts of all oomycetes were able to take up DNA and regenerate, with protoplast release as well as regeneration being most efficient in P. aphanidermatum. In addition to a good protoplast production system, more effective transformation vectors may improve stable transformation rates. We constructed, and evaluated 17 novel candidate transformation vectors for their ability to drive transient expression of the beta-glucuronidase (GUS) reporter gene in P. infestans and P. aphanidermatum. Five of the newly constructed vectors were also evaluated in P. sojae and P. citricola, and exhibited a similar pattern of transcriptional activity as in P. infestans and P. aphanidermatum. One of the newly constructed vectors, pDBHAMT35G, containing a chimeric promoter, supported the highest GUS expression in P. infestans and P. citricola, and could potentially be useful for future studies.