Genome analyses of an aggressive and invasive lineage of the Irish potato famine pathogen.

Pest and pathogen losses jeopardise global food security and ever since the 19(th) century Irish famine, potato late blight has exemplified this threat. The causal oomycete pathogen, Phytophthora infestans, undergoes major population shifts in agricultural systems via the successive emergence and migration of asexual lineages. The phenotypic and genotypic bases of these selective sweeps are largely unknown but management strategies need to adapt to reflect the changing pathogen population. Here, we used molecular markers to document the emergence of a lineage, termed 13_A2, in the European P. infestans population, and its rapid displacement of other lineages to exceed 75% of the pathogen population across Great Britain in less than three years. We show that isolates of the 13_A2 lineage are among the most aggressive on cultivated potatoes, outcompete other aggressive lineages in the field, and overcome previously effective forms of plant host resistance. Genome analyses of a 13_A2 isolate revealed extensive genetic and expression polymorphisms particularly in effector genes. Copy number variations, gene gains and losses, amino-acid replacements and changes in expression patterns of disease effector genes within the 13_A2 isolate likely contribute to enhanced virulence and aggressiveness to drive this population displacement. Importantly, 13_A2 isolates carry intact and in planta induced Avrblb1, Avrblb2 and Avrvnt1 effector genes that trigger resistance in potato lines carrying the corresponding R immune receptor genes Rpi-blb1, Rpi-blb2, and Rpi-vnt1.1. These findings point towards a strategy for deploying genetic resistance to mitigate the impact of the 13_A2 lineage and illustrate how pathogen population monitoring, combined with genome analysis, informs the management of devastating disease epidemics.

Recent Genotypes of Phytophthora infestans in the Eastern United States Reveal Clonal Populations and Reappearance of Mefenoxam Sensitivity.

Isolates of Phytophthora infestans (n = 178) were collected in 2002 to 2009 from the eastern United States, Midwestern United States, and eastern Canada. Multilocus genotypes were defined using allozyme genotyping, and DNA fingerprinting with the RG-57 probe. Several previously described and three new mulitilocus genotypes were detected. The US-8 genotype was found commonly on commercial potato crops but not on tomato. US-20 was found on tomato in North Carolina from 2002 through 2007 and in Florida in 2005. US-21 was found on tomato in North Carolina in 2005 and Florida in 2006 and 2007. US-22 was detected on tomato in 2007 in Tennessee and New York and became widespread in 2009. US-22 was found in 12 states on tomato and potato and was spread on tomato transplants. This genotype accounted for about 60% of all the isolates genotyped. The US-23 genotype was found in Maryland, Virginia, Pennsylvania, and Delaware on both tomato and potato in 2009. The US-24 genotype was found only in North Dakota in 2009. A1 and A2 mating types were found in close proximity on potato and tomato crops in Pennsylvania and Virginia; therefore, the possibility of sexual reproduction should be monitored. Whereas most individuals of US-8 and US-20 were resistant to mefenoxam, US-21 appeared to be intermediately sensitive, and isolates of US-22, US-23, and US-24 were largely sensitive to mefenoxam. On the basis of sequence analysis of the ras gene, these latter three genotypes appear to have been derived from a common ancestor. Further field and laboratory studies are underway using simple sequence repeat genotyping to monitor current changes in the population structure of P. infestans causing late blight in North America.

Interference by Mes [2-(4-morpholino)ethanesulfonic acid] and related buffers with phenolic oxidation by peroxidase.

While characterizing the kinetic parameters of apoplastic phenolic oxidation by peroxidase, we found anomalies caused by the Mes [2-(4-morpholino)ethanesulfonic acid] buffer being used. In the presence of Mes, certain phenolics appeared not to be oxidized by peroxidase, yet the oxidant, H(2)O(2), was utilized. This anomaly seems to be due to the recycling of the phenolic substrate. The reaction is relatively inefficient, but at buffer concentrations of 10 mM or greater the recycling effect is nearly 100% with substrate concentrations less than 100 microM. The recycling effect is dependent on substrate structure, occurring with 4'-hydroxyacetophenone but not with 3',5'-dimethoxy-4'-hydroxyacetophenone (acetosyringone). Characterization of the reaction parameters suggests that the phenoxyl radical from the peroxidase reaction interacts with Mes, causing the reduction and regeneration of the phenol. Similar responses occurred with related buffers such as Hepes [4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid] and Pipes [piperazine-1,4-bis(2-ethanesulfonic acid)]. Results from this work and other reports in the literature indicate that great care is required in interpreting any results involving these buffers under oxidizing conditions.

Alternate intron processing of family 5 endoglucanase transcripts from the genus Phytophthora.

Twenty-one homologs of family 5 endo-(1-4)-beta-glucanase genes (EGLs) were identified and characterized in the oomycete plant pathogens Phytophthora infestans, P. sojae, and P. ramorum, providing the first comprehensive analysis of this family in Phytophthora. Phylogenetic analysis revealed that these genes constitute a unique eukaryotic group, with closest similarity to bacterial endoglucanases. Many of the identified EGL copies were clustered in a few genomic regions, and contained from zero to three introns. Using reverse transcription PCR to study in vitro and in planta gene expression levels of P. sojae, we detected partially processed RNA transcripts retaining one or more of their introns. In some cases, the positions of intron/exon splicing sites were also found to be variable. The relative proportions of these transcripts remain apparently unchanged under various growing conditions, but differ among orthologous copies of the three Phytophthora species. The alternate processing of introns in this group of EGLs generates both coding and non-coding RNA isoforms. This is the first report on Phytophthora family 5 endoglucanases, and the first record for alternative intron processing of oomycete transcripts.

Involvement of acetosyringone in plant-pathogen recognition.

In this study, acetosyringone was identified as one of the major extracellular phenolics in tobacco suspension cells and was shown to have bioactive properties that influence early events in plant-bacterial pathogenesis. In our model system, tobacco cell suspensions treated with bacterial isolate Pseudomonas syringae WT (HR+) undergo a resistant interaction characterized by a burst in oxygen uptake several hours after inoculation. When the extracellular concentration of acetosyringone in tobacco cell suspensions was supplemented with exogenous acetosyringone, the burst in oxygen uptake occurred as much as 1.5h earlier. The exogenous acetosyringone had no effect on tobacco suspensions undergoing susceptible interactions with Pseudomonas tabaci or a non-resistant interaction with a near-isogenic mutant derivative of isolate P. syringae WT (HR+). Resistant interactions with isolate P. syringae WT (HR+) also produce an oxidative burst which oxidizes the extracellular acetosyringone. This study demonstrates that acetosyringone, and likely other extracellular phenolics, may have bioactive characteristics that can influence plant-bacterial pathogenesis.

Population changes in Phytophthora infestans in Taiwan associated with the appearance of resistance to metalaxyl.

In recent years, late blight, caused by Phytophthora infestans (Mont) De Bary, has increased in severity in many parts of the world, and this has been associated with migrations which have introduced new, arguably more aggressive, populations of the pathogen. In Taiwan, late blight has been endemic on outdoor tomato crops grown in the highlands since the early 1900s, but recent epidemics have been more damaging. To ascertain the present status of the Taiwanese population of P infestans, 139 isolates of the pathogen collected and maintained by the Asian Vegetable Research and Development Center (AVRDC) were characterized using mating type, metalaxyl sensitivity, allozyme genotype, mitochondrial haplotype and RFLP fingerprinting. Up to 1997, all isolates were found to belong to the old clonal lineage of P infestans (US-1 and variants), but in isolates from 1998 a new genotype appeared, and by 2000 this had apparently completely displaced the old population. This new genotype was an A1 mating type and has the dilocus allozyme genotype 100/100/111, 100/100 for the loci coding for glucose-6-phosphate isomerase and peptidase, respectively. These characters, together with RG57 fingerprinting, indicated that these isolates belonged to the US-11 clonal lineage, a minority (11%) being a previously unreported variant of US-11. Whereas metalaxyl-resistant isolates were not detected in the old population, 96% of the new genotypes proved resistant, with the remainder being intermediate in sensitivity. It may be inferred from this sudden, marked change in the characteristics of the Taiwanese P infestans that a new population of the pathogen was introduced around 1997-98 and that this may well have already been metalaxyl-resistant when it arrived, although a role for in situ selection cannot be excluded.