First Report of Late Blight Caused by Phytophthora infestans Clonal Lineage US-23 on Tomato and Potato in Atlantic Canada.

Phytophthora infestans (Mont.) de Bary has produced significant losses in potato and tomato yield and quality during recent late blight epidemics in North America. During the 1990s, more aggressive and genetically diverse P. infestans genotypes migrated to Canada and the United States (2). For example, US-8 became predominant and was found to be more aggressive in potato than previous clonal lineages of P. infestans. Recent P. infestans genotypes in potato and tomato plants from the United States and Canada include US-22, US-23, and US-24 representing clonal lineages with unique epidemiological characteristics (2,3,4). Characteristic phenotypic traits have been described for P. infestans clonal lineages US-8, US-22, US-23, and US-24 based on the mating type, mefenoxam sensitivity, pathogenicity, and rate of germination suggesting an association between phenotypic variations and the genotype (1,4). Analysis of P. infestans isolates collected in Canada during 2010 revealed the presence of the US-23 clonal lineage in four different areas of western Canada but not in eastern Canada (4). Isolates of P. infestans collected from eastern Canada for several years prior to 2011 were all US-8 A2 mating type. Isolation and analysis of 98 P. infestans isolates in 2011 from New Brunswick and Prince Edward Island followed standard procedures (2,3,4). Results confirmed the presence of the US-23 clonal lineage in Atlantic Canada on potato and tomato leaves with late blight symptoms, increasing the genetic complexity of P. infestans in eastern Canada. Allozyme banding patterns at the glucose-6-phosphate isomerase (Gpi) locus showed a 100/100 profile in 10 P. infestans isolates, consistent with the US-23 clonal lineage (2,3,4). Furthermore, in vitro mefenoxam sensitivity was observed in all 10 P. infestans US-23 isolates from New Brunswick and Prince Edward Island. Mating type assays confirmed the isolates were of the A1 mating type. RFLP analysis of EcoR1-digested genomic DNA using the multilocus RG57 sequence as a probe produced the DNA pattern 1, 2, 5, 6, 10, 13, 14, 17, 20, 21, 24, 24a, 25, indicative of US-23 (2,4). Microsatellite analysis using polymorphic markers on New Brunswick and Prince Edward Island P. infestans isolates produced the Pi4B 213/217 bp, D13 134 bp, and PiG11 140/155 bp profile of P. infestans US-23 (1). These results show the presence of the P. infestans A1 and A2 mating types in New Brunswick and Prince Edward Island, which increases the probability of sexual recombination. To our knowledge, this is the first report of P. infestans clonal lineage US-23 causing late blight in New Brunswick and Prince Edward Island, increasing the genetic diversity from previous years in eastern Canada and underscoring the annual fluctuation occurring in the population composition. References: (1) G. Danies et al. Plant Dis. 97:873, 2013. (2) S. B. Goodwin et al. Phytopathology 84:553, 1994. (3) C. H. Hu et al. Plant Dis. 96:1323, 2012. (4) M. L. Kalischuk et al. Plant Dis. 96:1729, 2012.

The role of a putative peroxisomal-targeted epoxide hydrolase of Nicotiana benthamiana in interactions with Colletotrichum destructivum, C. orbiculare or Pseudomonas syringae pv. tabaci.

Motif analysis among 30 EH1 and EH2 epoxide hydrolases from Solanaceaeous plants showed differences primarily in the lid region around the catalytic site. Based on in silico models of 3D structures, EH1 proteins lack a catalytic triad because of the orientation of one of the conserved lid tyrosines, while the orientation of that tyrosine in EH2 proteins fomed a catalytic triad inside a hydrophobic tunnel. Two similar EH2 protein genes from Nicotiana benthamiana, NbEH2.1 and NbEH2.2, have a predicted peroxisomal targeting sequence, catalytic triad, and structural similarities to a potato cutin monomer-synthesizing epoxide hydrolase. NbEH2.1 expression increased with infections by the hemibiotrophs, Colletotrichum destructivum, Colletotrichum orbiculare or Pseudomonas syringae pv. tabaci only during their biotrophic phases, while there was only a slight increase during the hypersensitive response to P. syringae pv. tabaci (avrPto). In contrast, among the four pathogens, NbEH2.2 expression increased only in response to P. syringae pv. tabaci. Virus-induced gene silencing of NbEH2.1 significantly affected only the interaction with C. destructivum, resulting in a delay in the appearance of necrosis that may be related to its biotrophic phase being restricted to single epidermal cells, which is unique among these pathogens. These results differed from that of a previously reported EH1 gene of N. benthamiana for these interactions, demonstrating specialization among EH genes in basal resistance.

Quantifying fungal infection of plant leaves by digital image analysis using Scion Image software.

A digital image analysis method previously used to evaluate leaf color changes due to nutritional changes was modified to measure the severity of several foliar fungal diseases. Images captured with a flatbed scanner or digital camera were analyzed with a freely available software package, Scion Image, to measure changes in leaf color caused by fungal sporulation or tissue damage. High correlations were observed between the percent diseased leaf area estimated by Scion Image analysis and the percent diseased leaf area from leaf drawings. These drawings of various foliar diseases came from a disease key previously developed to aid in visual estimation of disease severity. For leaves of Nicotiana benthamiana inoculated with different spore concentrations of the anthracnose fungus Colletotrichum destructivum, a high correlation was found between the percent diseased tissue measured by Scion Image analysis and the number of leaf spots. The method was adapted to quantify percent diseased leaf area ranging from 0 to 90% for anthracnose of lily-of-the-valley, apple scab, powdery mildew of phlox and rust of golden rod. In some cases, the brightness and contrast of the images were adjusted and other modifications were made, but these were standardized for each disease. Detached leaves were used with the flatbed scanner, but a method using attached leaves with a digital camera was also developed to make serial measurements of individual leaves to quantify symptom progression. This was successfully applied to monitor anthracnose on N. benthamiana leaves. Digital image analysis using Scion Image software is a useful tool for quantifying a wide variety of fungal interactions with plant leaves.

The involvement of two epoxide hydrolase genes, NbEH1.1 and NbEH1.2, of Nicotiana benthamiana in the interaction with Colletotrichum destructivum, Colletotrichum orbiculare or Pseudomonas syringae pv. tabaci.

Epoxide hydrolase hydrates epoxides to vicinal diols in the phyto-oxylipin peroxygenase pathway resulting in the production of epoxy alcohols, dihydrodiols, triols and epoxides, including many lipid epoxides associated with resistance. Two epoxide hydrolase genes from Nicotiana benthamiana L., NbEH1.1 and NbEH1.2, were amplified from coding DNA of leaves during a susceptible response to the hemibiotrophic pathogens, Colletotrichum destructivum O'Gara, Colletotrichum orbiculare Berk. and Mont. von Arx. or Pseudomonas syringae pv. tabaci Wolf and Foster, or the hypersensitive resistance response to P. syringae pv. tabaci expressing avrPto. Increases in expression of NbEH1.1 generally occurred during the late biotrophic and necrotrophic stages in the susceptible responses and before the hypersensitive response. NbEH1.2 expression was not significantly induced by C. orbiculare but was induced by C. destructivum, P. syringae pv. tabaci and P. syringae pv. tabaci expressing avrPto, although to a lesser degree than NbEH1.1. Virus-induced gene silencing of NbEH1.1 delayed the appearance of lesions for C. destructivum, reduced populations of P. syringae pv. tabaci and increased populations of P. syringae pv. tabaci expressing avrPto. The importance of epoxide hydrolase during pathogen attack may be related to its roles in detoxification, signalling, or metabolism of antimicrobial compounds.