First Report of Fusarium commune Causing Root Rot of Field Peas in Canada.

Abstract

A survey on field pea (Pisum sativum) root rot was carried out in Prince Edward Island (PEI) and New Brunswick (NB), Canada, between July and August, 2018. The average disease incidence was 75% and 78%, and severity was 3.5 and 2.8 on a 1 to 7 scale for NB and PEI, respectively (Schneider and Kelly 2000). Symptoms included seedling stunting, root rot, and wilting. Surface sterilized diseased root segments were incubated on water agar at 25°C for 5 days. Pure isolates were obtained by single spore culturing. A total of 210 isolates were identified as Fusarium spp. Five isolates were identified as F. commune by morphological and molecular characteristics (Leslie and Summerell, 2006; Skovgaard et al. 2003). The isolates on Potato Dextrose Agar produced whitish fluffy mycelia on the upper surface and grayish yellow coloration on the bottom surface of the colony cultured at 25°C in darkness. The isolates on carnation leaf agar at 25°C in darkness formed abundant chlamydospores and macroconidia but rare microconidia with 0 to1 septa, measuring 6.2 to 12.5 x 2.7 to 3.6 µm (n = 5). Macroconidia were typically fusiform with a slightly curved apical cell and a foot-shaped basal cell, bending equally toward both ends. Three-septate conidia were 26.8 to 39.3 x 3.6 to 4.5 µm (n = 20) and five-septate conidia were 56.2 to 64.3 x 4.5 to 5.4 µm in size (n = 10). Chlamydospores were smooth, terminal, and single, 7 to 12.5 µm in diameter (n = 20). Genomic DNA of the five isolates were used to amplify and sequence the translation elongation factor 1α (TEF-1α) and the mitochondrial small subunit ribosomal DNA (mtSSU rDNA) using EF1/EF2 and NSM1/NSM2 primer pairs (White et al 1990; O'Donnell et al 2000), respectively, which were used to define the F. commune (Skovgaard et al. 2003). The mtSSU rDNA sequences were deposited in GenBank, OP752229 to OP752232 and OP752234 for isolates GR11-8, GR11-9, GR1-21, FRDC11-1 and FRDC11-2, respectively, and the TEF-1α sequences were assigned OP831956 to OP831959 and OP831961 for GR11-8, GR11-9, GR1-21, FRDC11-1, and FRDC11-2, respectively. The sequence similarities of the five isolates with ex holotype culture NRRL 31076 ranged from 98.69% to 99.79% for the TEF-1α (AF362263.1), with the matching base pairs of 526/533, 523/528, 483/484, 497/502 and 527/533, respectively, and 99.83 to 100% for the mtSSU rDNA (AF362279.1), with the matching base pairs of 633/634, 633/634, 600/601, 633/634, and 621/621, respectively. The sequences of mtSSU rDNA and TEF-1α for the F. commune type species and related Fusarium species were retrieved from NCBI. A phylogenetic tree constructed using the combined sequences of mtSSU rDNA and TEF-1α showed the five isolates clustered with F. commune. Three isolates (GR11-8, GR11-9, GR1-21) were used for pathogenicity testing with four replicates of four plants each and the trial was repeated twice. Seeds of field pea (CDC Limerick) were soaked in 2% sodium hypochlorite for 2 minutes, washed three times with sterilized distilled water, and then were soaked in conidial suspension at 2 × 106 conidia / mL or in sterilized distilled water as a control for 16 h in darkness at 20°C. Seeds were placed in sterilized vermiculite in a greenhouse at 24 / 18°C day / night temperature with a 16 h photoperiod. Three weeks after planting, the tested isolates were observed to cause seed decay, root rot, and seedling stunting, with disease severity ranging from 5 to 7 based on 1 to 7 scale in repeated trials. No symptoms were observed on the control plants. F. commune isolates were re-isolated and confirmed by sequencing the mtSSU rDNA and TEF-1α. F. commune was reported in Alberta causing soybean root rot (Zhou et al. 2018) but this is the first report of F. commune causing root rot of field pea in Canada. Considering its high pathogenicity in field pea and in soybean, the prevalence, host range and geographic distribution of this pathogen need further study.