Evaluating the Pathogenicity of Pythium vexans Isolates from Fraser Fir in North Carolina

The pathogenicity of Pythium vexans isolates collected from fir samples with typical root rot symptoms in North Carolina was evaluated on Fraser fir seedlings (Abies fraseri). Two replicated pathogenicity trials involving seven treatments were conducted in the lath house and greenhouse. Although the P. vexans isolates examined in these trials were able to colonize Fraser fir root systems, they did not cause mortality or incite root rot symptoms. In comparison, Phytophthora cinnamomi, a known aggressive pathogen of Fraser fir, caused severe root rot symptoms in all plants. These experiments provided no evidence that P. vexans is a pathogen of Fraser fir. Introduction Christmas trees are an important nursery crop in the United States, representing a $211 million industry in 2006 (8), with North Carolina leading the nation in production of Fraser fir. Phytophthora root rot and stem canker, caused by several Phytophthora spp., has been associated with significant damage to Fraser fir since the 1960s (4). In North Carolina, Phytophthora cinnamomi, P. citricola (10), P. cactorum (2), and P. drechsleri (1) have been isolated from symptomatic roots and surrounding field soils, although P. cinnamomi accounted for most of the disease incidence in Fraser fir in a 1999 survey (2). During the 2005 growing season, 113 samples of Fraser and Canaan fir (Abies balsamea var. phanerolepis)) with typical root rot symptoms were collected throughout the state of North Carolina and submitted to the Plant Disease and Insect Clinic (PDIC) at NC State University for disease diagnosis by plating of root systems on selective media (P10ARPH) (5) for isolation of Pythiaceous organisms; diagnoses were as follows: Phytophthora species (51 samples), no pathogen (21 samples), Pythium vexans (13 samples), other pathogens or disorders (13 samples), other Pythium species (6 samples), and both Pythium and Phytophthora species (9 samples). Although Pythium undulatum has been previously reported to cause root disease on Noble fir (Abies procera) in Ireland (9), North Carolina Christmas tree specialists and agents were unsure how to respond to Abies samples confirmed with P. vexans. The objective of this research was to evaluate the pathogenicity of P. vexans on Fraser fir. Phytophthora cinnamomi, known as an aggressive pathogen on this host, was used as a standard for comparison. Preparation of Inoculum, Inoculation Procedures, and Pathogenicity Assays Seven treatments were evaluated in two separate trials. On 20 April 2006 (trial 1), greenhouse-grown Fraser fir plugs were individually transplanted in 3⁄4gal pots containing six parts pine bark to one part peat moss and placed in a 6 October 2008 Plant Health Progress randomized complete block design with 14 replications in a lath house at the NC State University Horticultural Field Lab in Raleigh, NC. On 19 October 2006 (trial 2), greenhouse-grown Fraser fir plugs were individually transplanted in Anderson band pots (OBC Northwest Inc., Canby, OR) with six parts pine bark to one part peat moss and placed in a randomized complete block design with 10 replications in a NC State University greenhouse in Raleigh, NC. For both trials, isolates were grown on CMA for one week, transferred to flasks containing autoclaved rice grains and incubated for 10 to 14 days (Table 1). About 3 to 4 weeks after transplanting (19 May 2006 for trial 1; 14 November 2006 for trial 2), two colonized rice grains were placed in each of three holes around the edge of the root system about 4 cm below the surface. Plants at the field lab were watered by sprinkler irrigation, while seedlings in the greenhouse trial were given drip irrigation (Fig. 1). Plants were observed for foliar symptoms of root rot and rated as: 1 = no disease; 2 = slight disease (slight needle chlorosis); 3 = moderate to severe (moderate chlorosis and/or necrosis); and 4 = dead plant. Plants were harvested 21 and 30 weeks after inoculation for trial 1 and 2, respectively, except as described below for trial 2. At harvest, top weight was assessed and root systems of each plant were rated on a scale of 1 to 5 (with 1 = healthy; 2 = up to 25% of root system necrotic; 3 = up to 50% of root system necrotic; 4 = more than 50% of root system necrotic; and 5 = dead plants, all roots necrotic). ANOVA analyses of mean top weight and root rot ratings were made following the GLM procedure in SAS (SAS v. 9.1, SAS Institute Inc., Cary, NC). Table 1. Source of Phytophthora cinnamomi and Pythium vexans isolates used in the pathogenicity trials * All isolates originated from Fraser fir plants growing in North Carolina and were identified by culture morphology and ITS sequence. Pathogenicity Results, Trial 1 Foliar symptoms of Phytophthora root rot began to show up on plants in the Phytophthora cinnamomi and Phytophthora cinnamomi plus Pythium vexans treatments about 6 weeks after inoculation (data not shown). Within 8 weeks of inoculation, only plants inoculated with P. cinnamomi or the combination with P. vexans had significantly higher foliar disease ratings than the non-inoculated Isolate (ID)* Source Pythium vexans (Ph565) G. Abad Pythium vexans (Ph564) G. Abad Pythium vexans (Ph546) G. Abad Pythium vexans (Buch1A1) B. Richter Phytophthora cinnamomi (2378) D. M. Benson Fig. 1. Set up of pathogenicity trial 2 conducted in the greenhouse. Dead seedling in the foreground had been inoculated with the combination of Phytophthora cinnamomi and Pythium vexans. 6 October 2008 Plant Health Progress control (Table 2, Fig. 2, Fig. 3). By 4 August 2006, all Fraser fir plants inoculated with Phytophthora cinnamomi were dead but fir inoculated with P. vexans appeared healthy. At harvest on 16 October 2006, top weight and root rot ratings of fir plants inoculated with P. vexans were not significantly different from those of non-inoculated plants. In comparison, plants inoculated with Phytophthora cinnamomi or Phytophthora cinnamomi with P. vexans in combination had smaller top weights and significantly higher root rot ratings in comparison to non-inoculated plants. Table 2. Mean disease ratings and top weights of Fraser fir seedlings among treatments evaluated in Trial 1w. w All values in table are the means of 14 replications. x Foliar rating scale: 1 = no disease; 2 = slight disease (slight chlorosis); 3 = moderate to severe (moderate chlorosis/necrosis); and 4 = dead plant. y At harvest on 16 October 2006, fresh top weight was assessed and root systems rated as: 1 = healthy; 2 = up to 25% of root system necrotic; 3 = up to 50% of root system necrotic; 4 = more than 50% of root system necrotic; and 5 = dead plants, all roots necrotic. z Means within a column followed by the same letter are not different according to the Waller-Duncan k ratio test, k = 100, P = 0.05. ns = not significant. For plants that had died by 28 August 2006, roots were rinsed to remove adhering potting mix, blotted dry, and cultured on P10ARP. For the Phytophthora cinnamomi treatment, the pathogen was recovered from 11 of 13 dead firs. No mortality was observed in fir inoculated with P. vexans isolates Ph565 and Ph564 by 28 August, although one plant was dead in each of the treatments with P. vexans isolates Ph546 and Buch1Al. P. vexans was recovered from each dead plant along with an unidentified semi-papillate Phytophthora sp., which may have been introduced from other trials in the area or on the original fir seedling. Treatment Isolate Foliar rating (1 to 4)x Harvesty