J. A. Percich

Characterization of Aphanomyces euteiches strains based on pathogenicity tests and random amplified polymorphic DNA analyses

Genotypic variation among 62 strains of Aphanomyces euteiches, four of A. cochlioides, and a Saprolegnia sp. was investigated using RAPD analysis. Pathogenicity assays on pea, bean, alfalfa, red clover, and sugarbeet were used to determine host preference among the strains of A. euteiches and A. cochlioides. Pathogenicity tests revealed six pathotypes of A. euteiches with host preferences for bean, alfalfa, pea, pea and alfalfa, red clover and alfalfa, and bean and alfalfa. Another group of strains was non-pathogenic on the five plant species. The host of origin tended to be the host on which each strain incited the highest disease severity. A. euteiches did not incite root rot symptoms on sugarbeet, and A. cochlioides was pathogenic only to sugarbeet. RAPD analyses provided a measure of genetic diversity in Aphanomyces. Fifty random decanucleotide primers were screened with five test strains from four hosts representing different pathotypes, and 32 primers amplified DNA fragments from all five strains. Eight primers were chosen for most of this study based on number and polymorphic nature of the bands generated. RAPD assays of 62 strains of A. euteiches with the eight primers yielded 159 polymorphic and no monomorphic, strongly amplified bands. Cluster analyses of RAPD data revealed genotypic differences among three groups of A. euteiches which corresponded to their host of origin and host preference for bean, alfalfa, and red clover/alfalfa. Strains nonpathogenic on all plants tested formed another genotypic group, corroborating results from the pathogenicity assays that indicated this is a discrete group. The bean and non-pathogenic groups were the most distinct. The A. cochlioides and Saprolegnia strains were genotypically distinct from the pathogenic, but not the non-pathogenic strains of A. euteiches. Reproducibility of RAPD assays was confirmed by replicated amplifications and DNA hybridization analysis. Results indicated that A. euteiches is composed of distinct subspecific groups based on genotype and host preference.

Genotypic and pathogenic diversity among pea-infecting strains of Aphanomyces euteiches from the central and western United States.

ABSTRACT Pathogenic and genotypic variability among four populations of Aphanomyces euteiches from individual fields in Minnesota, Wisconsin, and Oregon were investigated using pathogenicity and randomly amplified polymorphic DNA (RAPD) analyses. About 50 strains were isolated from each of two pea fields in Minnesota, and 11 and 6 strains from pea fields in Wisconsin and Oregon, respectively, using pea (Pisum sativum) as a baiting host. Pathogenic variability and host range were evaluated in greenhouse studies with five pea lines or cultivars having different levels of resistance to Aphanomyces root rot and one cultivar each of alfalfa and snap bean. All strains were pathogenic on one or more pea cultivars, and 18 and 14% were pathogenic on alfalfa and bean, respectively. Disease severity incited by different strains varied significantly on individual pea cultivars and on all hosts combined. The percentage of strains pathogenic on different hosts varied among locations. Genotypic variation among all 114 strains was evaluated with RAPD analysis. Ten decanucleotide primers detected 92 polymorphic bands. Cluster and principal coordinates analysis revealed one large group containing 102 of the 114 strains from all locations. Two closely related minor groups of strains (12 strains) were genotypically distinct, with about 55% similarity to the main group of 102 strains. The strains in the minor groups were all isolated from the Minnesota locations and were pathogenic on two disease-resistant pea breeding lines (MN313 and MN314). Estimates of genetic diversity based on RAPD analysis ranged from 0.24 to 0.33 within populations to 0.35 among all strains from all populations. A. euteiches populations were genotypically and phenotypically variable, but no distinct genotypic differences were identified among populations from the four isolated locations.

Variation in pathogenicity and genotype among single-zoospore strains of Aphanomyces euteiches

ABSTRACT The role of asexual reproduction in the production of pathogenic and genotypic variation in Aphanomyces euteiches was investigated. Variation was studied among three groups of 18 single-zoospore progeny of A. euteiches derived from each of three single-zoospore parental strains. Pathogenicity was assessed by evaluating disease severity (DS) on roots of five pea lines possessing different levels of resistance to Aphanomyces root rot and of a susceptible cultivar of snap bean and alfalfa. None of the single-zoospore progeny incited significantly higher DS levels than their parental strain on any of the seven hosts; however, 3 or 4 of the 18 progeny in each group incited significantly lower DS than their parental strains. The host range of the progeny either decreased or remained the same as compared with parental strains. Genotypic variation was assessed with randomly amplified polymorphic DNA (RAPD) analysis. Polymorphic RAPD markers that distinguished parental and progeny strains were detected within two of the three groups of strains with two of four RAPD primers used. Of 76 total RAPD markers that were detected among all strains in all groups, four (5%) were polymorphic. The polymorphic markers were not associated with the pathogenic variation.

Identification of Pisum sativum Germ Plasm with Resistance to Root Rot Caused by Multiple Strains of Aphanomyces euteiches

Aphanomyces root rot is a serious disease of pea (Pisum sativum), and additional sources of resistance are needed for development of disease-resistant cultivars. Accessions (n = 123) from the P. sativum Plant Introduction (PI) collection with the highest relative levels of resistance to one strain of Aphanomyces euteiches were previously identified from among approximately 2,500 accessions evaluated. The chosen 123 accessions were evaluated in this study for resistance to root rot caused by multiple strains of this pathogen. Five strains representing different US geographical locations and pathogenicity characteristics were used to evaluate pea seedlings in a greenhouse. Disease severity (DS) and percent loss of fresh biomass (inoculated vs. non-inoculated plants) were determined 15 days after inoculation. Significant differences (P = 0.05) in levels of DS and biomass loss (BL) occurred among the accessions after inoculation individually with the five strains. The relative rank of accessions based on DS and BL varied with the strain of A. euteiches used for inoculations. The 20 accessions with the lowest DS after inoculation with each strain were identified. Based on lowest DS, two accessions were among the 20 identified with all five individual strains, and four other accessions were among the 20 identified with four of the five strains. The results suggest that the P. sativum PI collection contains useful accessions for breeding programs aimed at developing pea varieties with resistance to A. euteiches.

Evaluation of methods for estimating inoculum potential of Aphanomyces euteiches in soil

Three methods were evaluated for determining inoculum potential of Aphanomyces euteiches inoculum in soil and sand: rolled towel (RT), most probable number (MPN), and soil indexing (SI) bioassays. The RT and MPN bioassays were evaluated using clay loam, loam, and sand that were artificially infested with a series of oospore concentrations ranging from 2 to 500/g. Oospore numbers added to soil and estimated inoculum potential measured using the RT and MPN were highly correlated (r=0.99 and 0.85, respectively). These two bioassays, however, yielded highly variable results among replications, and inoculum potential estimates for sand and soil infested with equal numbers of oospores were dissimilar

Interaction of temperature and moisture on infection of wild rice by Bipolaris oryzae in the growth chamber

Infection of wild rice (Zizania palustris) flag leaves by Bipolaris oryzae was studied at temperatures of 5 to 35°C and wet periods of 2 to 36 h after inoculation. Lesion densities (lesions/cm2) increased with increasing wet periods depending on optimum temperature. High rates of infection occurred at 25 and 30°C and generally increased with continuous wet periods of 16, 18, 24, and 28 h. There were no lesions at 5°C and few at 10 and 35°C. Lesion densities declined when wet periods of 2, 4, or 6 h were interrupted by dry periods of 4, 6, 8, 10, or 12 h followed by a final 14 h of wetness. Lesion densities decreased at all temperatures with increased dry periods regardless of the initial wet period. The interaction of dry period length × wet period length × temperature was significant at the 0.5% level. With continuous wet periods, lesion numbers were highest at 25 to 30°C.

First Report of Fusarium redolens Causing Root Rot of Soybean in Minnesota

Multiple Fusarium species have been found in association with soybean (Glycine max) plants exhibiting root rot in the United States (3). Soybean plants that lacked apparent foliar symptoms, but exhibited 2- to 5-mm brown, necrotic taproot lesions and lateral root necrosis were observed in Minnesota in one field each in Marshall and Otter Tail counties in July of 2007, as well as in one field in Marshall County in July of 2008. Sampling was conducted as part of a study investigating root rot in major soybean-production areas of Minnesota. Plants were arbitrarily dug up at the R3 growth stage. Root systems were washed, surface disinfested in 0.5% NaOCl for 3 min, rinsed in deionized water, and dried. Fusarium isolates were recovered from root sections with necrotic lesions embedded in modified Nash-Snyder medium (1). One resulting Fusarium colony from one plant per county was transferred to half-strength acidified potato dextrose agar (PDA) and carnation leaf agar (CLA) to examine morphological characteristics (4). Culture morphology on PDA consisted of flat mycelium with sparse white aerial mycelium. On CLA, thick-walled macroconidia with a hooked apical cell and a foot-shaped basal cell were produced in cream-colored sporodochia. Macroconidia ranged from 32.5 to 45.0 μm long. Microconidia were oval to cylindrical with 0 to 1 septa, ranged from 7.5 to 11.25 μm long, and were produced on monophialides. Chlamydospores were produced abundantly in chains that were terminal and intercalary in the hyphae of 4-week-old cultures. Morphological characteristics of the three isolates were consistent with descriptions of F. redolens (2,4). The identity of each isolate was confirmed by sequencing the translation elongation factor 1-α (TEF) locus (4). BLAST analysis of the TEF sequences from each isolate against the FUSARIUM-ID database resulted in a 100% match for 17 accessions of F. redolens (e.g., FD 01103, FD 01369). Each F. redolens isolate was tested for pathogenicity on soybean. Sterile sorghum grain was infested with each isolate and incubated for 2 weeks. Sterile sorghum was used for control plants. Soybean seeds of cv. AG2107 were planted in 11.4-cm pots ~1 cm above a 25-cm3 layer of infested sorghum or sterile sorghum. Two replicate pots containing four plants each were used per treatment and the experiment was repeated once. Root rot was assessed 28 days after planting. Each F. redolens isolate consistently caused taproot necrosis on inoculated plants, whereas control plants did not exhibit root necrosis. Isolations were made from roots of inoculated and control plants and the isolates recovered from inoculated plants were identified as F. redolens based on morphological characteristics and TEF sequences. Fusarium species were not isolated from control plants. To our knowledge, this is the first report of F. redolens causing root rot of soybean; however, it is possible F. redolens has been found previously and misidentified as F. oxysporum (2,4). Results from inoculations suggest that F. redolens may be an important root rot pathogen in Minnesota soybean fields. References: (1) J. C. Bienapfl et al. Acta Hortic. 668:123, 2004. (2) C. Booth and J. M. Waterston. No. 27 in: CMI Descriptions of Pathogenic Fungi and Bacteria. CMI, Kew, England, 1964. (3) G. L. Hartman et al. Compendium of Soybean Diseases. 4th ed. The American Phytopathological Society, St. Paul, MN, 1999. (4) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA, 2006.