First Report of Aphanomyces euteiches Race 1 and Race 2 Causing Aphanomyces Root Rot on Alfalfa (Medicago sativa) in South Dakota

Aphanomyces euteiches causes Aphanomyces root rot (ARR) in alfalfa (Medicago sativa), along with root rot on many other legumes, including pea, clover, and lentil (Malvick et al., 2009). In 2020, South Dakota (SD) planted the most acres of alfalfa in the United States, which demonstrates the importance of alfalfa to the state. Several SD growers reported alfalfa establishment problems likely to be associated with ARR. Soil samples were collected from 16 fields under commercial alfalfa production in Lake County, SD in June 2020. Composite soil samples based on 24 subsamples were collected in a W-shaped pattern at a depth of 15 cm. Collected soil was sieved, and 80 cm3 was placed in plastic pots (6 cm x 6 cm). Each pot was planted with 25 seeds, covered with an additional 15 cm3 soil, and placed in a growth chamber with a 16-hour photoperiod at temperatures of 24 and 19 ℃ (day and night). Alfalfa seedlings, including Saranac (susceptible to races R1 and R2), WAPH-1 (resistant only to R1), WAPH-5 (resistant to both R1 and R2), and Mustang 625 (resistant to both R1 and R2 and coated with mefenoxam) grew in collected soil for 7 days, followed by 4 days under flooded conditions. Trays were drained, and at 21 days after planting (DAP), roots were removed from soil, washed in distilled water, and rated to measure severity of disease symptoms (Samac et al., 2015). The average severity index (ASI) used a 1-5 disease severity scale, 5 being a dead plant and 1 being no symptoms present (http://www.naaic.org/stdtests/Aphano.html). Race was based on ASI where R1 included an ASI of ≥3 for Saranac and <3 for WAPH-1, and R2 included an ASI of >3.0 for Saranac and WAPH-1 and <3.0 for WAPH-5 (Malvick and Grau, 2001). Race-typing experiments were repeated twice with six replicate pots per alfalfa cultivar per experiment and determined the presence of both R1 and R2 in Lake County, SD. ASI values for Mustang 625 and WAPH-5 were similar across all fields evaluated, which indicates limited confounding effects of other root rotting pathogens. DNA was extracted from three symptomatic roots from each field and was PCR amplified using A. euteiches specific primers (Vandemark et al., 2002). A PCR product was observed in all 16 fields evaluated, and the absence of a product was observed when DNA was extracted from alfalfa roots grown in vermiculite. Following race-typing, infected alfalfa roots were surfaced sterilized and placed on Aphanomyces selective media consisting of mefenoxam and benomyl in cornmeal agar (CMA) (Pfender et al., 1984). Isolates were identified as A. euteiches based on hyphal morphology (Malvick and Grau, 2001). Alfalfa seedlings (Saranac) were grown in vermiculite under growth conditions used for the race-typing assay and inoculated 6 DAP with two isolates of A. euteiches. Inoculation was completed using half plates of one week old A. euteiches mycelium on CMA blended with one liter of water (Samac et al., 2015). At 35 DAP, control alfalfa seedlings inoculated with blended CMA and water remained asymptomatic, and alfalfa infected with A. euteiches displayed symptoms including honey-brown colored lesions. For confirmation of Koch’s postulates, DNA from three re-infected seedlings was again PCR amplified using A. euteiches specific primers and confirmed our previous work. This is the first report of either R1 or R2 of A. euteiches causing ARR on alfalfa in SD. To avoid future yield loss, SD growers should consider planting available alfalfa cultivars that have resistance to both races of A. euteiches.

Fusarium species associated with root rot of lentil (Lens culinaris L.) in North Dakota

Root rot of lentils is caused by a pathogen complex which includes several Fusarium species as well as Aphanomyces euteiches, Rhizoctonia solani and Pythium species. Surveys of lentil fields were conducted in 2016 and 2018 in North Dakota to identify Fusarium species associated with root rot. Isolations were performed from lentil roots exhibiting disease and isolates were identified via morphology and sequencing. In total, 114 fields were surveyed and 391 Fusarium isolates were collected. A diversity of Fusarium species was recovered as ten species were represented; however, F. oxysporum was the most frequently isolated species, present in 71% of fields where Fusarium root rot was detected. Based on correlation with disease severity data, it is likely that some of these isolates are pathogenic. F. avenaceum was relatively uncommon, unlike previous survey efforts on field pea in North Dakota. Further research is needed to determine how the Fusarium population responsible for root rot of lentil may be affected by cropping practices or seasonal climatic variation. Assessment of the virulence and host range of Fusarium species associated with root rot of lentil will further enhance our understanding of the root rot complex in lentil and the development and integration of effective management practices.

Identification, laboratory, greenhouse and field handling of Aphanomyces euteiches on pea (Pisum sativum L.)

Aphanomyces euteiches is a destructive soilborne plant pathogen, causing economic losses when adequate to excess soil moisture is available. This oomycete organism survives in the soil for decades and infects and degrades roots of plants from 11 families. Symptoms of Aphanomyces root rot are similar across the major economic hosts alfalfa (Medicago sativa subsp. sativa L.), lentil (Lens culinaris Medik.), and pea (Pisum sativum L.). Initial symptoms include the appearance of grey, water-soaked root tissue. Roots develop a golden-brown color, and lateral roots begin to disintegrate as the disease worsens. In the most severe cases, the entire root system is destroyed and aboveground chlorosis and necrosis are observed. A. euteiches isolation can be successful from both infested soil and infected plant tissue. Macroscopically, colony growth is generally nondescript and white in color. A. euteiches is self-fertile (homothallic) and produces sexual oospores in culture in addition to asexual zoosporangia and zoospores (primary and secondary) on hyphae lacking regular septa (coenocytic). Numerous molecular techniques have been developed for successful A. euteiches detection. A. euteiches can be stored in frozen soil or on agar slants, but viability should be evaluated regularly. Oospores or zoospores have been utilized for inoculation under greenhouse and field conditions; although the generation of a field site using soil infestation techniques can take several seasons of productions of a susceptible crop to be utilized to effectively screen for Aphanomyces root rot.

Characterization of Aphanomyces euteiches pathotypes infecting peas in Western Canada

Aphanomyces root rot, caused by the soil-borne oomycete Aphanomyces euteiches Drechs., has developed into a serious disease in the pea and lentil-producing areas of the Great Plains of North America. Based on six pea differentials previously used to differentiate 11 pathotypes in France, pathotypes were identified among field isolates from Saskatchewan (14) and Alberta (18). Four isolates from the USA and standard isolates for pathotypes I and III designated in the French study were also included. Each isolate was tested twice in replicated experiments by inoculating French pea differentials Baccara, Capella, MN 313, 902131, 552 and PI 80693, along with the Canadian susceptible pea cultivar CDC Meadow and partially resistant USDA line PI 660736 under controlled conditions. Pea plants grown in vermiculite were inoculated 10 days after seeding by pipetting 5 mL of a suspension containing 1 x 103 zoospores mL-1 to the base of each plant. Root discoloration was scored 10 days post-inoculation using a 0-5 scale. Testing revealed that 38 of the isolates, including standard pathotype I isolate RB84 belonged to pathotype I, 4 isolates including standard pathotype III isolate Ae109 were pathotype III, and USA isolate Ae16-01 was a pathotype II isolate. An alfalfa isolate from Quebec was avirulent on all pea genotypes. These findings indicate that pathotype type I is predominant on the Canadian prairies.

Mapping QTL Associated with Partial Resistance to Aphanomyces Root rot in Pea (Pisum Sativum L.) using a 13.2K SNP Array and SSR Markers

Aphanomyces root rot (ARR), caused by Aphanomyces euteiches Drechs., is a destructive soilborne disease of field pea (Pisum Sativum L.). No completely resistant pea germplasm is available, and current ARR management strategies rely on partial resistance and fungicidal seed treatments. In this study, an F8 recombinant inbred line (RIL) population of 135 individuals from the cross ‘Reward’ (susceptible) × ‘00-2067’ (tolerant) was evaluated for reaction to ARR under greenhouse conditions with the A. euteiches isolate Ae-MDCR1 and over 2 years in a field nursery in Morden, Manitoba. Root rot severity, foliar weight, plant vigor and height were used as estimates of tolerance to ARR. Genotyping was conducted with a 13.2K single-nucleotide polymorphism (SNP) array and 222 simple sequence repeat (SSR) markers. Statistical analyses of the phenotypic data indicated significant (P<0.001) genotypic effects and significant G×E interactions (P<0.05) in all experiments. After filtering, 3050 (23.1%) of the SNP and 30 (13.5%) of the SSR markers were retained for linkage analysis, which distributed 2999 (2978 SNP + 21 SSR) of the markers onto nine linkage groups representing the seven chromosomes of pea. Mapping of quantitative trait loci (QTL) identified 5 major-effect (R2 > 20%), 13 moderate-effect (10%<R2< 20%) effect and 10 minor-effect (R2 <10%) QTL. A genomic region on chromosome IV, delimited by the SNP markers PsCam037549_22628_1642 and PsCam026054_14999_2864, was identified as the most consistent region responsible for partial resistance to A. euteiches isolate Ae-MDCR1. Other genomic regions important for resistance were of the order chromosome III, II and VII.

Associations among the communities of soil-borne pathogens, soil edaphic properties and disease incidence in the field pea root rot complex

Background and aims Field pea production is greatly impacted by multiple soil-borne fungal and oomycete pathogens in a complex. The objectives of this research were to 1) identify the soil-borne pathogens associated with field pea in North Dakota and; 2) develop prediction models incorporating the occurrence of the soil-borne pathogen communities, soil edaphic properties and disease incidence. Methods Soil and plants were sampled from 60 field pea fields in North Dakota during 2014 and 2015. Plants (1500 across two years) were rated for both root rot and soil-borne pathogens isolated from roots. Soils were analyzed for edaphic properties. Indicator species analysis was used to identify soil-borne pathogen communities. Logistic regression was used to determine associations and develop prediction models. Results Survey results from 2014 and 2015 indicated that the most prevalent soil-borne pathogens identified in field pea fields were Fusarium spp. and Aphanomyces euteiches. Five soil-borne pathogen communities were identified; three of which had statistically significant associations characterized by (1) Fusarium acuminatum, (3) A. euteiches, and (4) Fusarium sporotrichioides. The occurrence of the three communities were associated with clay content, soil pH, Fe2+, and K+. Disease incidence was associated with the presence of either community 1 or 3 and K+. Conclusions The results generated from this research will contribute to the development of management strategies by providing a soil-borne pathogen community prediction tool.

Antifungal Activity of Plant Derived Essential Oils on Pathogens of Pulse Crops

Pulse crops such as chickpeas, lentils, and dry peas are grown widely for human and animal consumption. Major yield and quality limiting constraints include diseases caused by fungi and oomycetes. The environmental and health concerns of synthetic fungicides use for disease management, emergence of fungicide-resistant pathogens, and demand for organic pulse crop products necessitate the search for effective alternatives. Safe and environmentally friendly plant-derived essential oils (EOs) have been reported effective against some pathogenic fungi. Growth on EO amended growth medium and an inverted Petri-plate assay were used to determine the effects of 38 oils and their volatiles on mycelial growth and spore germination of important pathogenic fungi and oomycetes: Aphanomyces euteiches, Botrytis cinerea, Colletotrichum lentis, Didymella pisi, D. rabiei, D. lentis, Fusarium avenaceum, Stemphylium beticola, Sclerotinia sclerotiorum, and Pythium sylvaticum. Palmarosa, oregano, clove, cinnamon, lemongrass, citronella, and thyme oils incorporated in media inhibited mycelial growth of all the pathogens by 100% at 1:1,000 to 1:4,000 dilution. In addition, thyme oil (1:500 dilution) showed complete inhibition of conidial germination (0% germination) of F. avenaceum, and D. pisi. All the seven EO volatiles inhibited mycelial growth of all pathogens by 50 to 100% except for B. cinerea and S. sclerotiorum. Essential oil effects on mycelial growth were fungistatic, fungicidal or both and varied by E. Essential oils show potential for management of major crop diseases in organic and conventional production systems.