Phytophthora cactorum causing bleeding canker of Acer x freemanii in southern Idaho.

Since 2018, bleeding cankers have been observed on maple trees in multiple home gardens in southwest Idaho. The cankers ooze a dark sap and and are approximately 10 cm to 35 cm in diameter. Cankers typically occur on the main trunk but are also present on scaffold branches in severe infecrions. Symptoms of foliar chlorois, branch dieback, and premature autumn senescence were also associated with the disease. Phytophthora DNA was detected in symptomatic material from five trees using real-time PCR (Miles et al., 2017). In July 2019 recovery of a causal agent from a symptomatic Acer x freemanii tree was attempted. Excisions were made from the interface of healthy and diseased tissue around the cankers using a chisel. The tissue was then placed in sealed plastic ziplock bags at 4°C for 7 days. Hyphae were then removed with forceps and placed onto potato dextrose agar (PDA) amended with penicillin G (0.2 g/liter) and streptomycin sulfate (0.8 g/liter). Colonies resembling Phytophthora cactorum were consistently observed after 5 days at 21°C. Tentative P. cactorum identification was based on the presence of abundant papillate and caducous sporangia on a short pedicel; sporangia were approximately 30 µm long and 26 µm wide (Bush et al., 2006; Hudler, 2013). Individual hyphal tips were transferred to fresh PDA plates and sequencing of both the rDNA ITS region and Cytochrome c oxidase subunit I (COI) was completed for a representative isolate (D19-130). DNA extraction, PCR and sequencing were as previously described (Woodhall et al. 2013; Robideau et al., 2011). The resulting DNA sequences for rDNA ITS (MW315449) and COI (MW881040) were both 100% identical (723/723 bp and 728/728 bp) with sequences from cultures previously identified as P. cactorum (MH171627 and MH136858). To determine pathogenicity, 14 month-old maple (A. x freemanii) trees in individual containers with potting mix were wounded 15 mm above the soil line with a single 10 mm incision using a sterile razor blade and inoculated by placing a 10 mm2 fully colonized PDA plug of isolate D19-130 on the wound. The inoculum and wound were then covered with a damp cotton ball that was secured loosely with parafilm. Control plants consisted of uninoculated plants and wounded plants inoculated with a PDA agar plug. Each treatment was replicated five times and placed in a controlled environment chamber set at 24ºC and 90% relative humidity. All treatments were sprayed with water daily to ensure the cotton balls remained damp. After 8 weeks, black lesions, up to approximately 25 mm above the soil line, were observed on the stem base of all P. cactorum-inoculated plants. No black lesions were observed on non-inoculated plants or plants inoculated with a PDA agar plug. P. cactorum was isolated from lesions, as described above, except polystyrene foam boxes containing moist paper towels were used instead of bags. This report confirms P. cactorum as a causal agent of bleeding canker of maple in Idaho for the first time. It has been shown that several Phytophthora species can infect maple (Jung and Burgess, 2009; Huddler, 2013). P. cactorum has a wide host range but certain strains have been associated with lethal bleeding stem cankers in maple and other deciduous trees worldwide (Huddler, 2013). Knowledge of the causal agent of bleeding canker on maple will help determine appropriate disease management practices.

Rhizoctonia solani AG2-1 causing root rot of wasabi (Eutrema japonica) in the UK.

In 2014, glasshouse-grown wasabi (Eutrema japonica) grown in a compost based media displayed symptoms of poor growth and wilting. Visual assessment of the roots showed that 25% of the symptomatic plants sampled had raised black lesions on the roots affecting between 5 and 20% of the total root area. To isolate the causal agent, affected material (approximately 5 mm3) was surface disinfested in sodium hypochlorite (2%) for 30 s, rinsed twice in sterile water and plated on to water agar medium amended with penicillin G (0.2 g/liter) and streptomycin sulfate (0.8 g/liter). Plates were incubated at 20ºC until fungal colonies were visible. After three days, colonies of Rhizoctonia solani were identified based on the presence of septate hyphae with right-angle branching, a pure culture was obtained through hyphal tip transfer onto a new plate of PDA. DNA was extracted from a 7-day old plate of the isolate (WAS1) as described previously (Woodhall et al., 2013). The AG of WAS1 was determined as AG2-1 using a subgroup specific real-time PCR assay (Budge et al., 2009b) and confirmed by DNA sequencing as described previously (Lekuona Gomez et al., 2015). The sequence was 100% identical (587/587bp) to a previously identified AG2-1 isolate 1971 (GenBank accession FJ435126) (Budge et al., (2009a). Pathogenicity of the isolate was confirmed by inoculating three healthy one-year-old wasabi plants grown in loam based compost (John Innes No.3) each with four 5 mm fully colonised PDA plugs of isolate WAS1 placed at approx. 40 mm depth in the soil. Four sterile PDA plugs were place in each of three control plants. All six plants were placed in a greenhouse at 21°C, 18h:6h light: dark and watered as required. After 21 days, multiple black root lesions typically 3-5mm in length were observed on the roots of all inoculated plants. No lesions were observed on the control plants. From three lesions per plant, isolations were attempted as described above. Rhizoctonia solani was recovered from all isolations and the resulting cultures all tested positive for AG2-1 using the real-time PCR assay. Isolations were attempted from the roots of healthy control plants but Rhizoctonia was not recovered. Here we demonstrate that R. solani AG2-1 is associated with root necrosis of Eutrema japonica. Rhizoctonia solani AG2-1 has been reported previously in various Brassica crops in the UK (Budge et al., 2009a) and on Matthiola incana (Lekuona Gómez et al., 2015). It has also been reported causing disease in potatoes and as widely present in UK field soils (Woodhall et al., 2013). Although R. solani AG1 and AG4 of R. solani have been reported to infect Eutrema japonica in Japan (Takeuchi et al., 2003; 2008), this is the first finding that identifies AG2-1 as the causal agent. The potential presence of AG2-1 in soil and/or as plant debris should be considered prior to planting susceptible hosts.

First report of rubbery rot of potato caused by Geotrichum candidum in the United States.

Rubbery rot of potato caused by Geotrichum candidum Link is characterized by symptoms of damp, flaccid tubers that feel rubbery when squeezed (Humpreys-Jones 1969), similar in consistency to potato diseases such as pink rot (caused by Phytophthora erythroseptica) and Pythium leak (caused by species of Pythium). In November 2019, several symptomatic tubers of potato variety 'Ciklamen' that had been held in storage since harvest and originated from an over-head irrigated, sandy-loam production field in Bingham county, Idaho were submitted to the University of Idaho for diagnosis. Shipping-point inspection records indicated 4-9% of tubers were affected. External symptoms included irregularly shaped, randomly located sunken black-colored lesions on more severely affected rubbery-textured tubers. When cut, internal affected tissue developed a greyish appearance after several minutes. Lens-shaped cavities were apparent in two of the tubers, indicating an advanced infection. A sour-milk smell accompanied the sample. To isolate the pathogen, pieces of tuber tissue approximately 5 mm in diameter were collected from the margins of symptomatic areas and surface-sanitized in sodium hypochlorite (2%) for two minutes, rinsed twice in sterile water and plated onto tap water agar amended with penicillin G (0.2 g/liter) and streptomycin sulfate (0.8 g/liter). After three days at 21°C, colonies having distinct creamy white mycelia, a sweet, juniper-like odor, and hyaline hyphae were consistently associated with diseased tissue. Cylindrical to oval-shaped arthroconidia ranged in size from 6.6-11.0 × 3.2-5.9 µm (mean = 8.4 × 4.7, n=21), within dimensions as reported by Carmichael (1957). No other pathogens including species of Pythium and Phytophthora were recovered from the sample. Pure cultures were obtained by transferring hyphal tips to potato dextrose agar plates. Species identity was confirmed via rDNA ITS sequencing using primers ITS5/4 (White et al., 1990). DNA extraction and PCR conditions were as previously described (Woodhall et al., 2013). Resulting sequences (NCBI accession numbers MT893312 and MT893315) shared 99.4% identity with G. candidum Accession KY103453.1 on GenBank. To confirm pathogenicity, ten tubers (cv. Ciklamen) were inoculated by placing a 10mm2 plug of fully colonized PDA of G. candidum on the tuber surface, and ten tubers were mock inoculated with sterile PDA plugs. After 27 days at 21C in a dew chamber, tubers were examined for symptoms. Eight of the 10 inoculated tubers exhibited a rubbery texture and fluid leaking from tubers when cut, with two tubers also exhibiting a grey internal discoloration and the distinctive smell. Control tubers did not exhibit any symptoms. Isolations were attempted from four symptomatic tubers and G. candidum was successfully recovered from three tubers. The disease has been reported sporadically in the United Kingdom (Humphreys-Jones 1969) and Korea (Kim et al., 2011) and the pathogen occurs worldwide (Carmichael 1957). Though the fungus causes a tomato rot in the United States (US) (Pritchard and Porte, 1923; Bourret et al., 2013), and potatoes with rubbery rot originating from Australia were intercepted at a US port (Farr et al., 2020), the disease has not to our knowledge been documented on potato grown in the US. Because symptoms may be confused with pink rot and Pythium leak, it is critical for producers to obtain a correct diagnosis to facilitate appropriate management strategies.

Rhizoctonia solani AG 2-2 IIIB causing root rot of onion in Idaho.

In September of 2018, onion plants (Allium cepa cv. Joaquin) grown in one field in southwest Idaho were observed to have roots with brown discoloration over 10-20% of the total root surface area. Approximately 10% of plants over a 1 ha area were affected and these plants were about visually 50% smaller than the typical bulb size present in the field. To determine the causal agent, 3 mm pieces of symptomatic roots from four plants were placed in sodium hypochlorite (2%) for one minute, followed by two rinses in sterile water and plated on to water agar medium amended with penicillin G (0.2 g/liter) and streptomycin sulfate (0.8 g/liter). After 3 days at 21°C, fungal colonies with septate hyphae with right-angled branching resembling Rhizoctonia solani were observed in over half of the 16 isolations attempted. Species identity was confirmed through rDNA ITS sequencing, as described previously (Woodhall et al., 2013), with DNA obtained from a single representative hyphal tip culture grown on Potato Dextrose Agar (PDA) which was designated isolate ON3. The resulting sequence (MT672318), was 100% identical (678/678bp) to a sequence previously identified as R. solani AG 2-2 IIIB on GenBank (FJ492137). Pathogenicity of the culture was determined by inoculating ten 20-day-old plants (cv. Joaquin) grown in premium potting compost (Scotts) with a single, fully colonized 10 mm2 plug taken from a 2-week-old PDA culture of isolate ON3. A further nine plants were inoculated with sterile PDA plugs as controls. Plants were grown in the greenhouse at 21C in a 16-hour light regime. After 24 days, each plant was assessed for root rot disease as described previously (Misawa et al. 2017). Root rot was observed on nine of the inoculated plants. Mean diseased root area was 32% of the total root surface, with a minimum of 5% and a maximum of 100% diseased root area and a standard deviation equal to 39.6. No root browning was observed on any of the control plants. Isolations were attempted from nine symptomatic plants and R. solani was successfully isolated from seven plant samples onto water agar. Sequencing was used to confirm identity as AG2-2IIIB. To our knowledge, this is the first report of R. solani AG 2-2 IIIB affecting onions in Idaho. Previous work in the Pacific Northwest recovered R. solani AG2-1, 3, 4 and 8 and also BNR AG A from stunted onions (Patzek et al., 2013). In Japan, Misawa et al. (2017) found AG 2-2 IIIB to be pathogenic to Welsh onion (Allium fistulosum). In Idaho, R. solani AG 2-2 IIIB has was previously reported causing disease in sugar beets (Strausbaugh et al. 2011) and potatoes (Woodhall et al. 2012). Growers should consider crop rotation strategies or soil treatments if R. solani AG2-2IIIB is causing disease in their crops.

First report of Fusarium proliferatum causing necrotic leaf lesions and bulb rot on storage onion (Allium cepa) in southwestern Idaho.

In September 2014, a high rate of bulb rot (5-15% depending on producer) was reported across all cultivars developing early in the storage season in the onion producing region of southwestern Idaho. Spanish yellow onion bulbs cv. Vaquero displaying tan to light brown necrotic rot were obtained. The bulb rot originated in the neck and spread to successive scales (Figure 1). In August 2015, onion cv. Redwing and Vaquero were observed to have wet necrotic lesions developing on leaves in the field (Figure 2). Margins of necrotic tissue, 1-2 cm3, were excised, surface sterilized, plated on water agar medium and incubated at 24°C. Hyphal growth was sub-cultured from eight strains (A- D in 2014; E-H in 2015) to fresh potato dextrose agar to obtain pure cultures. Cultures were characteristic of Fusarium species as described by Nelson et al. (1983) with the presence of microconidia formed on polyphialides with macroconidia present. Primers ITS4-A1 and ITS5 primers (White et al. 1990); EF-1 and EF-2 (O'Donnell et al. 1998); and fRPB2-5F and fRPB2-7cR (Liu et al. 1999) were used to amplify regions of the ITS, elongation factor 1-α and the second largest subunit of DNA-directed RNA polymerase II. Amplicons were sequenced and analyzed using BLAST (https://www.ncbi.nlm.nih.gov/) and in combination using Pairwise DNA Alignment and Polyphasic Identification (http://www.westerdijkinstitute.nl/Fusarium/DefaultInfo.aspx?Page=Home) as described by O'Donnell et al. 2015. Analysis indicated that these strains are Fusarium proliferatum, which is part of the F. fujikuroi species complex (O'Donnell et al. 1998). Similarity (99.5%) was observed in pairwise analyses and the polyphasic identification clustering to representative F. proliferatum strain NRRL 22944 and others. Sequences were submitted to Genbank and registered accession numbers are found in Table 1. To complete Koch's postulates, cv. Vaquero onion bulbs were surface sterilized and injected with 3 × 105 microconidia into the shoulder of each bulb. Five bulbs were inoculated for each isolate, placed in a mesh bag, and incubated at 30°C in the dark. Five bulbs injected with sterile water and five non-inoculated bulbs served as controls. After 14 days, each bulb was sliced vertically down the center and inspected for rot. All eight strains induced tan to light brown necrotic rot symptoms in each inoculated bulb. No symptoms were observed for the water inoculated and the non-inoculated onion bulbs. A fungus was isolated from the necrotic tissue and confirmed to be F. proliferatum as described above. Ten µl aliquots containing 1 × 105 microconidia of F. proliferatum strains (C, E-H) were applied to leaves in triplicate of 12-week-old onion plants (cv. Vaquero) wounded with a 21-gauge needle. Water controls were included. Within three days lesions, with light chlorosis, began to form and quickly spread on the leaves. A fungus was isolated and confirmed to be F. proliferatum as described above. This is the first extensive description and identification of F. proliferatum causing bulb rot in storage in Idaho (Mohan et al. 1997). In addition, this is the first report of the fungus causing leaf infection in the field. These findings confirm F. proliferatum as the causal agent of the high incidence of bulb rot observed in 2014 and 2015. This bulb rot continues to occur in southwestern Idaho and since the pathogen can cause leaf infections growers are encouraged to be vigilant for both leaf lesions during the growing season and bulb rot in storage.