First Report of Phytopythium helicoides Causing Crown and Root Rot on Rhododendron pulchrum in China

During a 2019–2020 survey of plant pathogenic oomycetes in Nanjing, China, a cluster of five adjacent Rhododendron pulchrum plants in Xuanwuhu Park exhibited symptoms including crown and root rot and wilting. foliage blight caused due to collar and had rotting crown and root tissues resultingrot foliage blight. Diseased roots were rinsed in water, cut into 10 mm pieces, immersed in 70% ethanol for 60 sec, and plated onto clarified V8 juice agar (cV8A) containingamended with pimaricin (20 mg/liter), ampicillin (125 mg/liter), rifampicin (10 mg/liter), and pentachloronitrobenzene (20 mg/liter). After three3 days of incubation at 26°C, Ffive Pythium-like isolatescoloniesisolates were obtained using hypalhyphal-tipping after 3 days of incubation at 25°C. Ten agar plugs (2×2 mm2) of each isolate were growntransferred into 10 mLl of 10% clarified V8 juice (cV8) in a 100 -mm plate at 26°C to produce mycelial mats. After 3three days, cV8 was replaced with sterile water. To stimulate sporangial production, 3–5 drops of soil extract solution were added to each plate. Five isolates had identical morphological features. Sporangia were terminal, ovoid to globose, andmeasuring 34.2 ± 6.2 µm (24.0–42.5 µm range) in length and 30.7 ± 6.6 µm (20.9–41.1 µm range) in width. Oogonia were not observed. The following primers were used to amplify the rDNA internal transcribed spacer (ITS) region and the mitochondrial cytochrome c oxidase subunit 1 (cox1COI) and 2 (cox2COII) genes of from aA representative isolate, PH-C were amplified using the primer pairs ITS6 and ITS4 (Cooke et al. 2000), OomCoxI-Levup and OomCoxI-Levlo (Robideau et al. 2011) and Cox2-F and Cox2-RC4 (Hudspeth et al. 2000), respectivelyPhe-1. Isolate A xxx675 bp, xxx657 bp and 561xxx bp fragmentPH-C , respectively were amplified and had have identical sequences of the ITS (GenBank ACN. MT824568), and cox1 (MT834959), COI and cox2 COII genes the rDNA internal transcribed spacer (ITS) region and the mitochondrial cytochrome c oxidase subunit 1 and 2 genes (GenBank ACN. MT824568, MT834959, (MT834958, respectively) sequences identical to those of Phytopythium helicoides (MN541109, MK879709, KT595689, respectively). Based on the morphological and molecular characters, all five isolatesthe causal agent waswere identified the species represented by Phe-1 was identified as P. helicoides. One-year-old R. pulchrum plants (approx. 0.3 m in height) grown in 8×8 cm2 pots were used in to test the pathogenicity trials. Ten plants wasere carefully dug up to expose root ballsclusterballs. TenThree- days -old cultures of the isolate PH-Che-1 were used as the inoculum. Five The pplantss wereere inoculated by inserting 10 agar plugs into thee root ball of each plantcluster. For inoculatingfive control plants, sterile cV8A discsplugs were used. All inoculated plants were re-potted using original fresh potting mix and potsture .Ten 3-day-old cV8A cultural plugs (5×5 mm2) of Phe-1 were evenly insert into the root ball of each of five plants, while sterile cV8A plugs were used for five control plants. All were then planted into their original pots. Plants were maintained in a growth chamber set at 26°C with a 12/12 h light/dark cycle and irrigated as needed. After 21–25 days, the inoculated plants had symptoms identical to those in the field, while the controls remained asymptomatic. Identical outcomes were obtained from two repeated The pathogenicity trials. test was repeatedconducted twice . and the coutcome was identical. Phytopythium. helicoides (Phe-1) was reisolated from all symptomatic plants inemerging from the pathogenicity trials. Phytopythium helicoides was found causing diseases of Asian lotus (Yin et al. 2015), mandarin orange (Chen et al. 2016), and kiwifruit (Wang et al. 2015) plants in China. Phytopythium isolates with identical morphological features to those of Phe-1 were recovered from rotted crown and root tissues of all inoculated plants. In this note, P. helicoides causing crown and root rot on R. pulchrum is reported for the first time. Globally, this is the first report of P. helicoides causing crown blight and root rot of R. pulchrum. Additional surveys are being conducted forto mapping the distribution of P. helicoides in Nanjing, Province of China.