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

Abstract

During a survey of pathogenic oomycetes in Nanjing, China from June 2019 to October 2020, at least ten adjacent Rhododendron pulchrum plants at a Jiangjun Mountain scenic spot showed symptoms of blight, and crown and root discoloration . Symptomatic root tissues collected from three 6-year-old plants were rinsed with water, cut into 10-mm pieces, surface sterilized with 70% ethanol for 1 min, and plated onto 10% clarified V8 PARP agar (cV8A-PARP) containing pimaricin (20 mg/liter), ampicillin (125 mg/liter), rifampicin (10 mg/liter), and pentachloronitrobenzene (20 mg/liter). Four Pythium-like isolates were recovered after three days of incubation at 26°C, and purified using hyphal-tipping. Ten agar plugs (2×2 mm2) of each isolate were grown in 10 mL of 10% clarified V8 juice (cV8) in a 10 cm plate at 26°C for 3 days to produce mycelial mats, and then the cV8 was replaced with sterile water. To stimulate sporangial production, three to five drops of soil extract solution were added to each plate. Sporangia were terminal, ovoid to globose, and the size is 24 to 45.6 (mean 34.7) (n=10.8) in length x 23.6 to 36.0 (mean 29.8) (n=6.2) in width. Gametangia were not observed in cV8A or liquid media after 30 days. For colony morphology, the isolates were sub-cultured onto three solid microbial media (cV8A-PARP, potato dextrose agar, corn meal agar) . All isolates had identical morphological features in the three media. Complete ITS and partial LSU and cox2 gene regions were amplified using primer pairs ITS1/ITS4, NL1/NL4, and FM58/FM66 , respectively. The ITS, LSU, and cox2 sequences of isolate PC-dj1 (GenBank Acc. No. MW205746, MW208002, MW208003) were 100.00% (936/936 nt), 100.00% (772/772 nt), and 99.64% (554/556 nt) identical to those of JX985743, MT042003, and GU133521, respectively. We built a maximum-likelihood tree of Phytopythium species using the concatenated dataset (ITS, LSU, cox2) to observe interspecific differences. Based on the morphological characters and sequences, isolate PC-djl was identified as Phytopythium litorale . As the four isolates (PC-dj1, PC-dj2, PC-dj3 and PC-dj4) tested had identical morphological characters and molecular marker sequences, the pathogenicity of the representative isolate, PC-dj1, was tested using two inoculation methods on ten one-year-old R. pulchrum plants. For the first inoculation method, plants were removed from the pot, and their roots were rinsed with tap water to remove the soil. Each of these plants was placed in a glass flask containing 250 mL of sterile water and 10 blocks (10 x 10 mm2) of mycelial mats harvested from a three-day-old culture of P. litorale, while the other plant was placed in sterile water as a control, and incubated at 26°C. After three days, symptoms including crown rot, root rot and blight was observed on the inoculated plants whereas the control remained asymptomatic. For the second inoculation method, ten plants were dug up to expose the root ball. Ten three-day-old cV8A plugs (5×5 mm2) from a PC-dj1 culture or sterile cV8A plugs were evenly insert into the root ball of a plant before it was planted back into the original pots. Both plants were maintained in a growth chamber set at 26°C with a 12/12 h light/dark cycle and irrigated as needed. After 14 to 21 days, the inoculated plant had symptoms resembling those in the field , while the control plant remained asymptomatic. Each inoculation method was repeated at triplicate and the outcomes were identical. Phytopythium isolates with morphological features and sequences identical to those of PC-dj1 were recovered from rotted crown and root tissues of all inoculated plants. Previously, P. litorale was found causing diseases of apple and Platanus orientalis in Turkey, fruit rot and seedling damping-off of yellow squash in southern Georgia, USA. This is the first report of this species causing crown and root rot on R. pulchrum, an important ornamental plant species in China. Additional surveys are ongoing to determine the distribution of P. litorale in the city of Nanjing.

Oomycete communities in lowland tropical forest soils vary in abundance and are composed of saprophytes and pathogens of seeds and seedlings of multiple plant species.

“…All Phytopythium isolates tested were weakly pathogenic or not pathogenic on the seedlings of three tropical tree species but may still negatively impact seed and seedling survival. While Phytopythium are generally less aggressive pathogens compared to Phytophthora and Pythium , many species are increasingly being recognized as important root rot pathogens that impact long-term health of various plant species (Wang et al 2015, Fichtner et al 2016, Noireung et al 2020, Baysal-Gurel et al 2021, Li et al 2021). While these root and stem infections may not lead to seedling death in the short-term, they may affect the long-term fitness and survival of the seedling.…”

Characterization of Phytopythium Species Involved in the Establishment and Development of Kiwifruit Vine Decline Syndrome

“…In this work, we demonstrated for the first time the pathogenicity of P. litorale on kiwifruit plants and P. helicoides was reported for the first time on kiwifruit plants in Italy. P. litorale , P. helicoides and P. vexans were previously reported as pathogenic on other hosts, such as on Platanus orientalis [ 37 ], Rhododendron pulchrum [ 38 ], on citrus, apple, and pear [ 39 ], and on almond [ 40 ]. Moreover, P. helicoides was already reported as agent of root and collar rot on kiwifruit in China [ 41 ].…”

Rapid detection of Phytophthora cinnamomi based on a new target gene Pcinn13739

“…(2019) developed LAMP assays for detecting P. cinnamomi in soil and targeted a new target gene ( Pcinn100006 ) that had been identified from genomic sequencing data. Over recent years, there have been many reports of pathogens causing crown and root rot in Rhododendron pulchrum , including P. pini ( Xu et al., 2021 ), Phytopythium littorale ( Li et al., 2021 ), and Phytopythium helicoides ( Chen et al., 2021 ) except P. cinnamomi . Therefore, more isolates need to be used to verify the specificity of the RPA-LFD assay, especially those causing diseases in the same host.…”