First Report of Choanephora cucurbitarum Causing Wet Rot on Fresh Pea (Pisum sativum L.) in China

Pea (Pisum sativum L.) is one of the most important edible legume crops, and China is the largest producer of fresh pea in the world (https://www.fao.org/faostat/en/). In January 2024, severe pea wet rot was observed on nearly 30% fresh peas in the fields of Sanya City, Hainan Province (18.54°N, 108.88°E), resulting in an estimated yield loss of approximately 15%. Most of the infected stems and pods exhibited water-soaked lesions and were covered by white mycelium with brown sporangiophores. To isolate the causal agent of the disease, about 20 infected stem and pod tissues were collected from the fields and sterilized in 75% ethanol for 30 s and 2% sodium hypochlorite for 2 minutes, rinsed 3 times in sterilized distilled water, dried on sterilized filter paper, and incubated (in 90 mm petri dishes) on potato dextrose agar (PDA). Hyphal tips were transferred to fresh PDA plates. Five fungal isolates (d2a-d2e) were obtained and purified individually through hyphal tip isolation, with three plates recovered from each isolate. After 2 days of incubation, the colonies produced buff pigmentation with a covering of abundant white mycelium, which were non-septate and hyaline on the medium and emerged sporangiophores carrying both sporangiola and sporangia. The sporangiola were brown and ranged in shape from elliptical to oval, with longitudinal striations from 10.1 to 18.4 μm × 6.1 to 12.7 μm (n = 50). The sporangia were globose or sub globose, with diameters spanning from 44.8 to 165.2 μm, and contained ovoid sporangiospores that measured 12.5 to 24.6 μm× 7.4 to 14.8 μm (n = 50), with hyaline appendages at the ends. Based on the morphological and cultural characteristics, the fungus is presumed to be Choanephora cucurbitarum (Ryu et al. 2023). To further identify the pathogens, the polymerase chain reaction was performed with primer pair ITS4/ITS5 and NL1/LR3 for the internal transcribed spacer (ITS) region, large subunit rRNA 28S (LSU) regions, and translation elongation factor 1-alpha (EF-1α), respectively (Vilgalys and Hester 1990; White et al. 1990, O’Donnell et al. 2001). The ITS, LSU, and EF-1α sequences of five isolates were determined to be identical. The sequences of one representative isolate, d2e, were uploaded to GenBank with accession number PP621873 (ITS), PP621874 (LSU) and PQ834432 (EF-1α). The BLASTn search revealed that the sequence of ITS and LSU exhibited 100% identity with reported sequences of C. cucurbitarum (e.g., JN943006 and JN939195), whereas the EF-1α sequence shared 99.18% identity with the single available C. cucurbitarum reference sequence (AF157239) in GenBank. The ITS and LSU sequences of Choanephora spp. were retrieved from GenBank for constructing phylogenetic trees. The phylogenetic analysis revealed that the five isolates clustered together with other C. cucurbitarum isolates within the same clade in both phylogenetic trees. For pathogenicity tests, two 4-mm diameter edge mycelial plugs were cut from the edge of actively growing colony of six isolates and placed on and fourth node of the 10-day-old seedlings and pods (Sun et al., 2018). Additionally, to reproduce the disease as closely as possible to natural conditions, a spore suspension (2 × 10⁴ spores/mL) was sprayed onto the surface of the seedlings (Ryu et al. 2023). The pods and seedlings were inoculated with PDA discs as control. Four days after inoculation under humid conditions at 27°C with natural lighting, the white mycelia and sporangiola growth with a soaked lesion were observed on inoculated tissues, while the control pods and seedlings showed no symptoms. Re-isolation of the fungus from the inoculated tissues, showing visual morphological characteristics and sequences consistent with d2e, confirmed that the causal agent was C. cucurbitarum, fulfilling Koch’s postulates. The C. cucurbitarum has been reported as the causal pathogen of blossom blight and fruit rot in many other plants, such as quinoa and lettuce (Sun et al., 2018; Ryu et al. 2023). To our knowledge, this is the first report of C. cucurbitarum causing stem and pod rot on fresh pea in China. This disease is a major concern for producers, as infected pods can result in significant yield losses, making it essential to implement effective control measures.