First Report of Fruit Rot Caused by <i>Lasiodiplodia pseudotheobromae</i> on Ridge Gourd in India

Ridge gourd (Luffa acutangula Roxb L.), is an important vegetable crop of India (Shadrach et al. 2023) and thrives in a warm, humid climate. The crop is cultivated for its young, tender fruits, which are consumed as a fresh vegetable. In April 2023, disease symptoms were observed on L. acutangula (Eureka F1) in a 0.96-ha field at Latiacherra (23.640 N, 91.350E) and Dakshin Charilam (23.630 N, 91.320E), Sepahijala, Tripura, India with fruit rot incidence of 10 to 35% (n = 100 plants). Initial symptoms included light yellowing of young fruits followed by a browning discoloration that later on turned dark brown; the affected area gradually covered the entire fruit. The affected fruit became soft and rotten within 2 to 4 days. To isolate the pathogen, ten diseased fruits were randomly collected from ten individual plants and cut into small pieces (5.0 mm2) at the junction between diseased and healthy tissue. The pieces were first sterilized with sodium hypochlorite followed by rinsing three times with sterile water. Disinfected tissues were plated onto potato dextrose agar and incubated at 25±2°C. Ten isolates with similar morphology were purified using the hyphal tip method, from which two isolates (RG-1 and RG-4) were selected for further morphological and molecular identification. Colonies were fast growing, floccose with a grey surface above, yellow below. The hyphae were branched, septate, smooth- to spiculose/rough-walled, variable in dimensions 1 to 6.25 µm wide. Pycnidia were light olivaceous to dark brown, globose-shaped. Pseudoparaphyses were hyaline and cylindrical, with hyaline, smooth-walled, cylindrical (20.5–31.5× 4.75–9.5 μm (x̅ = 25.5 ×7.25 μm n= 30), conidiogenous cells were slightly swollen at the base. Conidia were hyaline, smooth walled, aseptate, ellipsoid to obovoid, and 13.5 to 32.5 × 10.5 to 14.5 μm (x̅ = 23.5 ×12.5 μm n= 30). Similar observations were consistent with those of L. pseudotheobromae (Guo et al. 2023). Additionally, genomic DNA was extracted from 7-day-old, freshly harvested mycelia using a fungus genomic DNA extraction kit (Biomiga, U.S.A.). The partial internal transcribed spacer (ITS) region, beta-tubulin (TUB), and translation elongation factor 1-alpha (TEF-1α) gene were amplified with primer pairs ITS1/ITS4 (White et al. 1990), BT2a/BT2b (Glass and Donaldson. 1995), and EF1-728F/EF1-986 (Carbone and Kohn. 1999), respectively. These were submitted to GenBank with accession nos. PP949460, PX106004 for ITS; PV435145, PX111739 for TUB2, and PV594014, PX111737 for TEF-1α. BLAST analysis of the sequences exhibited 99.41% identity (508/511 bp) with L. pseudotheobromae CBS 116459 (Type material; Sequence ID: NR_111264.1) based on the ITS gene region; 100% identity (432/432 bp) with isolate CBS 116459 (Sequence ID: EU673111.1) based on the TUB2 gene; and 100% identity (307/307 bp) with strain CBS 116459 (Sequence ID: EF622057.1) based on the TEF-1α gene. The pathogenicity test was conducted on five young ridge gourd fruits with two young fruits as controls for each treatment. Before inoculation, the fruits were surface-sterilized and rinsed thoroughly thrice with sterilized distilled water. Inoculation with a spore suspension (106 spores/ml) was carried out with sterile water used as control. After 5 days, inoculated fruits developed symptoms identical to field observations, while controls remained asymptomatic. The same pathogen was reisolated (isolate Tri RG-2) from the symptomatic fruits in three successive trials, and identified as L. pseudotheobromae, thus fulfilling Koch’s postulates. DNA sequences for ITS, TUB2, and the TEF-1α gene were submitted to GenBank with accession nos. PX106005 for ITS; PX111740 for TUB2, and PX111738 for TEF-1α. We have not seen any report of L. pseudotheobromae causing fruit rot on ridge gourd in India. This investigation revealed that L. pseudotheobromae may be an important pathogen and could cause more economic loss; hence, early detection and development of management strategies will be helpful to minimize the yield loss.