First Report of Rhizopus stolonifer Causing Fruit Rot of Ziziphus mauritiana Lamarck.

Indian jujube (Ziziphus mauritiana Lamarck) is one of the most popular fruits in South China. On 15 August 2024, a soft rot disease affecting Indian jujube fruits, with an incidence of 15%, was found in the market of Qujing City, Yunnan Province, China. The initial symptom was a localized brown rot, which extended to half of the fruits after three days, and eventually all the fruits turned brown and rotted, accompanied by the odor of alcoholic fermentation, with white mycelium overflowing from the rotting parts. The margins of the infected fruits were cut into 0.5×0.5 mm pieces, soaked in 75% ethanol for three minutes, then rinsed three times with sterile water, and placed on potato dextrose agar (PDA) in the dark, 28°C. After 12 hours, thin white mycelium grew around the tissues, and after 24 hours the diameter of the colony reached 7 cm. After 48 hours, the mycelium had grown to fill the 9 cm medium, and after 72 hours, the upper mycelium turning black, and the entire colony becoming grey-black. After transferring the mycelium to new PDA medium for 72 hours, grey-black colonies were formed again after the above process. Morphologically, the sporangiophores measured 1.04 to 1.89 mm in length and 8.88 to 33.12 μm in diameter, with rhizoids and stolons present at their bases. Sporangia were almost spherical, 122 μm (±64 μm, n=50) in diameter. Sporangiospores were round to ovoid, measuring 8.36 to 20.01 × 5.83 to 17.12 μm (mean 9.35 μm, n=50). The morphological characteristics of this isolate were analogous to Rhizopus stolonifer (Haque et al. 2023). To identify this pathogen molecularly, primers ITS1/ITS4 were used to amplify the ITS1-5.8S-ITS2 rDNA region (Zarrin et al. 2016), PCR products were purified and ligated to the vector (pClone007 Versatile Simple Vector), followed by introduction into competent cells (Trelief ® 5α Chemically Competent Cell), coated on LB plates and single clones were picked. Finally, the plasmids were sequenced with universal primers M13F-47/M13R-48 (Tsingke Biotech Co., Ltd., Kunming, China) (James et al. 2006; Woo et al. 2010). The sequence obtained was deposited with accession number PQ557265.1 and was 99.54% homologous with Rhizopus stolonifer var. stolonifer (OP863060.1) in the Genbank database. Five wounded and five non-wounded Indian jujube fruits were inoculated with mycelium grown for 2 days, Another set of five wounded and five non-wounded fruits were inoculated with PDA medium as a control, these were incubated in the dark at 28°C. The wounded fruits inoculated with R. stolonifer exhibited the same morbidity symptoms observed previously, and the entire fruits had become completely rotten after 96 hours. Non-wounded fruits inoculated with R. stolonifer, as well as the control, remained free of disease after 5 days. It is worth noting that when a non-wounded fruit is adjacent to an infected one, the mycelium invades the non-wounded fruit from where the pedicel has fallen off, leading to its infection. The pathogen was reisolated from symptomatic fruits and was identified as R. stolonifer, which fulfilled Koch's postulates. To our knowledge, this is the first report of R. stolonifer causing fruit rot of Indian jujube.