Mangosteen (Garcinia mangostana Linn.) is an evergreen fruit tree grown in Southeast Asia. The fruit of Mangosteen is dark purple spherical with a diameter of 5 to 8 centimeter, a hard rind and white flesh. The seeds and pericarps of the fruit have long been utilized as a traditional medicine in Southeast Asia (Ovalle-Magallanes et al. 2017). Surveys were conducted during the months from March to April 2019 of fruit markets located in Wujing Town, Shanghai (31°02′33″ N, 121°28′04″ E), one noteworthy postharvest disease was observed. Overall 116 fruit samples were collected from 5 different supermarkets, 10.3% (12) of the fruits were found infected with the same symptoms during cold storage. The infected fruits were subjected to pathogen isolation and characterization. The junction areas between diseased and healthy tissues were cut into small pieces of diameter 5 mm, which were surface sterilized with 0.5% NaClO solution for 3 min, followed by rinsing in sterile water thrice, and then the tissues were placed on sterilized potato dextrose agar (PDA) plates supplemented with 50 μg/ml streptomycin. The colony edges of resulted fungal cultures were cut and removed to new PDA plates for purification after 2 days incubation at 28°C. After 3 rounds of subculture, a total of 19 purified isolates were obtained and these isolates showed similar phenotypes in morphological characters. The fungal colonies were fast growing (25 mm per day at 28°C on PDA plates) and were circular shaped with massive aerial hyphae, white to ochraceous color. Non-septate hyphae were hyaline and the septa were found only at the branching points. Sporangiophores arose from hyphae or from stolons. Sporangiospores were hyaline, smooth-walled, mostly subspherical to ellipsoidal, and measured 1.34 to 2.73 µm (width) and 3.12 to 6.19 µm (length) (1.98 ± 0.36×4.46 ± 0.69, n=100)(Alvarez et al. 2011). Three independent isolates (GM-07, GM-16, GM-19) were selected used for further genetic analysis and pathogenicity test. The internal transcribed spacer (ITS) and the D1/D2 region of the nuclear large subunit ribosomal RNA gene (LSU) were amplified by primers ITS1/ITS4 and primers NL1/LR3, respectively (Walther et al. 2013). BLASTn analyses of ITS (MN081612, MN081613, MN087659) and LUS (MN080423, MN080422, MN080432) sequences exhibited 99 to 100% identify to Mucor irregularis strains HZ640, extr25 and CBS977.68 (Accession numbers KC461926, MH510278 of ITS and JX976214 of LUS). These observed morphological features and molecular identification indicated that the pathogen possessed identical characteristics as Mucor irregularis Stchigel, Cano, Guarro & Ed. Alvarez (Alvarez et al. 2011). To conduct a pathogenicity test (Al-Sadi et al. 2014), 20 µl of spore suspension (106 spores ml-1) were inoculated into five healthy mangosteen fruits by sterile syringes, meanwhile another five healthy fruits were injected with sterilized water as controls. All inoculated fruits were kept in sealed plastic boxes at 28°C with a 12L/12D illumination cycle and 90% relative humidity. After about 3 to 7 days, the inoculated materials showed similar symptoms to those on the original diseased fruits, while control samples remained symptomless. M. irregularis was successfully re-isolated from symptomatic fruits, fulfilling Koch’s postulates. Only three plant species that can be infected by M. irregularis have been reported so far, including key lime from Oman (Al-Sadi et al. 2014), corn from China (Peng et al. 2015), and sorghum from Brazil (Lima et al. 2018). To our knowledge, this is the first report of M. irregularis causing the postharvest rot on mangosteen fruit in China.
Isolation and Identification of Fungi Associated with Habanero Pepper (Capsicum chinense jacq) Using Basic Molecular Techniques
