Identification and first report of Colletotrichum fructicola causing fruit rot on Zizyphus mauritiana in China.

Abstract

Because of its high economic value and potential for adaptation to subtropical climates, Indian jujube (Zizyphus mauritiana Lam.) is one of the most important fruit crops introduced into south of Guizhou Province, China. In December 2020, approximately 10 to 15% of the harvested jujube (Z. mauritiana Lam. Wuqian) showed fruit rot symptoms after storage at 4°C for 10-15 days in Luodian county (25°34'N, 106°82'E). Symptoms of brown, circular, watery lesions were observed on the jujube fruits. Small pieces (c.a. 5 mm) at the margins of rot tissue were incubated on PDA plates at 25°C in darkness after surface sterilization in 1.5% NaClO for 45 s followed with triple washes using sterile distilled water. Two monoconidial isolates were obtained after incubation and identical colony morphologies were observed with olive grey, cottony aerial mycelium which became darker after 10 days growth. The colony reverse began white but turned brown with age. Conidia, produced in orange masses, were mainly cylindrical with the size of 9.2-16.8 µm (average 13.7 µm) × 3.8-6.2 µm (average 4.6 µm) (n = 50), typical of Colletotrichum spp. (Vieira et al. 2014). For further identification, DNA of these two isolates were extracted and were used for multi-locus genotyping. Five loci, including the ITS region, partial sequences of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), β-tubulin (BTU) and chitin synthase (CHS) genes, were amplified and sequenced with primers of ITS1/ITS4, GDF1/GDR1, ACT512F/ ACT783R, Bt2a/Bt2b and CHS79F/CHS354R, respectively. No differences was found between the isolates at any of the loci and one sequence for each locus was deposited in the Genebank database under accessions OL376803, OL404925, OL404926, OL404927 and OL404928, respectively. Blastn results indicated that the ITS, GAPDH, ACT, BTU and CHS sequences of the jujube isolates shared 100%, 98.56%, 96.62%, 99.48% and 99.33% similarity with those of ex-type strain ICMP 18581 of C. fructicola (GenBank Accession Nos. JX010165, JX010033, JX009501, JX010405 and JX009866). Phylogenetic analysis including published ITS, GAPDH, ACT, BTU and CHS data for C. fructicola and other Colletotrichum species was performed using MEGA 6.0. Based on morphological and molecular data, the jujube isolates were identified as C. fructicola. Pathogenicity was determined for both isolates on jujube fruits cultivar "Wuqian". Fruit surface was sterilized with 75% ethanol, air dried, and wounded with a needle by piercing into 2 mm depth. Ten microliters of a spore suspension (1 × 106 spores/ml) or sterilized water were applied to one of two wounds on the same fruit. There were six replicate inoculations for each isolate and the whole experiment was repeated twice. Treated fruit were maintained in a growth chamber with 80% relative humidity at 25°C. Symptoms of fruit rots, identical the original observations, developed around the infection sites at 3 days post inoculation. These began as light brown, circular lesions, which got darker with orange spore masses after 7 days and both isolates caused identical symptoms. However, the wounds inoculated with water remained asymptomatic. C. fructicola was successfully reisolated from the infected areas to fulfill Koch's postulates. To the best of our knowledge, this is the first report of jujube fruit rot caused by C. fructicola in China, which may become an emerging problem considering the area expansion of Z. mauritiana cultivation and transportation of its fruit. Funding: Funding was provided by Science and Technology Foundation of Guizhou Province (Guizhou Science Base [2020]1Y104), Talent Development Program of Guizhou Province (Qian Jiaohe KY [2021]080), Innovation and Entrepreneurship Training Program of Guizhou University (Guo Chuangzi [2020]017). Reference: (1) Vieira, W., et al. 2014. Fungal Divers. 67(1): 181-202.