First Report of the Root Rot of <i>Ardisia crenata</i> Caused by <i>Neofusicoccum parvum</i> in China

Ardisia crenata Sims, belonging to the family Myrsinaceae, has high medicinal and economic value. In July 2021, root rot disease was observed in plantations located in Ziyun (106°08'45″ E, 25°75'15″ N) and Xiuwen (106°46'53″ E, 26°54'01″ N) Counties, Guizhou Province, China, with an incidence rate of approximately 30% at fruit drop stage. The disease manifested as root softening, blackening, and phloem rot, while the aerial parts showed progressive yellowing, curling, and withering of leaves. Ultimately, the plant died. Nine symptomatic root segments were collected from fifteen infected plants, surface sterilized in 5% NaClO and 75% ethanol for 1 minute each, washed three times with sterile water, and incubated on potato dextrose agar (containing 10μg/mL of chloramphenicol) for five days at 28°C. The hyphal tip technique (Senanayake et al. 2020) was used to obtain pure cultures. Sixteen strains exhibiting similar morphological characteristics were isolated from the infected tissues. The colonies of these isolated cultures appeared white and turned light grey after 4 days. Five isolates were selected and grown on 2% water agar for 7 days for morphological characterization. Conidia were single-celled, hyaline, spindle-shaped to oval, and measured 12.04 to 19.47 µm long and 4.63 to 7.41 µm wide (n = 50). Based on these morphological features, the isolates were suspected to be Neofusicoccum parvum (Pavlic et al. 2009). For molecular identification, strain-7 was randomly selected as a representative individual to extract DNA. The internal transcribed spacer (ITS) region was amplified using primers ITS1/ITS4 (White et al. 1990). Additionally, the translation elongation factor 1-alpha (TEF1) gene was amplified using primers EF1-728F/EF1-986R (Rehner et al. 2005), and the β-tubulin (TUB2) gene was amplified using primers BT2a/BT2b (Glass et al. 1995). The sequences of strain-7 (accession number OR789487[ITS]; PV209690[TEF1]; PV209691[TUB2]) were deposited in GenBank, demonstrating a sequence homology of 99% to 100% (569/570, 265/265, 448/448) with N. parvum YBF5-1 (accession numbers PQ222752, PQ227810 and PQ227811). Based on morphological features and Neighbor-Joining method analyses of combined ITS, TEF1, and TUB2 gene sequences, strain-7 was identified as N. parvum. A pathogenicity test was performed using strain-7 by inoculating roots of 12-month-old A. crenata seedlings. The test was repeated seven times. Healthy seedlings were cut vertically with a sterile knife at 2 cm from the edge of one stem to create root damage. Spore suspensions (150 ml, 1 × 106 conidia/ml) of N. parvum were applied on the cut side, while the control group was watered with the same volume of sterile water. All plants were kept in the same glasshouse under natural conditions. After 7 days, some yellowish spots appeared on the leaf surface of some plants, with the edges turning dry and curled. The branches and leaves turned completely yellow, and the roots rotted extensively on the 14th day, whereas the control group remained asymptomatic. To satisfy Koch's postulates, N. parvum was reisolated from the inoculated plants but not from the control. N. parvum has been reported to cause leaf spot disease on Macadamia integrifolia and Vitis heyneanan in China (Li et al. 2023; Wu et al. 2015). This is the first report of N. parvum causing root rot in A. crenata in China. These findings provide a basis for the early detection of A. crenata root rot and the formulation of targeted control measures.