First Report of Fruit Rot of Grapes (Vitis vinifera) Caused by Cladosporium cladosporioides in Xinjiang, China.

Abstract

Xinjiang Province accounts for nearly 20% of the total grape (Vitis Vinifera L.), proles orientalis) (wine, table and raisin combined) production, being the largest production area in China. Fruit rot is the most common disease that impacts grape quality and yield. A new disease where the ripe grape berry surfaces were coated with brownish mildew was observed, and the disease mainly occurred on whole clusters or most of the berries in the cluster. In September 2019 and 2020, 125 diseased grape clusters were collected from 10 locations in northern Xinjiang where the disease incidence was 15.3% - 27.4% ((diseased clusters/ total clusters)*100). To identify the pathogen, symptomatic grape berries were disinfected with 1% NaClO for 2 min, followed by 70% ethanol for 30 s, and rinsed thrice in sterile distilled water. Three pieces of ~0.5 cm2 diseased grape skin with partial exocarp were placed on potato dextrose agar (PDA) amended with streptomycin sulfate and kanamycin (50 µg/mL each). The PDA plates were then incubated at 25℃ under light condition with the luminous intensity 3500 Lux for 7 days. Fungal colonies emerging from the plated tissue were subcultured and single-spored three times to obtain pure cultures. From 20 strains with similar colony phenotype and grey olive hue, flocculent, felt-like surface, six (Cc-Vivi-3, 7, 9, 11, 13 and 19) isolates were chosen for further characterization after 7 days of incubation. Conidia were either single or grew in chains, with around 4 conidia per chain. Conidia were ovoid, nearly spindle or globose with slightly smooth or irregular reticulate surface. Conidiophores were solitary, smooth, septate, erect or geniculate. These characteristics were consistent with the descriptions for Cladosporium cladosporioides. To confirm this identification, PCR was performed on the genomic DNA of the selected strains using primers for internal transcribed spacer (ITS) region ITS1/ITS4, actin (ACT) and translation elongation factor (TEF) (Schubert et al., 2007; Braun et al., 2003). Amplified ITS sequences provided a 100% match to C. cladosporioides (AY213641) in NCBI. Homology of ACT sequences to C. cladosporioides (HM148527 and MH047330) was 99.57% and 100%, respectively; and the homology of TEF sequences with C. cladosporioides (HM148258, HM148289, HM148260 and HM148266) was 97.56% ~ 100%. To further confirm the evolutionary relationship of strains from grapes with Cladosporium spp., phylogenetic analyses based on ITS, ACT and TEF conjoint sequences from the six experimental isolates, five C. cladosporioides strains, eight proximal Cladosporium species were analyzed. The phylogenetic tree showed that the six isolates from grapes clustered with C. cladosporioides strains, but not other proximal Cladosporium species. This confirmed that all six isolates evaluated were C. cladosporioides. Pathogenicity tests with one C. cladosporioides isolate (Cc-Vivi-3; accession No. ITS: MW556429, ACT: MW567144, TEF: MW567143) were carried out as follows: ripe and healthy grape clusters from cultivars Xinyu and Munag when total soluble solids were 20-21°Bx and 19-20 °Bx, respectively, were detached from the vines. Five berries of three clusters of each cultivar were punctured with a sterile syringe, then inoculated with a 20 μL conidial suspension (107 conidia/mL). And uninoculated, punctured berries in clusters treated with sterilized water served as controls. The experiment was repeated three times. Symptoms were recorded 15 days after incubation at 80% relative humidity and 25℃ with a 14 h light/10 h dark cycle. The olive green or blackish green mildew layer was produced on all inoculated berries. No symptoms were observed on the uninoculated berries. Koch's postulates were fulfilled by reisolating C. cladosporioides from all symptomatic tissues and identifying them by PCR targeting the ACT gene. This is the first description of C. cladosporioides causing grape fruit rot in Xinjiang, China. In recent years, worldwide reports of Cladosporium spp. damaging crops are increasing (Briceño et al., 2008; Walker et al., 2016; Meneses et al., 2018; Robles-Yerena et al., 2019; Ding et al., 2019; Yang et al., 2021). However, relatively few methods of management including some fungicides and biocontrol agents are available in different crops (Wang et al., 2018; Addrah et al., 2019). In view of the important role of Xinjiang in China agricultural production, that should arouse strong attention.