First Report of Root Rot Caused by Calonectria ilicicola on Blueberry in Yunnan Province, China

Abstract

Blueberry (Vaccinium spp.) has been cultivated in more than 20 provinces in China as reported in 2015. In July 2016, a root rot disease of blueberry causing plant death was found in Qujing (24°49′N; 103°59′E), Yunnan Province. Approximately 5% of plants in the field showed symptoms characterized by leaf chlorosis, black rot on roots and stems, and finally the whole plant wilted in about 20 days. In order to identify the causal agent of this disease, typical disease samples were collected. Twenty infected root tissues were sterilized and put on potato dextrose agar (PDA) at 25°C (Fang 1998) and nine fungal isolates were obtained. The isolate YNQJ01Aa was chosen for further testing. Its colony on PDA was white to pale yellow. The conidia were collected from SNA colony after growing for 10 days. Conidia presented with one to three septa (mostly one septa). Fifty conidia from one isolate measured 59.37 to 71.33 × 5.93 to 7.91 μm (avg. 62.25 × 6.22 µm). Ascospores were produced in perithecia on PDA and were 30.16 to 38.89 × 5.78 to 7.76 µm (avg. 34.97 × 6.52 µm). By DNA sequencing of the internal transcribed spacer (ITS) rDNA region (GenBank accession no. MF785081), the identity of the isolate (YNQJ01Aa) was confirmed to be the genus Calonectria (99% homologous to several Calonectria spp. strains). The partial β-tubulin (TUB) gene sequence was also analyzed (MF785082) and the result of a BLAST search showed that it was 100% homologous to C. ilicicola strain (CBS 190.50). Further species identification was analyzed by phylogenic tree based on TUB (partial β-tubulin gene, T1/Bt2b), tef1 (translation elongation factor 1 alpha gene, EF1-728F/EF1-986R), and HIS3 (histone 3 gene, H3-1a/H3-1b) (Chen et al. 2011; Crous 2002). Sequences obtained were deposited in GenBank (TUB: MF785082, tef1: MF785083, HIS3: MF785084). Alignments were made using CLUSTAL X v.2.0, and a maximum likelihood phylogram was generated by MEGA v.7.0. Isolate YNQJ01Aa clustered together with C. ilicicola reference strain CBS190.50 (98% bootstrap). Three healthy 1-year-old ‘O’Neal’ blueberry plants were used to conduct pathogenicity tests by wound inoculation. Each plant was inoculated with 10 ml of conidial suspension (1 × 10⁵ conidia/ml) by the root irrigation method. Three plants were inoculated with the same amount of sterile water as control. All blueberry plants were incubated at 25°C. After 20 days, the test plants showed typical symptom of black root rot and finally the whole plant wilted and died, while the control plants remained healthy. The same colonial fungus was reisolated. This is the first report of C. ilicicola (anamorph Cylindrocladium parasiticum) infecting blueberry in China. Root rot disease can be difficult to detect early and therefore it is important to identify the etiology to develop appropriate disease management strategies. This fungus was previously reported on Medicago sativa in China (Pei et al. 2015).