First Report of Rubber Tree Wilt Caused by Ceratocystis fimbriata in China.

Abstract

The rubber tree (Hevea brasiliensis) is an important economic resource for the rubber and latex industry. During November 2013 and June 2016, rubber trees showing typical wilt symptoms were found in Mengla, Xishuangbannan, Yunnan, China (N 21° 28', E 101° 33'). Symptomatic trees initially exhibited wilting of foliage on individual branches, then spread to the whole canopy, finally followed by death of the whole tree. Dark-blue to black discoloration was observed in the inner bark and affected xylem, a grayish layer of fungal growth and sporulation occasionally. The disease was detected on 20% of trees surveyed. The diseased tissues of three rubber trees were surface disinfected with 75% ethanol for 30 s and 0.1% mercuric chloride (HgCl2) for 2 min, rinsed three times with sterile distilled water, plated onto potato dextrose agar (PDA), and incubated at 25°C. After 7 days, a fungus was consistently observed growing from the tissue. Three single-spore isolates were obtained. In culture, colonies reaching 69 mm diam within 10 days, mycelium was initially white, then becoming celadon. After 5 days of perithecium formation, observed perithecia were black, globose (173.1 - 237.9 × 175.6 - 217.2 μm) and showed a long black neck (507.3 - 794.1 μm). Ascospore with outer cell wall forming a brim, hat-shaped at the tips of ostiolar hyphae (3.43 × 5.63 μm). Cylindrical endoconidia (10.5 - 39.7 × 3.5 - 6.6 μm) were hyaline. Chain of barrel-shaped conidia (7.2 - 9.5 × 4.1 - 6.2 μm) was found. Aleuroconidia were ovoid or obpyriform, and smooth (10.2 - 14.1 × 8.4 - 10.6 μm). Morphological characteristics of the fungus were consistent with the description of Ceratocystis fimbriata (Engelbrecht and Harrington 2005). The genomic DNA was extracted from isolates (XJm10-2-5, XJm8-2-5, XJm4) using the Chelex-100 method (Xu et al. 2020). The ITS region of rDNA was sequenced using the procedures of Thorpe et al. (2005). Analysis of ITS sequence data (GenBank accessions KJ511488, KJ511485, KT963149) showed that the isolates were 100% homologous to those of the isolates on Punica granatum and Colocasia esculenta from China (GenBank accessions KT963152, MH793673) by BLAST analysis. Neighbor-joining phylogenetic analyse were performed using MEGA 6.06 based on ITS sequences (Fig. 1). Analyses showed that all isolates located on the same clade with all C. fimbriata with a high bootstrap support. Therefore, the fungus was identified as C. fimbriata based on morphology and molecular evidences. Pathogenicity of C. fimbriata isolated from this study was tested by inoculation of three one-year-old pot-grown (3L) seedlings of rubber tree. The soil of three seedlings was inoculated by drenching with 30 ml spore suspension (2.0 × 106 spores / ml). Three control plants were inoculated with 30 ml of sterile distilled water. The experiment was repeated three times. The plants were kept in a controlled greenhouse at 25°C and watered weekly. After the inoculation for one month, all the isolates produced typical wilt symptoms, while control plants showed no symptoms. The original fungus was successfully re-isolated from inoculated trees and identified as C. fimbriata according to the methods described above. The pathogenicity assay showed that C. fimbriata was pathogenic to rubber trees. C. fimbriata was first reported on rubber tree in Brazil (Albuquerque et al. 1972; Silveira et al. 1985). To the best of our knowledge, this is the first report of C. fimbriata causing wilt of rubber tree in China. This finding contributes to understanding the diversity of this pathogen, and it appears to be a significant threat to rubber trees in its ecosystem.