First report of Cophinforma tumefaciens causing stem swelling, canker, and dieback of Tea plant (Camellia sinensis) in China.

Abstract

The tea plants (Camellia sinensis), one of the earliest cultivated woody crops with numerous varieties, have a rich history of cultivation in China. However, tea plantations (23°55'11″N, 116°42'4″E) in Chaozhou, Guangdong Province, a leading tea production and export region, face a severe disease outbreak. Approximately 50-60% of tea plants in this area exhibit stem swelling, canker, and eventual dieback symptoms. Ten diseased stem samples were collected from the affected plantations to investigate the causal agent. Isolation was carried out within three days of collection. Hyphae grown out from the xylem of the infected tissues were recovered and incubated on potato dextrose agar (PDA) plates for four weeks in the dark. A total of 53 isolates were obtained from diseased branches. The colonies were villous; initially, the colony's center was white, and the edge was olive. As it grew, dark pigmentation was produced, which caused the center to turn olive and the edge to turn greenish-black. When the sporulation in PDA media was absent, a piece of mycelial disk was picked from the colony's edge and inoculated on autoclaved custard apples (Annona squamosa) (Cardoso et al. 2002). Pycnidia were observed over the surface of whole fruits after incubation for four weeks at 25°C. Conidiophores were olive-colored, unicellular, winding, and ellipsoid to obovoid, with rare spore production. Conidia were hyaline, ellipsoid to fusiform, 5.0 to 7.0 (avg. 6.3) μm in length and 1.8 to 2.5 (avg. 2.0) μm in width. No sexual structure was observed. DNA extraction was conducted from the hyphae grown on the PDA of five representative isolates using the PrepMan® Ultra Sample Preparation Reagent (Applied Biosystems, USA). The internal transcribed spacer (ITS) and translation elongation factor-1α (TEF-1α) regions were amplified using primers ITS5/ITS4 (White et al. 1990) and EF1-728F (Carbone and Kohn 1999) /EF-2 (O'Donnell et al. 1998), respectively. The PCR reaction was in a 25 μL mixture system, including 1 μL of DNA template, 0.5 μL of each primer (10 μM), 0.5 mL of Mytaq polymerase (Bioline, USA), 5 μL of MyTaq buffer, and 17.5 μL PCR grade water (Yin et al. 2020). PCR conditions were an initial denaturation step at 95°C for 3 min, followed by 35 cycles of 95°C for 30 s, 55-52°C for 30 s, and 72°C for 1 min, and a final elongation at 72°C for 10 min. The sequences obtained from this study were deposited in GenBank under the accession numbers OQ565301, OQ565302, OQ565303, OQ565304, OQ565305 (ITS), and OQ570636, OQ570637, OQ570638, OQ570639, OQ570640 (TEF-1α). The BLASTn results of ITS and TEF-1α sequences indicated that all isolates were 100% identical to Cophinforma tumefaciens isolate IMI 76762 (accession no. MW810287 and MZ073950, respectively). The combined ITS and TEF-1α dataset of Cophinforma and related genera obtained from Genbank was analyzed using the maximum likelihood method, applying the GTR+I+G model, with 1,000 bootstrap replications conducted in MEGA 11. Phylogenetic analysis confirmed the identification of these isolates as C. tumefaciens (Cardoso et al. 2019, Phillips et al. 2013, Zhao et al. 2021). Pathogenicity tests were conducted on fifty seedlings (two-year-old, 40 cm in height, 1.5-2 cm in diameter) with five representative isolates. Each isolate was used to inoculate ten seedlings. The stem surface was disinfected with 75% ethanol for 30 seconds, rinsed with sterilized water, and wound to remove part of the phloem. A 1 cm diameter mycelial plug was taken from the margin of a 14-day-old PDA plate and placed on the wound. The inoculated wounds were covered with sterile medical gauze to prevent desiccation and contamination. For comparison, control plants were inoculated with non-colonized PDA plugs. All the plants were incubated in field conditions. After four weeks, a raised mass appeared, and browning was observed at the inoculation site, whereas plants used as controls remained symptomless. The one-way ANOVA statistical analysis was conducted in the pathogenicity test. The average lesion lengths of the testing group were 16-24 cm, while the lesions in the control group showed almost no expansion during pathogenicity tests (P<0.05). The same fungus was reisolated from the lesions of inoculated plants, fulfilling Koch's postulates. This is the first report of C. tumefaciens causing stem swelling, canker, and dieback of Ca. sinensis in China. The widespread nature of this disease and its potential impact on tea production underscore the need for further research into its outbreak mechanisms and effective control measures.