First Report of Collar Canker and Dieback of Camellia sinensis Caused by Fusarium solani Species Complex in Henan, China.

Abstract

Tea (Camellia sinensis) is a significant commercial crop cultivated in China. A collar canker and dieback disease of tea has recently been reported in Sri Lanka (Sinniah et al. 2017). During the Spring of 2020, tea bushes with symptoms of fewer leaves, yellowing of leaves with cankers having cracks and peeling of bark, premature falling in the early stage and progressive dieback of branches followed by death of the whole tree that were partially covered with white-colored mold were observed in tea plantations over a 200 hm2 area in Xinyang, Henan Province, China (Figure 1A). To determine the causative agent, dead tea plant stems with white-colored mold were collected, cut into small pieces and surface-sterilized with 75% ethanol, followed by rinsing twice with sterile distilled water and then air-drying. The pieces were placed on potato dextrose agar (PDA) and incubated at 28℃ for 7 days. A total of 19 fungal colonies were isolated and transferred to fresh PDA. Single-spore isolates were made for each and all were observed to produce white flocculent mycelium with the reverse side being pale yellow on PDA. The appearance of a representative strain, XYWLD-07, on PDA is shown (Figure 1B and 1C). Macroconidia (14.26 to 42.13 × 3.53 to 6.79μm) and microconidia (6.93 to 11.85 × 2.72 to 6.33μm) were observed in the cultures (Figure 1E). The genomic DNA from XYWLD-07 was extracted and the internal transcribed spacer region (ITS) of ribosomal DNA, beta-tubulin, translation elongation factor 1-alpha (TEF- 1α), RNA polymerase I beta subunit (RPB1) and RNA polymerase II beta subunit (RPB2) genes were amplified using the primer pairs ITS1/ITS4 (5'- CTTGGTCATTTAGAGGAAGTAA -3'/5'- TCCTCCGCTTATTGATATGC -3') (Gardes & Bruns 1993) , Bt-F/Bt-R (5'- AACATGCGTGAGATTGTAAGT -3'/5'- TCTGGATGTTGTTGGGAATCC -3'), EF-1/EF-2 (5'- ATGGGTAAGGARGACAAGAC -3'/5'- GGARGTACCAGTSATCATGTT -3'), RPB-1F/1R (5'- TTTTCCTCACAAAGGAGCAAATCATG -3'/5'- GTTCACCCAAGATATGGTCGAAAGCC -3') and RPB2-5F2/11aR (5'- GGGGWGAYCAGAAGAAGGC -3'/5'- GCRTGGATCTTRTCRTCSACC -3') (O'Donnell et al. 2010), respectively. The PCR products were sequenced and deposited in GenBank (Accession Nos. MZ411431, MZ439920, OL982608, OM069738 and OM265212), respectively. BLASTn against NCBI nr and Fusarium-ID database revealed that the ITS sequence had 99.65% homology with BLAST sequences of F. solani (GenBank Accession Nos. JN882257.1 and JN882255.1), the beta tubulin sequence had 96.92% similarity with BLAST sequences of F. solani (KU983876.1 and MN692929.1), TEF- 1α gene sequence had 99.15% and 99.29% homology with BLAST sequences of F. solani (KM096405.1 and KM096409.1), RPB1 gene sequence had 99.32% and 99.41% homology with BLAST sequences of F. solani (KU974331.1 and MT305119.1) and RPB2 gene sequence had 98.15% and 98.20% homology with BLAST sequences of F. solani (MT305182.1 and MT305177.1), respectively. Furthermore, TEF- 1α, RPB1 and RPB2 gene sequences had more than 98.52% match with F. solani sequences in Fusarium-ID databases. To test for pathogenicity, the inoculum was grown for 10 days at 25°C on 200g sterilized boiled wheat grain and sand (3:1, v/v) with 20-ml spore suspension (107 conidia /ml) of strain XYWLD-07. Two-year tea seedlings were planted in pots of autoclaved soil with 5% (m/m) strain XYWLD-07 inoculum but controls were grown in autoclaved soil with only sterile wheat/sand mix. The experiment was performed five times. After 30 days, collar canker symptoms appeared on inoculated tea branches, whereas the control was asymptomatic (Figure 1F). The fungal pathogen was reisolated from the symptomatic tea tissues and again identified as F. solani based on the above analysis of the five gene sequences (Figure 1D). F. solani has been previously identified as causal agent of collar canker and dieback of C. sinensis in Sri Lanka (Sinniah et al. 2017) and India (Sarmah et al. 2017).To our knowledge, this is the first report of F. solani causing collar canker and dieback of C. sinensis in China. As this disease could result in death of tea bushes, effective management strategies should be investigated and applied.