RESUMEN
Kadsura coccinea (Lem.) A. C. Smith is an evergreen liana widely cultivated in China for its economic importance in traditional medicine. Many phytochemical studies on the stems and roots of K. coccinea have shown a variety of biological activities, such as anti-hepatitis, anti-HIV, and anti-tumor (Yang et al. 2020). In July 2021, symptoms of leaf spot were observed in a plantation of K. coccinea in Longan (23°03´N, 107°54´E), Guangxi province, China. The incidence of this disease was 36%, and severity varies from approximately 20 to 40% of leaf surface coverage. Symptoms began as small brown spots that expanded into irregular to nearly flower-shaped lesions. To isolate the pathogen, leaves with spots were collected, sterilized with 75% ethanol for 15 s followed by 2% sodium hypochlorite for 120 s, rinsed three times in sterilized distilled water, cut into 5 × 5 mm pieces, and placed onto potato dextrose agar (PDA) plates. The plates were kept in an incubator at 26°C in the dark for at least 2 days. A total of 27 fungal colonies of similar morphology out of 30 pieces of infected tissues were isolated. Four representative isolates (HBB1 to HBB4) were selected to study for further characterization. Fungal colonies were initially grayish-white and then turned greenish-gray on PDA. The black pycnidium and immature conidia appeared over PDA plates after 18 days. The immature conidia were colorless and transparent, elliptical, and had a single-cell structure. After 5 days, the immature conidia gradually become black and develop into mature conidia. The mature conidia were dark brown and two-celled with longitudinal striations, 20.41-29.93 × 12.42-17.19 µm (average 26.07×14.51 µm; n = 100). For DNA-based identification, the internal transcribed spacer (ITS) region, translation elongation factor 1 alpha (EF1-α), and ß-tubulin (TUB) genes of the isolates were amplified and sequenced using the primers ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. Sequences were submitted to GenBank (Accession nos. MW045412 to MW045415 for ITS, MW065559 to MW065562 for EF1-α, and MW065555 to MW065558 for TUB). A phylogenetic analysis was conducted using the Maximum Likelihood method on concatenated sequences of the three genes, which showed that the four Chinese isolates from K. coccinea were clustered with reference isolates of Lasiodiplodia theobromae including the ex-neotype CBS 164.96. Pathogenicity tests were performed on young, fully expanded leaves of 2-year seedlings. A 10 µL conidial suspension (1×106 conidia/mL) was inoculated on each wound on the left-half leaf and a 10 µL sterile water was inoculated on each wound on the right-half leaf (control). Each treatment was repeated three times. Inoculated leaves were wrapped in plastic bags for 5 days and plants were maintained in a growth chamber at 27°C, 85% relative humidity. Brown leaf spots appeared 5 to 6 days after inoculation, whereas the control leaves treated with sterile water showed no symptoms. All re-isolations from spots produced colonies with the same morphological characters as L. theobromae, completing Koch's postulates. To our knowledge, this is the first report of L. theobromae causing leaf spot on K. coccinea in China and worldwide. Severe leaf disease caused by L. theobromae threatens K. coccinea production. The disease threatens K. coccinea growth, and effective control measures should be identified to reduce losses.
RESUMEN
Sacha inchi (Plukenetia volubilis L.) belongs to the family Euphorbiaceae. It is a perennial wooden oilseed crop, and also exhibits a good source of polyunsaturated fatty acids, protein and other bioactive compounds, such as tocopherols, carotenes and phytosterols (Chirinos et al. 2013). During 2017-2018 survey, canker disease showing greyish-brown sunken lesions was observed on the branches of sacha inchi in Danzhou campus, Hainan University, China. The disease incidence is less than 5%. However, it can lead to leaf yellowing, wilt, and eventually the whole plant death. In Nov. 2017, twelve branches showing the typical canker symptoms were collected and covered with parafilm at both ends of all samples to prevent desiccation and placed in black plastic bags keeping at 4°C until isolations were made. Samples were rinsed with tap water and dried with paper towels. Fragments, 5mm in length and cut from the junction of diseased and healthy parts of branches, were surface-sterilized with 2% sodium hypochlorite solution for 2 min, rinsed with sterilized distilled water for 5 times, dried by sterilized filter paper, plated on PDA medium amended with 100 µg/mL streptomycin (PDA-str) and incubated in the dark for 4 days at 28°C. Pure cultures of fungal isolates were obtained by transferring mycelial fragments from colony margins onto fresh PDA plates and incubated as described before. The colonies of cultures were initially white, and eventually turned black after 4 days on PDA medium (Fig S1A). The morphology characterization of conidia produced by the isolates was initially hyaline and aseptate (Fig S1B), and a single median septum formed in the mature conidia (Fig S1B). The average size of 50 conidia was 16.39±1.46â ¹ 8.52±0.92µm for J6, and 15.64±1.73â ¹ 8.94±0.86µm for J3. Three genes were used for phylogenetic analysis (Alves et al. 2006). ITS regions and the partial of TUB (ß-tubulin gene) were amplified using the primer pairs ITS1 and ITS4 (White et al. 1990), Bt2a and Bt2b (Glass and Donaldson 1995), respectively, and EF1-688F/EF1-1251R for J3 and EF1-728F/EF1-986R for J6 were used to amplify TEF (translation elongation factor 1-alpha) (Alves et al. 2008). The sequences of ITS, TUB and TEF from J3 and J6 were deposited in Gene-Bank (Table S1). The blast searches in Gene-Bank with ITS, TUB and TEF amplified from isolates J3, respectively, revealed 100, 99, and 100% identities with L. pseudotheobromae, and isolate J6 showed 100, 100 and 99% of identity with L. theobromae. The phylogenetic analysis of the combined ITS, TUB and TEF sequences of J3, J6 and 28 reference strains retrieved from Gene-Bank was performed using the program MEGA 6.0 evaluated by 1000 bootstrap replications, and the result was consistent with the conclusion above (Fig S2). With the phylogenic studies supported by morphological characters, J3 was identified as L. pseudotheobromae and J6 was L. theobromae. For the pathogenicity test, J3 and J6 were used to inoculate 4-week-old healthy sacha inchi potted seedlings. One wound about 5 mm in depth per seedling stem was made using a sterile blade. A 5-mm-diameter mycelium plug of each isolate taken from the edge of 4-day-old culture growing on PDA was placed to the freshly wound of each plant stem and the inoculated area was wrapped with Parafilm. Sterile PDA plugs were placed onto the wounds of control seedlings. Nine healthy seedlings were inoculated with each isolate or PDA plugs in a completely randomized design. After inoculation, plants were placed in a greenhouse at room temperature (26 to 30°C, 80% RH) and were irrigated when needed. The experiment was conducted twice. Five days later, black or dark-brown canker lesions formed on the stems of inoculated plants, and expended upward and downward from the inoculation points. Pycnidia produced on the necrotic regions and were used to to observe the morphology of conidia (Fig S3). The fungus L. pseudotheobromae or L. theobromae can be re-isolated from the inoculated plants, but not from the control ones. L. pseudotheobromae was recorded to be collected from dead leaves of P. volubilis in Yunnan Province, China, but did not prove this fungus to be pathogenic (Tennakoon et al. 2016). This is the first report that L. theobromae and L. pseudotheobromae causing stem canker in sacha inchi in Hainan, China. The results pave the way for the development of management strategies for canker disease in sacha inchi.