First report of Lasiodiplodia brasiliensis causing leaf blight on rambutan

Authors: AVILES-NORIEGA, ASHLEY - University Of Puerto Rico; Serrato Diaz, Luz; Goenaga, Ricardo; RIVERA-VARGAS, LYDIA - University Of Puerto Rico; BAYMAN, PAUL - University Of Puerto Rico

Submitted to: Journal of Agriculture of the University of Puerto Rico
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/13/2023
Publication Date: 1/1/2022
Citation: Aviles-Noriega, A., Serrato Diaz, L.M., Goenaga, R.J., Rivera-Vargas, L.I., Bayman, P. 2022. First report of Lasiodiplodia brasiliensis causing leaf blight on rambutan. Journal of Agriculture of the University of Puerto Rico. 106(1):119-122. https://doi.org/10.46429/jaupr.v106i1.21060.
DOI: https://doi.org/10.46429/jaupr.v106i1.21060

Interpretive Summary: Rambutan (Nepehelium lappaceum L.) is a tropical fruit tree native of Southeast Asia and commercially grown in the United States in Hawaii and Puerto Rico. Leaf blight has been a problem on rambutan in commercial and experimental orchards and greenhouses throughout the island. According to farmers and personnel of agricultural extension service, leaf blight is caused due nutritional deficiencies, but biotic factors have been not considered. To learn the causative agent of leaf blight, diseased leaves with leaf blight symptoms were collected from 2014 to 2016. Diseased leaves were disinfested with ethanol 70% and plated on potato dextrose agar (PDA) and Oatmeal agar (OA). Two isolates of Lasiodiplodia brasiliensis, A1 and A2, were identified based on morphology and the sequencing of three genes: ITS region, partial sequence of EF1-a, and ß-tubulin. Sequences were compared using BLASTN tool with sequences in GenBank. Sequences of L. brasiliensis were submitted to GenBank with accession numbers: MT784900 and MT784901 for ITS region; MT796209 and MT796210 for EF1-a; and MT796213 and MT796214 for ß-tubulin. Pathogenicity tests were conducted on six 1-year-old rambutan seedlings, using six healthy non-detached leaves per isolate. Two rambutan seedlings per isolate were inoculated using 5mm mycelial disks from pure cultures grown on PDA. Leaves were kept in a humid chamber using plastic bags for up to 8 days under greenhouse conditions. Untreated controls leaves were inoculated with PDA disks. The experiments were repeated twice. Fourteen days after inoculations (DAI), isolates of L. brasiliensis caused leaf blight. For both isolates, diseased leaves turned from light to dark brown starting at the apex and spreading through the lamina. Necrotic lesion size ranged from 10mm to 40mm. Untreated controls showed no symptoms and no fungi were re-isolated from these tissues. To conclude the pathogenicity tests, L. brasiliensis was re-isolated from diseased leaves, fulfilling Koch's postulates. L. brasiliensis has been reported causing dieback and corky bark on rambutan. To our knowledge, this is the first report of L. brasiliensis causing leaf blight on rambutan. The identification of L. brasiliensis as the causative agent of leaf blight is important for rambutan growers to develop strategies to control this new disease in Rambutan worldwide.

Technical Abstract: Rambutan (Nephelium lappaceum L.) is a tropical fruit tree belonging to the family Sapindaceae. Native to Southeast Asia, it has been cultivated on commercial orchards at Mayagüez, Puerto Rico since 1998. During a disease survey from 2014 to 2016, leaf blight was observed in greenhouses, commercial and experimental orchards. To isolate the fungal pathogen, diseased leaves were disinfected with 70% ethanol, rinsed with sterile, distilled water, and plated on potato dextrose agar (PDA) and Oatmeal agar (OA). Two isolates of Lasiodiplodia, A1 and A2, were obtained from rambutan leaves, purified, and identified based on cultural characteristics and conidial morphology (Netto et al. 2014). On PDA and OA, colonies were initially gray with aerial mycelia that turned dark brown with age. Pycnidia were dark brown to black. Immature, hyaline, one-celled conidia were sub-ovoid to ellipsoid, apex rounded, truncate at the base and thick-walled. When mature, conidia became dark brown, two-celled with irregular longitudinal striations. Conidia (n = 50) of isolates A1 and A2 measured an average of 26.1 µm long by 13.7 µm wide. PCR amplification of three genes: ITS region, partial sequence of EF1-a, and ß-tubulin, were used to support morphological identification. Genes were sequenced and compared using BLASTN with sequences in GenBank. The pathogen was identified as L. brasiliensis with GenBank accession numbers: MT784900 and MT784901 for ITS region; MT796209 and MT796210 for EF1-a; and MT796213 and MT796214 for ß-tubulin. Pathogenicity tests were conducted on six rambutan 1-year-old seedlings, using six healthy non-detached leaves per isolate. Two rambutan seedlings per isolate were inoculated using 5mm mycelial disks from pure cultures grown on PDA. Leaves were kept in a humid chamber using plastic bags for up to 8 days under greenhouse conditions. Untreated controls leaves were inoculated with PDA disks. The experiments were repeated twice. Eight and 14 days after inoculations (DAI), isolates of L. brasiliensis caused leaf blotch and leaf blights, respectively. For both isolates, diseased leaves turned from light to dark brown starting at the apex and spreading through the lamina. Necrotic lesion size ranged from 10mm to 40mm. Untreated controls showed no symptoms and no fungi were re-isolated from these tissues. To conclude the pathogenicity tests, L. brasiliensis was re-isolated and sequenced from diseased leaves, fulfilling Koch's postulates. These new sequences were submitted to GenBank with the following accession numbers: MT784902 for ITS region; MT796211 and MT796212 for EF1-a; and MT796215 for ß-tubulin. L. brasiliensis has been reported causing dieback and corky bark on rambutan (Serrato-Diaz et al. 2019). To our knowledge, this is the first report of L. brasiliensis causing leaf blight on rambutan.