First report of Lasiodiplodia theobromae Causing Dieback, a Destructive Disease on Mango trees in Saudi Arabia.

Mango (Mangifera indica L.) is a popular tropical fruit crop in Saudi Arabia. However, susceptibility to diseases is a major factor that restrict the development of mango trees, reducing the yield and production (Ploetz, 2003). In December 2021, a survey was conducted for mango trees which were showing symptoms of decline in the field located in the district Al-Jumum of Makkah Province, in western Saudi Arabia (21°46'18.9"N 39°35'21.2"E). The disease severity was approximately 40% with 15% incidence of mango trees showing symptoms of twig dieback, leaf necrosis, leaf fall, and internal tissue necrosis as well as darkening within the vascular tissue upon splitting the infected branches. As the disease progressed, the affected branches were turned black-brown and dried up (Supplementary Figure S1). To isolate the pathogen, 20 symptomatic branches were arbitrarily sampled from different parts of the field and washed with tap water. Diseased branches were cut into 4 × 4 mm portions (between symptomatic and healthy tissues), submersed in 70% alcohol for 20 s, surface sterilized with 1% sodium hypochlorite solution for 3 min, rinsed with sterile distilled water, and cultured on potato dextrose plates (PDA). The plates were incubated at 25°C in darkness for 3-4 days, and then pure culture of the fungus was obtained by hyphal tip isolation technique. After 3 days of culturing at 25°C on PDA medium, the fungal colonies were grayish-white with uneven edges, and becoming dark grey to black colored after 5 days. After 21 days at 25 ℃ in constant light, the colonies produced dense aerial mycelium at which stage numerous dark colored pycnidia were formed and conidia were observed. Immature conidia were unicellular, hyaline, elliptical or ovate, and truncated at the base, becoming dark brown, thick-walled, one-septate, and longitudinal striation at maturity. Mature conidia measured 22.4±1.6 to 28.7±2.8 μm long and 12.8±1.3 to 15.6±2.4 μm width (n=40). The morphological characteristics of the colonies were consistent with to Lasiodiplodia theobromae (Pat.) Griff. & Maubl. (syn. Botryodiplodia theobromae Pat.) (Zambettakis, 1954; Sutton, 1980). Fifteen isolates were obtained, and a single representative isolate (LPT07-KSU) was used for further study. To further confirm the pathogen identification, genomic DNA was extracted from a single-spore culture using the DNeasy Plant Mini kit (QIAGEN, Hilden, Germany). The internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) and translation elongation factor 1-α, (tef1-α) were PCR amplification and sequencing with the following primers: ITS4 and ITS5 (White et al. 1990); and EF-1 and EF-2 (O'Donnell et al. 2008), respectively. The resulting ITS, and TEF1-α, sequences were submitted in GenBank under accession numbers ON192029, and ON209443, respectively. BLASTn analysis of these genes revealed ≥99% identity with the corresponding sequences of L. theobromae in GenBank (MH644067 for ITS region and MZ502303 for tef1-α gene). The result of phylogenetic analysis also showed that the pathogen was identified as L. theobromae, confirming the morphological identification. A pathogenicity assay was carried out on healthy 1-year-old mango cv. "Haden" seedlings. Infection followed the method of Saeed et al., (2017), consisting of excising a 5-mm-diameter tissue bark out of branches (~ 10 to 15-cm of the apical tip) and replacing it with a 5 mm PDA plugs colonized with L. theobromae from 20-days-old-culture or non-colonized plugs (controls). The area of inoculation was covered with parafilm to avoid dehydration. All seedlings were kept under greenhouse conditions (27°C, 16/8-h day/night, 70% RH) and monitored for disease development. Five replicates were used for inoculated and control plants. After 28 days, all inoculated plants displayed similar symptoms to those observed in the field, whereas control plants remained symptomless. Koch's postulates were fulfilled when typical colonies of L. theobromae were successfully re-isolated from the from symptomatic tissues. The test was repeated twice. This pathogen was reported to affect mango cultivation in China (Li et al., 2013), United Arab Emirates (Saeed et al., 2017), and Mexico (Bautista-Cruz et al., 2019). However, to the best of our knowledge, this is the first report of L. theobromae causing dieback disease on mango in Saudi Arabia. The occurrence of manage dieback highlights the importance of disease surveillance in the region. Effective control strategies are need to be established to reduce the losses.