First Report of Colletotrichum gloeosporioides and Colletotrichum siamense, Causing Anthracnose Disease on coffee trees, in Saudi Arabia.

Coffee (Coffea arabica L.) is a promising agricultural commodity in many countries including Saudi Arabia, but crop production is often constrained by diseases. In December 2021, coffee trees had symptoms of anthracnose disease (CAD) were observed in Jazan Province, Saudi Arabia (17°19'00.8"N 43°11'26.8"E), and the incidence was 55%. Affected trees showed dieback and leaves necrosis. On green and ripening berries, slightly sunken and dark brown lesions were occurred; the berries finally become mummified (Fig. S1). For pathogen isolation, symptomatic tissues (4×4mm) of 30 diseased branches and berries samples were surface-sterilized in 1% sodium hypochlorite for 2 min, followed by 70% ethanol for 20 s, rinsed in sterile distilled water and placed on potato dextrose agar (PDA). Cultures were incubated at 26℃ for 8 days in the dark. Eighteen isolates were recovered, and 2 representative single spore isolates (KSU-CgM17, KSU-CsM42) were used for further study. PDA culture of KSU-CgM17 had aerial white mycelium at first and later became gray to grayish black; light salmon to orange conidial masses were observed on the mycelium plate surface as the cultures aged (Fig. S2). Colony produced by KSU-CsM42 was off-white to gray with cottony mycelia and grayish-white on the undersides of the culture after 10 days at 28° (Fig. S2). Conidial shape of these two isolates were both aseptate, cylindrical to nearly straight, hyaline, rounded at both ends. Conidia (n = 50) measurements were 16 to 18.0 µm long × 4.8 to 6.4 µm wide for KSU-CgM17 and 12.6 to 17.5 µm long × 3.2 to 4.5 µm wide for KSU-CsM42. The microscopic and culture features fitted those for Colletotrichum gloeosporioides species complex (Weir et al. 2012). To further identify these isolates, four genomic DNA loci including the partial ITS rDNA region, and CAL, TUB2, and GAPDH genes were amplified and sequenced (Hu et al., 2015). All sequences were deposited into GenBank under accession numbers: OQ791412 & OQ791413 (ITS), OQ786847 & OQ786851 (CAL), OQ786849 & OQ786850 (TUB2), and OQ786848 & OQ786852 (GAPDH) for KSU-CgM17and KSU-CsM42, respectively (Tables S1& S2). A BLAST search of GenBank showed that these pathogens were identified as C. gloeosporioides (KSU-CgM17) and C. siamense (KSU-CsM42). The pathogenicity was tested on detached coffee leaves or green and red berries (Coa et al., 2019). For inoculation, healthy leaves and berries were wounded with a sterilized needle, placed inside petri dishes containing moist filter paper, and then inoculated with a 10-µl droplet of conidial suspension (106 spores/ ml). Sterile distilled water was used as a negative control. Six replicates were tested per isolate and the experiment was repeated once. The inoculated materials were incubated at 25°C and 100% relative humidity for 8 days. Necrotic lesions developed on 100% of the inoculated coffee materials 6 days later, whereas the negative controls were asymptomatic (Fig. S2). Koch's postulates were fulfilled when typical colonies of these species were successfully re-isolated from the from symptomatic tissues. These pathogens were reported previously to affect coffee in Vietnam (Nguyen et al., 2010), China (Cao et al., 2019), and Puerto Rico (Serrato-Diaz et al., 2020). To our knowledge, this is the first record of C. gloeosporioides and C. siamense causing CAD in Saudi Arabia. Further studies on the epidemiology of CAD on arabica coffee plantations as well as effective strategies for managing this disease are needed.

Molecular epidemiology of the citrus bacterial pathogen Xanthomonas citri pv. citri from the Arabian Peninsula reveals a complex structure of specialist and generalist strains.

Molecular epidemiology studies are essential to refine our understanding of migrations of phytopathogenic bacteria, the major determining factor in their emergence, and to understand the factors that shape their population structure. Microsatellite and minisatellite typing are useful techniques for deciphering the population structure of Xanthomonas citri pv. citri, the causal agent of Asiatic citrus canker. This paper presents a molecular epidemiology study, which has improved our understanding of the history of the pathogen's introductions into the Arabian Peninsula, since it was first reported in the 1980s. An unexpectedly high genetic diversity of the pathogen was revealed. The four distinct genetic lineages within X. citri pv. citri, which have been reported throughout the world, were identified in the Arabian Peninsula, most likely as the result of multiple introductions. No copper-resistant X. citri pv. citri strains were identified. The pathogen's population structure on Mexican lime (their shared host species) was closely examined in two countries, Saudi Arabia and Yemen. We highlighted the marked prevalence of specialist pathotype A* strains in both countries, which suggests that specialist strains of X. citri pv. citri may perform better than generalist strains when they occur concomitantly in this environment. Subclade 4.2 was the prevailing lineage identified. Several analyses (genetic structure deciphered by discriminant analysis of principal components, RST-based genetic differentiation, geographic structure) congruently suggested the role of human activities in the pathogen's spread. We discuss the implications of these results on the management of Asiatic citrus canker in the region.

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.