First Report of Fusarium oxysporum f. sp. fragariae Causing Fusarium Wilt on Strawberry (Fragaria × ananassa) in Saudi Arabia.

Recently, Saudi growers have expanded their production of organic, soilless-grown strawberries (Fragaria × ananassa Duch.), but their production shows many difficulties associated with disease susceptibility. In October 2021, 45% of strawberry plants cv. "Festival" organically cultivated in Dammam city, Saudi Arabia (26°31'34.5"N 50°00'51.0"E) showed wilting symptoms. Typical symptoms were yellowing, rapid wilting, death of older leaves, stunting, and decreased roots. Vascular bundle necrosis and crown and root rot were also observed; plants eventually collapsed and died. Twenty symptomatic strawberry plants were sampled to isolate the pathogen. Pieces (4 × 4 mm) of the symptomatic tissues from crowns and roots were sanitized with 1% NaOCl (90 s), submerged in 70% alcohol (20 s), rinsed with sterile water (2x 30 s), placed on potato dextrose agar (PDA; Scharlau Chemie, Spain) and incubated at 25°C for 6 days. Next, we prepared single-spore cultures on PDA and synthetic nutrient-poor agar (SNA). On PDA media, pure cultures produced abundant aerial mycelium, with light pink or purple pigmentation in the medium after incubation at 25°C for 7 days. On SNA media, aerial microconidia were abundant cylindrical to ellipsoid hyaline with zero to one septate (3.8 - 5.9 × 1.3 - 2.5 µm, n = 50). Macroconidia were few, hyaline and falcate, with slightly curved apexes and 2 to 4 septate (18.9 - 27.5 × 3.3 - 4.6 µm, n = 50). Chlamydospores were roundish and terminal or intercalary. As Leslie and Summerell (2006) described, such morphological characteristics are typical of F. oxysporum. The isolates' identities were established by extracting DNA using the DNeasy Plant Mini kit (QIAGEN, Hilden, Germany). This was followed by amplification and sequencing of the internal transcribed spacer (ITS) (White et al., 1990), elongation factor 1-α (TEF1-α) (O'Donnell et al., 1998), and the ribosomal RNA intergenic spacer (IGS) (Canizares et al., 2015). The ITS, TEF1-α, and IGS sequences of an isolate Fof-10 were submitted to GenBank (PP564462, PP703242, and PP784894, respectively). BLAST analysis confirmed 99.71 and 100% identities to the ITS, TEF-1α, and IGS sequences of F. oxysporum (KU931552.1, OR640020.1, and FJ985519.1), respectively. All isolates tested were confirmed at the forma specialis fragariae, level using the specific primers FofraF/FofraR (Suga et al. 2013). The ∼239 bp amplicon was sequenced and submitted to GenBank (PP703243). Two-month-old healthy strawberry plants of cultivars "Festival," "Marquis," and "Monterey" were inoculated by dipping the roots in the spore suspension (107 conidia ml-1) for 15 min (Henry et al. 2017). There were five replicates for each cultivar. Plants dipped in water were used as a control treatment. The plants were transplanted in sterilized soil and placed in a greenhouse at 30/26°C (day/night). Within 4 to 6 weeks, inoculated plants showed severe wilting and discoloration of the internal crown tissue, while control plants were symptomless. The pathogen was re-isolated from the discolored vascular tissue onto PDA and identified morphologically and molecularly as the original one, thus fulfilling Koch's postulates. The test was repeated twice. This report confirms F. oxysporum f. sp. fragariae as a causal agent of Fusarium wilt of strawberries in Saudi Arabia. This pathogen was previously reported to cause the Fusarium wilt of strawberries in California (Dilla-Ermita et al., 2023). This disease has been observed in several hydroponic strawberry greenhouses in Saudi Arabia, with incidence ranging from 25% to 45% across multiple locations. Given this, proper strategies are needed to manage this disease and to be compatible with organic farming.

First Report of Basal Rot of Onion Caused by Fusarium oxysporum f. sp. cepae in Saudi Arabia.

Onion (Allium cepa L.) is a globally important crop worldwide including Saudi Arabia. In November 2020, 2-month-old onion plants (cv. Redwing) in commercial fields within the Sajir area of Riyadh region (∼ 1.4 ha), showed symptoms of yellowing, wilting, stunting, bulb discoloration, rot in the basal parts of bulb and decrease in roots. In the advanced stages, the affected plants collapsed and died. The incidence of symptomatic plants ranged from 30 to 65% in the surveyed fields. To isolate the pathogen, symptomatic onion plants (n =20) were sampled. Diseased tissues from roots and bulbs were cut into small pieces (4 × 4 mm), sanitized with 1% sodium hypochlorite solution for 2 min, submerged in 70% alcohol for 20 s, then rinsed with sterile water, before plating on potato dextrose agar (PDA) medium. The plates were incubated at 25°C for 6 days. Subcultures of the mycelia grown out of the diseased tissues produced purplish pink fungal colonies on PDA. On carnation leaf agar, cultures were characteristic of Fusarium oxysporum as described by Leslie and Summerell (2006), with the presence of unicellular microconidia (3.8 to 7.8 × 1.7 to 2.5 µm, n= 50) without septa in false heads or short monophialides and slightly curved macroconidia (16.3 to 28 × 4.2 to 6.1 µm, n= 50) with two to four septa. Older mycelia developed many chlamydospores that were single or in short chains. To further confirm the pathogen identification, DNA was extracted from single-spore cultures of three representative isolates using the DNeasy Plant Mini kit (QIAGEN, Hilden, Germany). Three different fungal nuclear regions of internal transcribed spacer (ITS), elongation factor 1-α, (TEF1-α) and the second largest subunit of DNA-directed RNA polymerase II (RPB2) DNA were amplified by PCR and sequenced with the following primers: ITS4 and ITS5 (White et al. 1990); EF-1 and EF-2 (O'Donnell et al. 2008); and fRPB2-5F and fRPB2-7cR (Liu et al. 1999), respectively. Phylogenetic analysis based on the alignment of the ITS, TEF1-α, and RPB2 sequences using MEGA7 placed these isolates in the F. oxysporum clade. The ITS, TEF1-α, and RPB2 sequences of an isolate FOC-OR9 were submitted to GenBank (OL721757, OL764494, and OL764495 respectively). To confirm the forma specialis cepae, a fragment of the F. oxysporum f. sp. cepae gene Secreted In Xylem 3 (SIX3) was amplified by PCR (Kalman et al. 2020). The SIX3 amplicon (∼ 277-bp) was sent for sequencing, and the sequence was submitted to GenBank (OL828265). BLASTn analysis of the sequences showed 100% identity with F. oxysporum f. sp. cepae (KP746408). To fulfill Koch's postulates, pathogenicity tests were performed with healthy onion bulbs cv. "Redwing" of 100-150 g each. Prior to inoculation of onion bulbs, the dry bulb scales, one of the fleshy inner scales, as well as the roots were removed. Bulbs were then surface sterilized (as described above) and injected with 20 µl of a conidial suspension (106 spores/ ml) into the basal plate of each bulb and approximately 1 cm deep into the tissue. Six bulbs were inoculated for each isolate, placed in a mesh bag, and incubated at 28 °C in the dark. Six bulbs injected with sterile water and six non-inoculated bulbs served as controls. At the 4th week post inoculation, necrotic rot symptoms and brown discoloration were observed on the basal plates of these inoculated bulbs (similar symptoms to those observed in the field), while control treatments showed no symptoms. The pathogen was re-isolated from the basal plates onto PDA and identified morphologically and molecularly as F. oxysporum f. sp. cepae, thus fulfilling Koch's postulates. The test was repeated twice. This pathogen was previously reported causing onion basal rot in United Kingdom (Taylor, et al., 2013). To our knowledge, this is the first report of basal rot in onion caused by F. oxysporum f. sp. cepae in Saudi Arabia. It is recommended that preventive management should be considered as this disease may cause significant economic losses for onion growers in Saudi Arabia. Also, Fusarium mycotoxin contamination of onion bulb could pose a public health risk.

First Report of Fusarium Root and Stem Rot Caused by Fusarium oxysporum f. sp. radicis-cucumerinum on Greenhouse Cucumbers in Saudi Arabia.

Cucumber (Cucumis sativus L.) is an important vegetable crop in Saudi Arabia. During May 2018, 45 - 60% of 5-month-old cucumber plants showed symptoms of a previously unknown wilt in commercial greenhouses around Al Kharj area of Riyadh region. Symptoms consisted of crown and root rot, wilting and stem disintegration, along with yellowish brown to brown external discoloration extended throughout the affected tissues. As the disease progressed, a pinkish-orange mycelial growth was often observed at the basis of affected stems while vessels were discolored. Subsequently, the affected plants were collapsed and died. Crown, stem, and root fragments (4 × 4 mm) were cut from symptomatic tissues, surface sterilized in 2.5% NaOCl, cultured on potato dextrose agar (PDA) with 25 mg/liter of streptomycin sulfate, and incubated at 26°C in darkness for 6 days. Single-spored cultures produced white mycelium with pink, white, or purple pigmentation in the center. The mycelium produced sporodochia. Macroconidia were mainly slightly curved with three to five septa. Microconidia were single-celled oval and produced on short lateral phialides. Chlamydospores were single or in short chains. Morphologically, the isolated fungus was characterized as Fusarium oxysporum (Leslie and Summerell 2006). To further confirm the fungus identification, DNA was extracted from a single-spored culture. Three different fungal nuclear regions of internal transcribed spacer (ITS), elongation factor 1-α, (TEF1-α) and the second largest subunit of DNA-directed RNA polymerase II (rpb2) with the following primers: ITS4 and ITS5 (White et al. 2017), EF-1 and EF-2 (O'Donnell et al. 2008), and fRPB2-5F and fRPB2-7cR (Liu et al. 1999), respectively. The ITS, TEF1-α, and rpb2 sequences of the isolate FCKSU17 were submitted to GenBank (MT232918, MW471131, and MW449833 respectively). Phylogenetic analysis based on the alignment of the ITS, TEF1-α, and rpb2 sequences using MEGA7 placed this strain in the F. oxysporum clade. To confirm the forma specialis radicis-cucumerinum, amplification with the specific primers ForcF1/ForcR2 was conducted (Lievens et al. 2007). The amplified fragment (∼ 250-bp) was sent for sequencing, and the sequence was submitted to GenBank (MW471132). BLASTn analysis of the sequences showed 100% identity with F. oxysporum radicis-cucumerinum (KP746408). To fulfill Koch's postulates, pathogenicity test was conducted on 7-day-old plants of cucumber cultivar Beit Alpha grown into pots filled with soil mix (2:1 sandy loam-peat moss, vol/vol). The plants were inoculated through drenching with 100 ml of conidial suspension in sterile distilled water (106 spores/ml) per pot. Control plants were treated with sterile distilled water. Each treatment included 10 replicates (pots), with two plants per pot. The pathogenicity test was repeated once. Cucumber plants inoculated with the fungus showed early wilting symptoms within the first 2 weeks post inoculation. At the 6th week post inoculation, 90 to 100% of the inoculated plants developed typical symptoms. No symptoms were observed on the control plants. The pathogen was successfully re-isolated from the inoculated wilted plants and identified morphologically. To our knowledge, this is the first report of F. oxysporum f.sp. radicis-cucumerinum on cucumber in Saudi Arabia. It is recommended that preventive management should be considered as this disease may cause significant economic losses on cucumbers in Saudi Arabia.