First report of Fusarium proliferatum on date palm (Phoenix dactylifera L.) in Jordan.

Date palm (Phoenix dactylifera L.) is one of the world's oldest cultivated fruit crops. In Jordan, date palm farming started in the 1990s. The major date palm planting areas are Jordan valley, Aqaba, and Azraq (Al Antary et al., 2015). 'Medjool' and 'Barhi' are the two major cultivars in Jordan. In early 2018, some 18- to 24- month old date palm trees (cv 'Medjool') showed light brownish discoloration and dryness symptoms on the leaves and branches of infected date palm trees at the Jordan University Agricultural Research Station (JUARS) at the Jordan Valley (GPS coordinates 32.086871, 35.597219) (Figure 1). All the leaf parts including leaf base, spines, and leaflets were wrinkled and malformed. The infection led to a loss of 1-2% out of 1100 total Medjool trees at the station. Similar symptoms were observed in many date palm farms in the Jordan Valley. Diseased samples from rachis tissue from the JUARS were collected, surface sterilized with 5% sodium hypochlorite for five minutes, rinsed with distilled water for three times, dried, and plated on potato dextrose agar (PDA) medium (HIMEDIA). The plates were incubated at 25°C for seven days. After that, different fungal colonies were purified using the hyphal tip method. Mycelium of a representative isolate (FpDP2018JO-01) was harvested, DNA extracted using the CTAB protocol (Doyle and Doyle, 1990), amplified with three primers: ITS1/4 (White et al., 1990), β-tubulin and the elongation factor 1-alpha (EF1) gene regions. Amplicons were sequenced at Macrogen Inc, South Korea. Sequences were edited via MEGA 7 software (Kumar et al., 2016) and Blastn at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov) which was used to search for similar accessions. The sequences were submitted to the GenBank and accession numbers were received for ITS1/2 (MK522076), β-tubulin (MK720958) and elongation factor 1 alpha (MW533146). The sequences were further used at the Fusarium MLST (https://fusarium.mycobank.org/) for identity confirmation. ITS1/4 and β-tubulin could not discriminate the species Fusarium proliferatum but EF1 - alpha could (Figure 2a-c; Supplement 1). For morphological identification, four representative F. proliferatum isolates (FpDP2018JO-01- FpDP2018JO-04) were used. Mycelium were white to dark purple in color, macroconidia (20.5 - 44.5 × 3.3 - 7.5 μm) were thin, slender, with 3-5 septa, and microconidia (4.3 - 12.1 × 2.5 - 4.3 μm) were thin and aseptate (Figure 3). Koch's postulate was performed on one-year-old seedlings according to Abdalla et al., 2000 method using the same sequenced isolate (FpDP2018JO-01). Five plants were inoculated by injecting 2 mlof inoculum into the crown area using a hypodermic needle and syringe. The inoculum was prepared according to Abdalla et al. (2000). The control set of seedlings (n=5) were injected with sterile distilled water. The experiment was arranged in a CRD design. Symptoms were evaluated three months after inoculation. On seedlings, yellowing of leaflets, discoloration of spines and rachis, and dryness of leaves were observed. Control seedlings showed no symptoms. Re-isolation form the detached leaves and infected seedlings was conducted to satisfy Koch's postulates. Fusarium sp. was confirmed to be F. proliferatum based on their microscopic characteristics. To our knowledge, this is the first record of F. proliferatum on date palm in Jordan. Date palm in Jordan especially 'Medjool' is an important cash crop. Fusarium spp. is an important pathogen that could cause huge losses on date palm and other crops. In Jordan, the pathogen has been isolated from samples from six farms so far, but detailed studies have not been conducted. It would be of importance to survey date palm farms for fungal diseases, test their pathogenicity using several isolates, and characterize them for proper management strategies. F. proliferatum was isolated from roots and leaves of declining date palm trees from many regions of Saudi Arabia and caused symptoms similar to those of F. oxysporum f. sp. albedinis, the causal agent of Bayoud (Abdalla et al. 2000; Saleh et al. 2016). Notonly that, but F. proliferatum was found to have the highest colonization abilities on date palm leaflets and is becoming serious pathogen on date palm (Saleh et al. 2016.