Forests of Jordan are located in the northern and southern parts of the country with 60% and 40%, respectively. Natural forests constitute about 75% in the northern part and 25% in the southern part. There are many types of forest trees in Jordan including pines (Pinus spp.), juniper (Juniperus), cypress (Cupressus), oak (Querus), acacia (Acacia), and Christ's thorn jujube (Ziziphus). There are three species of Pinus: P. halopensis (native), P. pinea (introduced), and P. canariensis (introduced) (Ministry of Agriculture, 2013). P. pinea is considered one of the most important components of Jordan's natural forests. Due to its adaptability, lack of environmental requirements and ease of cultivation, its cultivation has been expanded in all parts of Jordan. P. pinea cultivation prevent soil erosion and combating desertification. P. pinea seeds are used in making sweets and many popular foods. In the end of 2019, wilting and damping-off symptoms were noticed in 50 % of P. pinea seedlings nurseries (personal communication, November 2019). Six-month old P. pinea seedlings with visible symptoms of damping-off were collected between May and July 2020 from a pine nursery located in Amman Province, Jordan (32° 0′ 40.4316″ N, 135° 52′ 20.3628″ E). Thirty-two seedlings with different severities of the disease were selected for the isolation of root pathogens. Two root samples from each seedling were surface-sterilized using 1% sodium hypochlorite for 3 - 5 minutes and then rinsed with sterile distilled water. Root samples were subsequently cut into small pieces (1- to 2 cm long sections) and then placed on potato dextrose agar (PDA) supplemented with 5 mg/L streptomycin sulphate. Petri dishes were incubated in a growth chamber at 25±2°C for seven days and sub-cultured by hyphal tipping. The cultural and conidial morphology of 7-day old mycelia were observed for the isolates using an optical microscope (LEICA, ICC50 HD, Switzerland). For morphological identification of Fusarium, 200 measurements of microconidia, macroconidia and chlamydospores were conducted. The Fusarium isolates had a color of purple-violet mycelium growth in a PDA culture medium (Figure 1 A and B). Macroconidia had 3-5 septa with a foot- shaped basal cell. They were sickle-shaped, hyaline, and thin-walled with dimensions of 20-42 x 3.2-5.5 μm (Figure 1C). Microconidia were short, elliptic to oval unicellular, and with sharp unbranched monophyalides with an average dimension of 5.0–17.0×2.3–5.1 μm (Figure 1D, 1E). Older mycelia developed a large number of terminal chlamydospores (7.2 to 14.1 µm) that were intercalary and rough-walled (Figure 1F). All the characteristics agreed with those recorded by Leslie et al. (2006) and Nelson et al. (1983) for Fusarium oxysporum. Representative isolate (FoxypineJO2020-01) was selected for molecular identification. The DNA was extracted, amplified using the translation elongation factor 1-α (EF1α) gene (EF1/EF2) (O’Donnell et al., 1998), and sequenced at Macrogen Inc, South Korea. Forward and reverse sequences were received, assembled and consensus sequence was produced using BioEdit sequence alignment editor (Hall, 1999). The consensus sequence was BLASTn on the NCBI website (https://www.ncbi.nlm.nih.gov) and was 100% similar with F. oxysporum accession KC622308.1. Phylogenetic analysis was conducted using MEGA 7.0.26 (Kumar et al. 2016) with 1000 bootstrap values and correlated the representative isolate with the accession KC622308.1 (Figure 2). The isolated sequence was deposited in the GenBank and accession number was assigned (MW057934). Koch's postulates were fulfilled using FoxypineJO2020-01 isolate to confirm the Fusarium oxysporum as the causal agent of Pinus pinea damping-off. One-month-old seedlings of P. pinea were soaked in spore suspension of 1 × 106 spores/ml for 10 minutes. Seedlings were placed in 25cm x 20cm deep plastic pots filled with a sterile mixture of peat moss, perlite, and vermiculite (60:20:20). Controlled by thirty seedlings of P. pinea soaked in distilled water. Planted seedlings were incubated at 25 ± 2°C with a 12: 12 hrs light/dark period. Seedlings of P. pinea inoculated with spores gradually showed symptoms similar to those of naturally diseased infected plants (Figure 3, 4). The inoculated pathogen was successfully re-isolated from roots of the diseased seedlings. The uppermost leaves began to wilt (Figure 4c), and the roots had darkened at 25 days after inoculation (Figure 4d). By 40 days after inoculation, the entire seelings were discolored and dead (Figure 4e). Furthermore, the roots became dark and peeled (Figure 4f). These symptoms matched those described by (Machón et al., 2009) and (Luo and Yu 2020). Control P. pinea seedlings remained asymptomatic (Figure 4a, b). To our knowledge, this is the first report of F. oxysporum on P. pinea in Jordan. No previous disease notes were reported on P. pinea seedlings in Jordan. The pathogen can cause significant economic losses to P. pinea as well as to other types of Pinus spp. whether in nurseries or forests in Jordan. Therefore, for disease control in nurseries, it is extremely important to determine the onset time, decrease the incidence (Gordon et al. 2015) and identify the infection source (Morales-Rodriguezv et al. 2018). Future surveys need to be conducted on forest trees in selected forest and biosphere reserves that show tree decline to identify major forest fungal pathogens in Jordanian forests.
First report of Sydowia polyspora
causing disease on Pinus pinea
shoots
