First report of expansion of virulence in Puccinia striiformis f. sp. tritici to wheat resistance genes Yr10 and Yr24 in the Middle East.

Over the last decade, field assessments of the yellow rust differential lines for resistance genes Yr10 and Yr24 and race analysis in the Middle East have demonstrated efficient yellow rust control by Yr10 and Yr24 (=Yr26). Yellow rust samples collected during 2018-21 in Central West Asia & North and sub-Saharan Africa underwent race analysis at the Regional Cereal Rust Research Center in Izmir, Türkiye. The infected leaf segments were subjected to rehydration at 20°C for three hours. Subsequently, the leaf segments were rubbed on the first leaves of seedlings of susceptible cultivar Morocco. Inoculated seedlings were placed at 10°C in dark conditions with 95% humidity for 24 hrs, then moved to a growth chamber with a 16-hr light (220 µmolm-2s-1) cycle at 15°C and an eight-hour dark period at 12°C. Urediniospores were collected 15 days post-inoculation. A set of yellow rust differential lines including Morocco, Avocet 'S', Avocet 'R', Yr1/6* Avocet 'S', Kalyansona (Yr2), Vilmorin 23 (Yr3), Hybrid 46 (Yr4), Yr6/6* Avocet 'S', Yr7/6* Avocet 'S', Yr8/6* Avocet 'S', Yr9/6* Avocet 'S', Yr10/6* Avocet 'S', Moro (Yr10+), Yr17/6*Avocet 'S', Yr24/6* Avocet 'S', TP1295 (Yr25), Yr27/6* Avocet 'S', YrSp/6* Avocet 'S', Spalding Prolific (YrSP), Strubes Dickkopf (YrSD), Tres/6*Avocet'S', Cham 1, and Ambition was used in race analysis. A mixture of 2 mL Soltrol® and 0.5 mg fresh urediniospores was used to inoculate 10-day-old seedlings of the 23 differential varieties. Pre-inoculation, incubation, and post-inoculation conditions were the same as above. Seedling infection types (ITs) were recorded 15 days post-inoculation on a scale of 0 to 9 (McNeal et al. 1971), where ITs 0 to 6 are classified as low infection types (LITs= avirulent) and ITs 7 to 9 categorized as high infection types (HITs= virulent). HITs of 7 to 9 were observed for the first time on Yr10/6* Avocet 'S', Yr24/6* Avocet 'S', as well as on Moro (Yr10+) for 25 sample of the total 50 isolates from Lebanon and Türkiye in 2018. During the race analysis in 2019 to 2021, virulence for Yr10 and Yr24 was identified among tested samples from Egypt, Lebanon, Jordan, Syria, and Türkiye, indicating the expansion of virulence for Yr10 and Yr24 into new regions. HITs were observed for the durum wheat cultivar Cham 1 and wheat cultivar Ambition in all races. Virulence for YrA, Yr2, Yr6, Yr7, Yr8, Yr17, and 32 was common within the Yr10 and Yr24 virulent races, and virulence for YrSp and Yr27 were observed in low frequency. Molecular genotyping of 209 isolates, including the Yr10 virulent races, was performed using 19 microsatellite markers (Ali et al. 2017; Rodriguez-Algaba et al. 2017) and aligned with the Puccinia striiformis nomenclature system of the Global Rust Reference Center (GRRC). The results showed that 66 isolates were identical to the genotyping lineage "ME2018" identified in Egypt in 2018 by GRRC. This genetic lineage has now been designated as PstS17 (Hovmøller et al. 2023). The durum wheat cultivars have always been resistant to yellow rust in the Middle East. Seedling tests of 50 durum advanced lines from CIMMYT's International Durum Wheat Yield Nursery showed LITs in 45 accessions (90%) against an avirulent race for Yr10 and Yr24 (PstS2), but only 12% remained resistant while tested with a PstS17 (virulent for Yr10 and Yr24). This observation provides compelling evidence of the Yr10 and/or Yr24 presence within tested durum wheat germplasm. Continued monitoring of virulence and resistance of wheat germplasm to yellow rust is critical for successful breeding for rust resistance.

First Report of Fusarium verticillioides Causing Fusarium Ear Rot of Corn in Jordan.

Sweet corn (Zea mays L.) is one of the most popular crops grown in Jordan. Fusarium verticillioides (Sacc.) is a major pathogen of corn and a producer of mycotoxin fumonisins (Blacutt et al. 2018). During September and October 2019, ear rot symptoms were observed on Ì´30% of the sweet corn variety Mis Dolce grown in the Jordan Valley. The disease caused substantial losses, including damage to greater than 50% of the kernels within 15-20 days after harvesting. A total of 350-corn kernels were randomly taken from 70 plants distributed in five fields with a total area of 2 ha. About 35% of the samples showed typical symptoms of the disease. Discolored corn kernels were surface sterilized with 5% NaOCl solution for 1 min, then rinsed three times with sterilized distilled water (SDW), plated on potato dextrose agar (PDA) at 25°C, and incubated in the dark for 7 days. Twelve putative isolates of the genus Fusarium were hyphal-tipped on new PDA plates. Isolates were cultured on synthetic low-nutrient agar (SNA) with a ca. 1 × 2-cm strip of sterile filter paper on the agar surface (Nirenberg 1976). Cultures were incubated for 10 to 14 days at 20°C in dark conditions. When sporulation was observed, agar blocks were mounted on a microscopic slide with a drop of lactophenol cotton blue and examined under the microscope at 400x. Colonies grew rapidly with abundant pink to violet aerial hyphae. Sporodochia formed on the agar, and the aerial conidiophores branched sparsely, often alternately or oppositely, terminating with up to three verticillate phialides. Macroconidia were abundant, falcate to straight, three- to five-septate, with a distinct foot cell, 27 to 73 × 3.1 to 5.6 µm. Microconidia produced on polyphialides and aggregating in heads, were unicellular, ovoidal, or ellipsoidal, 4.4 to 17 × 1.5 to 4.5 µm (Fig. 1A, B, C, D, E, and F). Based on morphological characteristics, isolates were tentatively identified as F. verticillioides (Al-Hatmi et al. 2016; Guarro 2013). Two representative isolates were DNA extracted and the translation elongation factor 1-α gene (TEF1) was amplified (O'Donnell et al. 1998), and sequenced from both directions at Macrogen Inc, South Korea. The consensus sequences of the two isolates Fvcorn2021JO-03 (OK040159) and Fvcorn2021JO-04 (OK040160) were used as BLASTn query on the NCBI website and were 100% and 99% similar with F. verticilloides JF740717 and JF740737 accessions, respectively. Similarly, the two isolates were 100% and 99.85% similar with F. verticilloides reference sequences MH582332 and MH582327 on the Fusarium MLST database, respectively. The pathogenicity of the two isolates was tested on 15 cobs by injecting 2 ml of a 2.5 × 105 conidia/ml suspension into the silk channel and into kernel wounds of the primary ear (three replicates) for each treatment (Reid and Hamilton 1996). Inoculated kernels were incubated at 25°C for 2 weeks in plastic boxes. The healthy kernels were injected with 2 ml of SDW as a negative control. Grains started to rot after 2 weeks, in the form of a thick, cottony, crimson-looking growth between the ear and its covers, with only some grains or a group of a adjacent grains rotting, and then white lines appear on the outer skin of the grain, yielding symptoms similar to those in the field (Fig. 2A, B, C, and D). The fungus was re-isolated from the inoculated kernels and was morphologically identified as F. verticilloides thus fulfilling Koch's postulates. The fumonisins-producing potential of the isolated F. verticillioides was confirmed using the AgroQuant Total Fumonisins Assay (Romer Labs, Singapore). To our knowledge, this is the first report of F. verticillioides causing Fusarium ear rot on corn in Jordan. Further investigation is needed to gain a better understanding of the spatio-temporal dynamics of this novel pathogen.

First Report of Strawberry Wilt Caused by Fusarium oxysporum Schltdl. in Jordan.

Strawberries (Fragaria ananassa Duch.) are grown in Jordan year round due to the diversity of climatic conditions and the possibility of growing local or foreign varieties. More than six thousand greenhouses are planted with strawberries in the highlands and the Jordan Valley. About 12 thousand tons of strawberry are produced annually and 2000 tons are exported to European and Arab countries. In April and May 2019, symptoms of wiltand whole plant collapse were observed on approximately 30% of commercial strawberry cv. Deli gent crop in the Jordan Valley (Dayr Alla long.35.6188766, lat. 32.227465). Plants were either dead or showing symptoms including vascular wilt, external and internal discoloration of the stems, and dead shoots. Forty symptomatic plants were collected from 10 greenhouses, and stem fragments were surface sterilized and plated on potato dextrose agar (PDA). Six fungal isolates showed morphological characteristic of Fusarium oxysporum. Colonies on PDA were purple-violet, floccose, with abundant aerial mycelium; colony margins were irregular. Macroconidia were falcate, apical cells had a blunt or papillate shape, basal cells were foot shaped, three- to five-septate, hyaline, smooth, thin-walled, and 37 - 42 × 3 - 6.0 µm in diameter. Aerial microconidia were abundant, hyaline, ellipsoidal, zero to one-septate: 5 - 11 × 2 - 4.0 µm. Chlamydospores were globose to subglobose, intercalary or terminal, with an average diameter of 12 µm (Figure 1: A, B and C) (Nelson et al, 1983; Leslie and Summerell, 2006). Four representative isolates (FoSB2021JO-02, FoSB2021JO-06, FoSB2021JO-08 and FoSB2021JO-09) were DNA extracted, amplified with the translation elongation factor 1-α (EF1α) gene (EF1/EF2) primers (Geiser et al., 2004), and sequenced at Macrogen Inc, South Korea. Forward and reverse sequences were received, assembled and consensus sequence were produced using the BioEdit sequence alignment editor. Consensus sequences of the four isolates were used to conduct BLASTn queries of NCBI website (https://www.ncbi.nlm.nih.gov) and were 100%, 99.9%, 99.6%, 99.9% identical to F. oxysporum accessions MN417194.1, MK968948.1,MK968948.1, and MK968952.1, respectively. A phylogenetic tree with 1000 bootstraps was created using MEGA 7 software (Kumar et al. 2016) (Figure 2). Similarly, the four isolates were 99.5%, 100%, 100%, and 100% identical F. oxysporum reference accessions AF008507, FJ985275, FJ985275, and FJ985278 in the Fusarium MLST database, respectively. Consensus sequences of the four isolates were submitted to GenBank and accession numbers were assigned (OK040155 - OK040158). For pathogenicity tests on strawberries, a spore suspension of 1 × 105 conidia/ml was prepared separately for six isolates. Roots of identical 2-month-old healthy strawberry seedlings (15 plants of cv. Deli-gent) were cut and dipped in the spore suspensions for 30 min. They were then planted in 25 x 20 cm deep plastic pots filled with a sterile mixture of peat - moss, perlite, and vermiculite (60:20:20v/v). Control strawberry plants were soaked in water prior to planting. All plants were placed in a greenhouse at 25°C ±2 along with 15 uninoculated control plants. After 30 days, inoculated plants displayed similar symptoms to those observed in the green house, whereas control plants were symptomless. Roots from symptomatic plants were cultured on PDA and F. oxysporum was recovered and identified morphologically as F. oxysporum. To our knowledge, this is the first report of Fusarium wilt of strawberry in Jordan. The pathogen can cause significant economic losses to strawberries in Jordan and worldwide. Therefore, it is extremely important for disease control in nurseries to determine the infection source and possible factors that increase the incidence of infection to control the disease.

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), ß-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), ß-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 ß-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.

First report of damping off disease caused by Fusarium oxysporum on Pinus pinea in Jordan.

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.