Diversity of Genetic and Vegetative Compatibility Group of Colletotrichum coccodes Isolates from Chile Using Amplified Fragment Length Polymorphism Markers.

Colletotrichum coccodes (Wallr.) Hughes is an asexual fungus with five vegetative compatibility groups. It was postulated that C. coccodes was isolated at the center of origin of potato at one time, and due to the movement of potato around the globe, the fungus was established on each continent but became bottlenecked and genetically unable to form stable heterokaryons via vegetative compatibility grouping (VCG) studies. The objectives of this study were (i) to determine if the VCGs around the world are related to the VCGs in Chile, (ii) to determine the diversity of C. coccodes populations in Chile, and (iii) to find any evidence for a cryptic sexual life cycle for this fungus. Worldwide C. coccodes populations have been found to be genetically correlated and belong to one or more C. coccodes-identified VCGs. The most distributed VCG in Chile was VCG2, which is the most common VCG in North America. We hypothesize that one or more VCGs had spread from Chile to the rest of the world. Precautions and further studies should be investigated by using other molecular markers and gene sequencing.

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.

Production of Xylanase by Trichoderma Species Growing on Olive Mill Pomace and Barley Bran in a Packed-Bed Bioreactor.

Xylanases are hydrolytic enzymes that have tremendous applications in different sectors of life, but the high cost of their production has limited their use. One solution to reduce costs and enhance xylanase production is the use of agro-wastes as a substrate in fungal cultures. In this study, olive mill pomace (OMP) and barley bran (BB) were used as carbon sources and possible inducers of xylanase production by three species of Trichoderma (atroviride, harzianum, and longibrachiatum), one major xylanase producer. The experiments were conducted under a solid-state fermentation system (SSF) in flask cultures and a packed-bed bioreactor. Cultures of OMP and BB were optimized by examining different ratios of OMP and BB, varied particle sizes, and inoculum size for the three species of Trichoderma. The ratio of 8:2 OMP and BB yielded the highest xylanase activity, with a particle size of 1 mm at 29 °C and an inoculum size of 1 × 107 spores/mL. Studying the time profile of the process revealed that xylanase activity was highest after seven days of incubation in flask SSF cultures (1.779 U/mL) and after three days in a packed-bed bioreactor (1.828 U/mL). The maximum percentage of OMP degradation recorded was about 15% in the cultures of T. harzianum flask SSF cultures, compared to about 11% in T. longibrachiatum bioreactor cultures. Ammonium sulfate precipitation and dialysis experiments showed that Xylane enzyme activity ranged from 0.274 U/mL in T. harzianum to 0.837 U/mL in T. atroviride when crude extract was used, with the highest activity (0.628 U/mL) at 60% saturation. Xylose was the main sugar released in all purified fractions, with the G-50 and G-75 fractions showing the maximum units of xylanase.

First report of Fusarium culmorum (W.G. Sm.) Sacc causing crown rot on wheat in Jordan.

Fusarium crown rot (FCR) is a disease caused by numerous Fusarium species, primarily F. culmorum (W. G. Sm.) Sacc., F. pseudograminearum (O'Donnell & T. Aoki), and F. graminearum Schwabe (Paulitz et al., 200). FCR on wheat is a worldwide distributed disease that causes significant yield losses. In the Middle East, FCR was reported in Iraq (Motallebi et al., 2015; Matny et al., 2019) and Syria (Motallebi et al., 2015). In Jordan, Fusarium occurrence on wheat was documented in a checklist publication in 1984 (Mamluk et al., 1984) without further identification of the causative species and its pathogenicity level. There have been no other reports of Fusarium on wheat in Jordan since then. Symptoms of Fusarium crown rot were observed in 2016-2022 (Alananbeh et al., 2018) across Jordan through annual surveys of wheat diseases. The disease severity was higher in the dry seasons such as that of 2017 and 2021. Very severe symptoms were noted on wheat planted at the University of Jordan experimental wheat plots (n=4) in 2016-2022. A total of 40 symptomatic plants were randomly collected from these plots. Roots and stems of the 40 plants were then cut into small sections, disinfected in 0.5% hypochlorite for 5 minutes, 70% ethanol for one minute, and finally rinsed in sterile distilled water three times. The sections were dried under the laminar flow, plated on potato dextrose agar (PDA), and incubated for 10 -14 days at 25 ℃. The fungal cultures were purified by hyphal tipping. At least one pure isolate exhibited a typical morphology of F. culmorum was recovered from each plant. The colonies of pure cultures grew rapidly on PDA with fluffy floccose aerial mycelium and dark red to reddish brown pigment diffused in the agar. The isolates produced monophialidic conidiogenous cells. The formed marcoconidia were slightly curved, with pointed apical and foot cells, 3-5 septated, on average 28.5 - 46.5 X 4.5-7.0 µm, indication the cultures as Fusarium spp. (Figure 1). Chlamydospores were intercalary in hyphae and microconidia were absent. Two representative isolates (Iso-1 and Iso-2) identified putatively as F. culmorum, based on their morphological features, were sent to Macrogen Inc., South Korea to Sanger sequence a portion of the translation elongation factor 1-α gene using the EF1/EF2 primers (Geiser et al. 2004). Raw sequences were used to create consensus sequences using the BioEdit sequence alignment editor. The consensus sequences for the two representatives isolates were used to conduct BLASTn queries of NCBI (https://www.ncbi.nlm.nih.gov) which revealed they are 99.67% and 100% identical to MW233082.1, a TEF11-α sequence of the ex-epitype of F. culmorum (NRRL 25475, Crous et al. 2021). The two sequences generated herein were accessioned in GenBank (accession numbers: OQ785278 and OQ785279). Combined with the morphological and molecular analysis, the Iso-1 and Iso-2 were identified as Fusarium culmorum. The pathogenicity of the isolates was tested on two wheat cultivars using two methods: in vitro on seeds grown in sterile dishes and on seedlings. A 4 X 104 macroconidia suspension was prepared from 10 day-old culture of the isolate grown on PDA at 28 ºC. Seeds of two wheat cultivars, Hourani and Norsi were surface sterilized in 1% (v/v) bleach and rinsed in sterile distilled water three times. For the first method, seeds were soaked in the F. culmorum conidia suspension for 15 min and then dried using filter paper. The seeds were plated onto sterile paper towels in sterile plastic boxes and placed in a growth chamber. Three replicates with 10 seeds/replicate were used. Control Mock treatments used seeds treated with sterile distilled water. The germination percentage, coleoptile length, radicle length, longest seminal root length, and number of seminal roots were measured after 5 days. For the seedling-based pathogenicity test, seeds were planted in seedlings trays filled with sterilized 1:1:1 peat moss: sand: soil. 5 mL conidia suspension was drenched following seedling emergence. Ten replicates with one seed/replicate were used. Plants were watered when necessary to maintain appropriate soil growth conditions. The control seedlings were drenched with sterile distilled water. Disease symptoms were rated by the disease severity index (CRI) described by Mitter et al. (2006) after 35 days of inoculation. The in vitro test showed a reduction of germination and other seeds measurements in the presence of F. culmorum as compared to the control (Table 1 and Table 2, Figure 2). Similarly, the seedling's height, length of discoloration, disease score, disease severity index and germination percentage were all reduced in F. culmorum treated seedlings compared to the control. The two experiments showed that Cv. Norsi was more susceptible to FCR than Hourani (Table 1, Figure 2). F. culmorum was re-isolated from the roots of inoculated plants of both cultivars. The present study is the first report of the crown rot pathogen, F. culmorum on Jordanian wheat. Fusarium culmorum can cause significant economic losses and current research is ongoing to survey FCR-associated Fusarium spp. in Jordan, their genetic diversity, and QTL mapping for resistance genes in wheat landraces.

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.

Isolation and Characterization of Halotolerant Plant Growth Promoting Rhizobacteria From Durum Wheat (Triticum turgidum subsp. durum) Cultivated in Saline Areas of the Dead Sea Region.

Plant growth promoting rhizobacteria (PGPR) are beneficial microorganisms that can be utilized to improve plant responses against biotic and abiotic stresses. In this study, 74 halotolerant bacterial isolates were isolated from rhizosphere and endorhizosphere of durum wheat (Triticum turgidum subsp. durum) plants cultivated in saline environments in the Ghor region near the east of the Dead Sea. 16S rDNA partial sequences and phylogenetic analysis of 62 isolates showed clear clustering of the isolates into three phyla: Firmicutes (61.3%), Proteobacteria (29.0%), and Actinobacteria (9.7%). At the genus level, the majority of them were grouped within the Bacillus, Oceanobacillus, and Halomonas genera. The isolates, which possessed plant growth promoting traits including nitrogen fixation, ACC deaminase activity, auxin production, inorganic phosphate solubilization and siderophore production, were selected. The effect of the inoculation of selected PGPR strains on growth of salt sensitive and salt tolerant durum wheat genotypes under high salt stress conditions was evaluated. Six halotolerant PGPR strains were able to improve survival in inoculated plants under high salinity stress conditions as reflected in higher germination percentages and seedling root growth when compared with non-inoculated plants. Furthermore, three halotolerant PGPR strains were able to improve durum wheat tolerance to water deficit stress. In addition, antagonistic effect in four halotolerant PGPR strains against an aggressive pathogenic isolate of Fusarium culmorum that causes crown rot disease was observed in a dual culture assay. In conclusion, the halotolerant PGPR strains described in this study might have great potential to improve durum wheat productivity under different stress conditions.

Evaluation of aerial microbial pollutants in Al-Haram Al-Nabawi during pilgrimage of 2013.

Al-Madinah Al-Munawwarah is the second holiest site in Islam. The possibility of new emerging microbes is valid due to the increased number of pilgrims. The objectives of the current study were to estimate the numbers of fungi and bacteria inside and outside Al-Haram Al-Nabawi and to find whether new bacterial and fungal species have emerged compared to previous studies. Air samples were collected twice a day from 12 spots and four directions during the pilgrim year of 2013 for four consecutive weeks by using the sedimentation method. Thirty five genera and fifty eight species were identified. The most recovered bacterial genera were Staphylococcus, Micrococcus, Bacillus, and Dermacoccus with 32.47%, 18.18%, 12.85%, and 11.23%, respectively. Fifty nine isolates of fungi were molecularly identified. Aspergillus species had the highest percentage (78%). The other fungal genera identified (Alternaria triticina, Emericella nidulans, Emericella striata, Mucor circinelloides, Penicillium chrysogenum, Penicillium minioluteum, Rhizopus arrhizus, Rhizopus oryzae, and Syncephalastrum racemosum) had less than 5% frequency. In places such as Al-Haram Al-Nabawi, a large and crowded public (millions) exist especially during pilgrimages and Ramadan, thus, exposure to microorganisms is high. On the other hand, microorganism infectivity depends on many factors including their virulence, landing site, and person's immunity. For those reasons, many aspects should be considered to avoid aerosol contaminants.

Phomopsis Stem Canker: A Reemerging Threat to Sunflower (Helianthus annuus) in the United States.

Phomopsis stem canker causes yield reductions on sunflower (Helianthus annuus L.) on several continents, including Australia, Europe, and North America. In the United States, Phomopsis stem canker incidence has increased 16-fold in the Northern Great Plains between 2001 and 2012. Although Diaporthe helianthi was assumed to be the sole causal agent in the United States, a newly described species, D. gulyae, was found to be the primary cause of Phomopsis stem canker in Australia. To determine the identity of Diaporthe spp. causing Phomopsis stem canker in the Northern Great Plains, 275 infected stems were collected between 2010 and 2012. Phylogenetic analyses of sequences of the ribosomal DNA internal transcribed spacer region, elongation factor subunit 1-α, and actin gene regions of representative isolates, in comparison with those of type specimens, confirmed two species (D. helianthi and D. gulyae) in the United States. Differences in aggressiveness between the two species were determined using the stem-wound method in the greenhouse; overall, D. helianthi and D. gulyae did not vary significantly (P≤0.05) in their aggressiveness at 10 and 14 days after inoculation. These findings indicate that both Diaporthe spp. have emerged as sunflower pathogens in the United States, and have implications on the management of this disease.

Cultivation of oyster mushroom Pleurotus ostreatus on date-palm leaves mixed with other agro-wastes in Saudi Arabia.

Promoting the use of agricultural waste is one of the newly prepared water and environment friendly agriculture strategies in the Kingdom of Saudi Arabia (KSA). The objective of this research was to study the efficiency of cultivating oyster mushroom (Pleurotus ostreatus) on date palm wastes mixed with other agricultural wastes available in KSA. Four agricultural wastes were mixed with date palm leaves at different ratios, with two supplements and three spawn rates were used. Wheat straw mixed with date palm at ratio of 25 (date palm): 75 (agro-waste) showed the best results in most of the parameters measured. Corn meal was superior over wheat bran as a supplement in all treatments. Parameter values increased with the increase of the spawn rate of P. ostreatus. Treatments with date palm leave wastes contained higher carbohydrates and fibers. No significant differences were found among the fruiting bodies produced on the different agro-wastes studied for the different proximates analyzed. Analyses of metal concentration showed that potassium was the highest in all the treatments tested followed by Na, Mg, Ca, and Zn. This is the first study that reported the success of growing oyster mushroom on date palm leaf wastes mixed with other agro-wastes obtainable in KSA.