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.