First Report of Fusarium falciforme (FSSC 3+4) Causing Rot of Industrial Hemp (Cannabis sativa) in California

Industrial hemp (Cannabis sativa) is a newly legal crop in California that is grown for cannabidiol oil, fiber and seed. In August 2019, whole plant decline and root rot were observed affecting <5% of plants in two industrial fields in Fresno County, CA. Symptoms included chlorotic, collapsed foliage, stem vascular discoloration, and root rot with abundant mycelial growth. Stem and root segments (1-2 cm) from three to five diseased plants were agitated in 0.1% tween-20 and soaked in 70% ethanol for 30 s and 1% NaOCl for 2 min. After incubating for 5 to 7 days on 1:10 potato dextrose agar (PDA) amended with tetracycline, Fusarium selective medium (FSM), and PARP (pimaricin + ampicillin + rifampicin + pentachloronitrobenzene [PCNB] agar) medium, white to pale cream aerial mycelium emerged from tissue of all plants on PDA and FSM but not PARP. Isolates cultured on 0.1% potassium chloride agar formed heads of microconidia on long monophialides consistent with the Fusarium solani species complex (FSSC) (Leslie and Summerell 2008). To obtain pure cultures of two isolates (CS529 and CS530), a single-hyphal tip was excised and grown on PDA. DNA was extracted from actively growing mycelium (PrepMan Ultra kit). The translation elongation factor gene (EF-1α) was amplified via PCR using EF1/EF2 primers (O’Donnell et al. 1998). Sequences of the two isolates were identical and deposited under accession number MW892973 in GenBank. The 599 bp sequence was 99.33% identical to FSSC 3 + 4 (Fusarium falciforme) accessions FD_01443_EF-1a based on FUSARIUM-ID BLAST analysis. To evaluate pathogenicity, stems of hemp plants (cv. ‘Berry Blossom’; n=8 plants per isolate) were wounded by penetrating the epidermis in an area about 0.5-cm square by 1-mm deep and 8-inches above the soil line. A 0.5 cm-diameter plug of 7-day old F. falciforme-colonized PDA was placed against the wound. Inoculation sites were loosely wrapped with parafilm for 2 days. A negative control consisted of a sterile PDA plug (n=3). Treatments were arranged in a completely randomized design in a greenhouse. The experiment was conducted once, due to regulatory restrictions at campus facilities. At 61 days post-inoculation, external stem lesions were significantly larger in diameter (P < 0.05; Tukey’s HSD) in plants inoculated with CS529 (8 ± 1 mm) compared to the control (2 ± 0 mm), and larger but not significant for CS530 (6 ± 1 mm). Internal stem lesions (i.e., rot in stele) were observed in plants inoculated with CS529 (9 ± 3 mm); stem rot was very minor in plants treated with CS530 (1 ± 1 mm) and nonexistent for control plants. No other disease symptoms were observed. F. falciforme was isolated from stems of CS529- and C530-inoculated plants. Sequences of re-isolates matched 100% with accession MW892973. These results suggest that F. falciforme causes rot in hemp in California. These studies specifically confirm stem rot abilities; field observations of root rot indicate root rotting abilities, but further tests are needed for confirmation. This is the first report of F. falciforme causing disease in industrial hemp. FSSC was described as causing foot rot in hemp in Italy (Sorrentino et al. 2019), but these isolates belonged to phylogenetic species 5 (F. solani) not F. falciforme. In addition, F. falciforme was reported as causing root rot in hydroponically grown cannabis (Punja and Rodriguez 2018). These studies provide the foundation for development of management tools for hemp disease.

Detection and Identification of Novel Genes from Fungi Isolated from Wastewater

The environment in Mosul city is very rich, containing a wide variety of microorganisms which have not been recognised for a long time. Five new fungal genes were identified and registered for the first time in the gene bank. These included Fusarium falciforme 2020-06-MIK-F1 genes for 5.8S rRNA with Accession no. LC555741, Nectriaceae sp. 2020-06-MIK-F2 genes for ITS1 with Accession no. LC555742, Trichoderma asperellum MIK3 genes for 5.8S rRNA with Accession no. LC575020, Penecillum sp. MIK4 genes for 5.8S rRNA with Accession no. LC575021, and Neurospora crassa MIK5 genes for 5.8S rRNA with Accession no. LC575022. These fungal genes were isolated from wastewater of Khosr river in Mosul city/ Iraq, which has many contamination sources.

First report of Fusarium falciforme (FSSC 3+4) causing wilt disease of Phaseolus vulgaris in Mexico

Bean (Phaseolus vulgaris) is the second most important crop in Mexico after corn due to the high consumption of beans in all regions of the country. In the winter (January 2016), bean plants showing wilting, root discoloration and necrosis were observed, with an incidence of approximately 30% in different fields (<1 ha) in Tecoanapa, Guerrero State, Mexico. Symptomatic fine roots (<2 mm) were cut into 0.5 cm long pieces, washed with tap-water, surface disinfected with 1.5% NaOCl for 3 min, and rinsed with sterile distilled water. Thirty-five pieces were placed on potato dextrose agar (PDA, Difco) and incubated at 25 ℃ for seven days. Then, single-spore isolates were obtained. Colonies on PDA showed abundant white aerial mycelium and a growth rate of 4.5 mm/day, and in reverse, colonies were white/pink with a brown centre. Microconidia were cylindrical to ellipsoid, aseptate, hyaline and 7.8-(6.0)-4.7 × 2.7-(2.1)-1.6 µm. On carnation leaf agar, macroconidia were 37.8-(29.4)-23.5 × 4.1-(3.5)-2.6 µm, hyaline, falcate, with slightly curved apexes, and 3-5 septa. Chlamydospores were round, intercalary, hyaline, single or in chains (Boot 1971). A representative strain (CSAEGRO-AyDi-Ef) was analyzed by PCR and the translation elongation factor 1-alpha (tef1) gene (GenBank accession number MK945757) was sequenced using the EF-1/EF-2 primers (O’Donnell 2000). FUSARIUM-ID (Geiser et al. 2004) analysis showed 100% similarity with the Fusarium solani species complex (FSSC 3+4) strain NRRL28562. In addition, Bayesian phylogenetic analysis placed this strain in the Fusarium falciforme clade. A pathogenicity test was performed by immersing healthy plant roots (cv. Negro Jamapa) in 200 mL of a conidial suspension (50×106 conidia mL-1) for 10 min, and then transplanting the plants into pots. Control plants were immersed in sterile distilled water. Similar symptoms as those in the field were observed at 10 days after inoculation, and the controls were healthy. The fungus was reisolated from infected plants and showed the same morphology and tef1 sequence as the original isolate, fulfilling Koch’s postulates. Recently, F. falciforme was reported to cause wilting of P. vulgaris in Cuba (Duarte et al. 2019); however, this is the first report of F. falciforme (FSSC 3+4) causing wilt disease of P. vulgaris in Mexico. This species was previously reported in Mexico affecting onion (Tirado-Ramírez et al. 2018), papaya, tomato (Vega-Gutiérrez et al. 2019a, b), and maize (Douriet-Angulo et al. 2019), suggesting an ample host range in the country.