First report of Fusarium clavum causing garlic bulb rot in Mexico

In Mexico, the main states for garlic (Allium sativum L.) production are Zacatecas, Guanajuato, and Puebla. In the 2020 crop season, garlic cultivation encompassed 6,794 ha, yielding 85,505 tons (SIAP, 2021). In February 2020, 35 garlic samples showing basal rot symptoms were collected from the garlic-growing regions in the states of Zacatecas and Aguascalientes in the municipalities of San Antonio Tepezala 22°13'13.5''N, 102°15'55.3''W, Rincón de Romos 22°17'44.9''N, 102°13'06.8''W, and Calera 22°58'39.4''N and 102°41'29.9W, respectively. The sampling carried out was random sampling by conglomerates, dividing each field in groups with plants that showed similar symptoms. The infected plants were stunted in growth, with reddish dying leaves. The stalks and bulbs were soft, and their root system was poorly developed. The collected samples were placed in polyethylene bags and taken to laboratory. The roots and bulbs of 35 plants were cleaned, portions of the diseased tissue was cut into 0.5 cm pieces and disinfected in 1% sodium hypochlorite for 3 minutes. The samples were rinsed twice with sterile distilled water and dried on sterile paper towels. The tissues were cultured on Potato Dextrose Agar (PDA) medium and incubated in the dark at 25°C. Seven days after incubation, pure cultures were obtained using monoconidial cultures technique on Spezieller Nährstoffmmarmer agar (SNA) and subcultured on carnation leaf agar (CLA). Ten isolates were obtained that grew slowly, showing a white coloration, then turning yellow with abundant aerial mycelia. Microscopic traits of 30 characterized spores included slender macroconidia that were curved dorsiventrally, tapering towards both ends, with five to seven thin septa, measuring 36.4-56.6 µm × 4.0-4.9 µm in size and chlamydospores that were abundant, globose to oval, subhyaline and terminal or intercalary in chains measuring 8.8-4.5 µm in diameter. Microconidia, were single-celled, hyaline, nonseptate, and ovoid. The morphological traits matched the description of Fusarium clavum (Xia et al. 2019). To confirm the strain's identity, DNA was extracted from six monoconidial cultures and used as template to amplify translation elongation factor (TEF) gene 1α, RNA polymerase largest subunit (RPB1), and RNA polymerase second largest subunit (RPB2) (O'Donnell et al. 2010). The products were sequenced and deposited in GenBank as ON209360, OM640008 and OM640009, the homology analysis using BLASTn was similar to F. clavum with 99.46%, 99.49% and 98.82% respectively with E VALUE 0.0 in all cases with access numbers OP48709, HM347171 and OP486686. Koch postulate was performed to confirm the pathogenicity of the six isolates. Variegated garlic cloves were planted after being disinfected with sodium hypochlorite at 3% w/v in 2-kg pots under the greenhouse conditions. When the garlic plants developed 4 or 5 true leaves, their basal stalks were inoculated by pouring uniformly with 1 mL of a spore suspension at 108 conidia/mL prepared from 1-week-old colonies (Lai et al. 2020). Twenty-four plants were inoculated with six isolates (four plants per isolate), and four control plants were treated with sterile distilled water. Symptoms appeared 20 days post-inoculation. The leaves were reddish, and the stalks were soft. The leaves eventually developed foliar dieback disease symptoms, their root system showed brown lesions and rot, and all water-inoculated controls remained asymptomatic. Isolations were made on the diseased plants, and the inoculated pathogen was recovered and confirmed morphologically and molecularly by DNA extraction and PCR reactions. Koch's postulate was repeated twice, obtaining the same results. To our best knowledge, this is the first report of F. clavum infecting Allium sativum L. in Mexico. bulb rot caused by F. clavum is a severe threat to garlic cultivation, and identification of this pathogen is important for effective disease management and control.