First report of Clonostachys rosea causing root rot on garlic in Mexico

Garlic (Allium sativum) is an important crop worldwide and it is widely grown and used in different industries to manufacture food, pharmaceutical, and insecticidal products. (Shang et al., 2019, Velsankar et al., 2020). According to what was reported by SIAP in 2020, more than 87 ha of the crop were lost in Mexico due to various problems, including the diseases that attack this crop such as basal rot, white rot and root rot, among others. During the 2019 fall/winter season, garlic plants of Perla and Piedra Blanca cultivars were collected from Aguascalientes and Zacatecas states in San Antonio Tepezala, Rincon de Romos, and Calera municipalities. The commercial fields encompassed 10 ha with 20% disease incidence and 35% severity, approximately. The sampling focused on diseased plants with symptoms of root rot, foliar wilt, stunting, and small bulbs. The roots of 25 plants were cleaned, and portions of the diseased tissue were cut and disinfected in sodium hypochlorite at 1% for three minutes. They were rinsed twice with sterile water and dried with paper towels. The plant tissue was plated onto potato dextrose agar (PDA) and incubated at 25°C in the dark for 72 hours. Pure cultures were obtained after observing mycelial growth using monosporal culture. We obtained 16 isolates including three identified as Fusarium oxysporum, one as Fusarium solani and another 12 as Clonostachys rosea. The latter isolates were white at the beginning before turning yellow. The mycelia had a felt-like cotton texture. The conidia formed verticillate and penicillate conidiophores. The primary conidia were abundant, hyaline, smooth, and sub-globous. They were 5.1-7.7 X 8.3-8.9 µm (n=50) long and 2.0-2.9 X 3.2-3.5 µm wide (n=50). The conidiophore stipe length ranged from 70 to 180 µm, and the base width was 3.3-5.4 µm. Secondary conidiophores were penicillate and stiped with a length of 58 to 106 µm; the base measured 3.3-6.1µm. The secondary conidia measured 4.1-5 X 5.3-5.6 µm long and 2-2.3 X 2.6-2.9 µm wide (n=50) (Sun et al., 2020). The identity of six isolates was molecularly confirmed by DNA extraction and PCR reactions using ITS1/ITS4 primers and gene TEF 1α EF1-728F/TEF 1α EF1-986R. The resulting products were sequenced and compared with the National Center for Biotechnology Information (NCBI) database using BLAST. The results showed Clonostachys rosea at 99.56 and 100% with access numbers MN548399 and KX185000. The sequences were deposited at Genbank database under access number OK263088 and OL700031. Pathogenicity tests were carried out with the following procedure. A conidial suspension of five isolates (5×105 conidia/ml) in sterilized water was prepared from 1-week-old colonies. The garlic cloves were planted after being disinfected with sodium hypochlorite at 1% in sterilized soil. When the healthy garlic plants were 30 days old, we inoculated a spore suspension in soil through irrigation, to 10 plants. Likewise,10 control plants were inoculated with sterile distilled water. After 25 days, the plants were wilted and had dry leaves; their root system showed light-brown lesions and rot. These plants were stunted versus the control healthy plants. The inoculated strain was recovered and was morphologically and molecularly identified as C. rosea, thus confirming its pathogenicity towards garlic. There are reports of C. rosea causing root rot to Fabaceae crops such as Glycine max L. and Vicia faba L., (Bienapfl et al., 2012; Afshari and Hemmati, 2017) in addition to affecting orchid crops (Gastrodia elata) in Korea (Lee et al., 2020). This is the first report of C. rosea causing root rot on garlic (Allium Sativum) in Mexico, thus presenting a potential risk to this crop.