In Vitro Assay Using Proboscidea parviflora W. and Phaseolus lunatus L. Plant Extracts to Control Pythium amazonianum.

Avocado tree wilt is a disease caused by Phytophthora cinnamomi Rands. Recently, this disease has been associated to Pythium amazonianum, another causal agent. Avocado tree wilt is being currently controlled with synthetic fungicides that kill beneficial microorganisms, polluting the environment and leading to resistance problems in plant pathogens. The current research work aims to provide alternative management using extracts from Proboscidea parviflora W. and Phaseolus lunatus L. to control the development of mycelia in P. amazonianum in vitro. Raw extracts were prepared at UAAAN Toxicology Laboratory, determining the inhibition percentages, inhibition concentrations and inhibition lethal times. Several concentrations of the plant extracts were evaluated using the poisoned medium methodology, showing that both extracts control and inhibit mycelial development, in particular P. lutatus, which inhibits mycelial growth at concentrations lower than 80 mg/L, being lower than P. parviflora extracts. These extracts are promising candidates for excellent control of Pythium amazonianum.

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.

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.