First Report of White Mold Caused by Sclerotinia sclerotiorum on Brussels Sprouts in Mexico.

The Brussels sprout (Brassica oleracea var. gemmifera) is a cruciferous vegetable with high health-promoting value and Mexico is one of the most valuable exporters worldwide (Data Mexico 2023). From September to November 2021, white mold symptoms (Rimmer et al. 2007) were observed in Brussels sprouts (cv. Confidant) fields in Tonatico, Estado de México, Mexico. Irregular, necrotic lesions were observed on leaves, whereas abundant white mycelium, and later black sclerotia were produced outside and inside of stems. Disease incidence ranged from 20 to 40% in five fields. For fungal isolation, symptomatic stem pieces were surface sterilized with 2% sodium hypochlorite for 2 min, rinsed in sterilized distilled water twice, placed on PDA medium, and incubated at 25°C in darkness for 3 days. Sclerotinia-like colonies were consistently obtained and six isolates were purified by the hyphal-tip method. Fungal colonies were white and fluffy. Irregular, black, and small (3 to 6 mm diameter) sclerotia were produced at the edge of colonies after 5 days of incubation. The morphological characters were consistent with those of Sclerotinia sclerotiorum (Saharan and Mehta 2008). Two representative isolates were selected for molecular analysis and pathogenicity tests. The isolates were deposited in the Culture Collection of Phytopathogenic Fungi at the Colegio Superior Agropecuario del Estado de Guerrero under the accession numbers CSAEG50 and CSAEG51. For molecular identification, genomic DNA was extracted, and the internal transcribed spacer (ITS) region was amplified by PCR and sequenced using the primer pair ITS5/ITS4 (White et al. 1990). The sequences were deposited in GenBank (accession nos. OQ878510 and OQ878511). BLASTn searches in GenBank showed 100% identity with the available sequences of Sclerotinia sclerotiorum (accession nos. OQ891471, OQ891472, HQ833448, and MT177216). A phylogenetic analysis using the Maximum Likelihood method placed isolates CSAEG50 and CSAEG51 in the same clade as S. sclerotiorum. Pathogenicity tests were performed by inoculating 10 healthy Brussels sprout seedlings (cv. Confidant) grown in pots. A mycelial plug was directly placed on the stem of each plant. Five uninoculated Brussels sprout seedlings were used as control. All plants were placed in a moist chamber at 25°C with a 12-h photoperiod for 2 days. White mold symptoms appeared on inoculated plants after 3 days, whereas control plants remained symptomless. The fungi were reisolated from the infected plants and found to be morphologically identical to the isolates used for inoculation, fulfilling Koch's postulates. Pathogenicity test was performed twice with similar results. Sclerotinia sclerotiorum has been previously reported to infect Brussels sprouts in the USA (Campbell 1947). To our knowledge, this is the first report of Sclerotinia sclerotiorum causing white mold of Brussels sprouts in Mexico. The disease is widely distributed in Brussels sprouts fields in the central region of Mexico, therefore additional studies are needed to develop effective disease-management strategies.

First Report of Colletotrichum jiangxiense Causing Avocado Anthracnose in Mexico.

Mexico is the largest avocado (Persea americana) producer and exporter in the world. In January of 2019, typical symptoms of fruit anthracnose were observed on approximately 90% of avocado trees in backyards localized in Leonardo Bravo municipality in Guerrero, Mexico. Lesions on avocado fruits were circular, necrotic, and sunken, whereas the mesocarp showed a soft rot with dark brown discoloration. To perform fungal isolation, small pieces from adjacent tissue to lesions of five symptomatic fruits were surface disinfested by immersion in a 1% sodium hypochlorite solution for 2 min, rinsed in sterile distilled water, and placed in Petri dish containing potato dextrose agar (PDA). Plates were incubated at 25 ºC for 5 days in darkness. Colletotrichum-like colonies were consistently isolated and seven monoconidial isolates were obtained. An isolate was selected as a representative for morphological characterization, molecular analysis, and pathogenicity tests. The isolate was deposited in the Culture Collection of Phytopathogenic Fungi at the Colegio Superior Agropecuario del Estado de Guerrero (Accession No. CSAEG-CJ19). After 8 days on PDA, the colonies were gray on the upper surface, and with orange conidial masses. Conidia (n= 100) were cylindrical, hyaline, aseptate, with rounded ends, 14.4 to 18.5 × 4.5 to 6.2 µm. Based on morphological features, the isolate was tentatively identified in the C. gloeosporioides species complex (Weir et al. 2012). For molecular identification, genomic DNA was extracted and the internal transcribed spacer (ITS) region of rDNA, and partial sequences of actin (ACT), ß-tubulin (TUB2), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified by PCR, and sequenced with primers ITS5/ITS4 (White et al. 1990), ACT-512F/ACT-783R (Carbone and Kohn 1999), Bt2A/Bt2B (Glass and Donaldson 1995), and GDF/GDR (Templeton et al. 1992), respectively. BLAST analysis of the obtained sequences of the ITS, ACT, TUB2, and GAPDH genes revealed 100%, 99.63%, 99.77% and 100% identity with those of isolate LF687 of C. jiangxiense in GenBank (Accession numbers KJ955201, KJ954471, KJ955348, and KJ954902). A phylogenetic tree based on Bayesian inference and including published ITS, ACT, TUB2, and GAPDH data for Colletotrichum species was constructed. The multilocus phylogenetic analysis clearly distinguished the isolate CSAEG-CJ19 as C. jiangxiense separating it from all other species within the C. gloeosporioides species complex. The sequences were deposited in GenBank (accession numbers ITS:MT011397; ACT:MN968784, TUB2:MN968786, and GAPDH:MN968785). To conduct Koch's postulates, 20 healthy avocado fruits (cv. Hass) were wounded with a sterile toothpick (2 mm in depth) and a drop of 15 µl of conidial suspension (1 × 105 spores/mL) was placed on each wound. Ten control fruit were wounded and treated with sterilized water. All the fruits were kept in a moist plastic chamber at 25°C for 8 days. All inoculated fruits developed circular and necrotic lesions (12 to 18 mm in diameter), 5 days after inoculation, whereas control fruits remained healthy. The fungus was consistently re-isolated from the inoculated fruits. Previously, C. jiangxiense has been reported as a pathogen on Camellia sinensis and Citrus sinensis in China (Farr and Rossman 2020). To our knowledge, this is the first report of C. jiangxiense causing anthracnose on avocado worldwide. This study shown another species in the C. gloeosporioides complex associated with avocado diseases in Mexico. Therefore, it is necessary to explore the diversity of Colletotrichum species in detail through subsequent phylogenetic studies as well as to monitor the distribution of this pathogen into other Mexican regions.

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.