In January 2025, symptoms of charcoal rot were observed in approximately 40% of Lagenaria siceraria cv. Opo Squash plants at the vegetative-to-flowering developmental stages, about three months after seedling transplanting, in a 5-ha commercial field located in Culiacán, Sinaloa, Mexico (24°15'42.2"N, 107°12'02.2"W). Affected plants also showed stunting, wilting, chlorosis, necrosis, and defoliation of aerial parts, along with crown and root rot below ground. Twenty tissue fragments (1 mm2) were collected from ten randomly selected symptomatic plants and disinfected with a 1% sodium hypochlorite solution, followed by two one-minute rinses with sterile distilled water. The samples were dried on sterile absorbent paper and incubated at 28 °C for seven days on Potato Dextrose Agar (PDA) plates supplemented with streptomycin sulfate (0.3 g L⁻¹), with five fragments placed in each Petri dish. Of the five isolates obtained, MACROCH12CLNSIN and MACROCH9CULSIN were selected for subsequent identification. Microscopic examination of the two isolates revealed numerous microsclerotia of irregular shape, initially hyaline and progressively darkening as they mature. Morphometric analysis based on 10 measurements per isolate revealed that microsclerotia of MACROCH12CLNSIN ranged from 73.12 to 90.84 µm in width, with an average of 82.46 µm, whereas those of MACROCH9CULSIN ranged from 60.22 to 77.65 µm, with a mean width of 68.94 µm. These morphological features are consistent with the description of Macrophomina euphorbiicola, the causal agent of charcoal rot, as reported by Sanabria-Velázquez et al. (2023). Molecular characterization of both isolates was performed by PCR using the primer sets ITS1/ITS4 and EF1-728F/EF1-986R to amplify the internal transcribed spacer (ITS) region and a portion of the EF1-α gene, following the protocols described by White et al. (1990) and Carbone and Kohn (1999), respectively. GenBank accession numbers PV628714.1 and PX380965.1 correspond to the ITS sequences of isolates MACROCH12CLNSIN and MACROCH9CULSIN, respectively, while PX395931.1 and PV651778.1 correspond to their EF‑1α sequences. BLAST analysis showed that ITS sequences PV628714.1 and PX380965.1 shared 99.4% (498/501 bp) and 99.8% (508/509 bp) identity, respectively, with the previously reported M. euphorbiicola isolate OM956153.1. Similarly, EF‑1α sequences PX395931.1 and PV651778.1 showed 99.6% identity (270/271 bp and 272/273 bp, respectively) with the reference sequence KU058898.1. On the other hand, phylogenetic analyses using the Neighbor-Joining method with EF-1α sequences, employing a bootstrap of 1000 replications on MEGA 11, generated a well-supported clade grouped with the species M. euphorbiicola. These results confirm the molecular identity of the isolates as M. euphorbiicola. To confirm pathogenicity, ten-day-old L. siceraria cv. Opo Squash seedlings were inoculated using the toothpick method. Sterile wooden toothpicks colonized by each M. euphorbiicola isolate on PDA until fully overgrown were used to create small wounds at the stem base of ten plants per isolate. Controls received sterile, non‑colonized toothpicks. Pathogenicity assays were conducted three times; in each assay, plants were maintained in a growth chamber under a 12‑h photoperiod, 70 ± 5% relative humidity, and 28 ± 2 °C. Two weeks after inoculation, all inoculated plants developed symptoms identical to those observed in the commercial field, while control plants remained symptomless. The fungus was re‑isolated from symptomatic root tissue and identified as M. euphorbiicola, thereby fulfilling Koch’s postulates. To date, M. euphorbiicola has been documented on a limited number of hosts, including Ricinus communis and Jatropha gossypiifolia in Brazil (Machado et al. 2019) and Stevia rebaudiana in Paraguay (Sanabria‑Velázquez et al. 2023). To the best of our knowledge, this study, conducted in Mexico, is the first worldwide report of M. euphorbiicola causing disease on L. siceraria. This finding expands the known host range and geographic distribution of M. euphorbiicola and will support further research into disease management strategies for charcoal rot in L. siceraria.
Castro-Diego et al. (Mon,) studied this question.