First Report of Charcoal Rot of <i>Lagenaria siceraria</i> Caused by <i>Macrophomina phaseolina</i> in Mexico

In February 2025, symptoms of charcoal rot were detected in approximately 40% of four-month-old Lagenaria siceraria plants at the vegetative-to-flowering developmental stages, cultivated in a 2-ha commercial field located in Culiacán, Sinaloa, Mexico (24°15'06.7"N, 107°11'39.3"W). Diseased plants additionally exhibited above-ground symptoms such as stunting, wilting, leaf yellowing, partial foliage necrosis, and defoliation, while below-ground symptoms included crown and root rot. Thirty small tissue fragments (10 × 10 mm) were excised from ten randomly selected diseased plants and surface-disinfected in a 1% sodium hypochlorite solution, followed by two rinses with sterile distilled water, each lasting one minute. After drying on sterile absorbent paper, the tissue samples were incubated at 28 °C for seven days in Petri dishes containing Potato Dextrose Agar (PDA) supplemented with streptomycin sulfate (0.3 g L⁻¹), with five tissue fragments placed per dish. Among six isolates generated, three representative isolates were selected for identification, MACROCH7CLNSIN, MACROCH10CULSIN and MACROCH13CULSIN. Microscopic examination of the three isolates revealed numerous microsclerotia of irregular shape, initially hyaline and progressively darkening with maturation. Morphometric analysis based on 10 measurements per isolate showed that microsclerotia of isolate MACROCH7CLNSIN ranged from 71.59 to 91.99 μm in width, with an average of 81.79 μm; those of MACROCH10CULSIN ranged from 58.93 to 78.48 μm, averaging 68.71 μm; whereas MACROCH13CULSIN exhibited widths between 52.19 and 75.03 μm, with a mean of 63.61 μm. These morphological are consistent with the description of Macrophomina phaseolina, the causal agent of charcoal rot (Marquez et al. 2021). PCR analysis was performed on the three isolates using the primer sets ITS1/ITS4 and EF1-728F/EF1-986R to amplify the internal transcribed spacer (ITS) region (White et al. 1990) and a portion of the EF1-α gene (Carbone and Kohn 1999), respectively. The sequences were deposited in GenBank under accession numbers PV628711, PV628712, and PV628713 for the ITS region, and PV651775, PV651776, and PV651777 for the EF-1α gene, corresponding to isolates MACROCH13CULSIN, MACROCH7CLNSIN and MACROCH10CULSIN, respectively. BLAST analyses revealed that sequences PV628711, PV628712, and PV628713 (ITS) shared 100% identity with the reference sequence KJ609175, with alignment lengths of 504/504 bp, 515/515 bp, and 507/507 bp, respectively. On the other hand, sequences PV651775, PV651776, and PV651777 (EF-1α) exhibited 98–99% identity with the reference sequence MK447854, with alignment lengths of 217/218 bp, 217/218 bp, and 214/219 bp, respectively. These results confirm the molecular identity of isolates as M. phaseolina. Phylogenetic analyses performed using the Neighbor-Joining methods with EF-1α sequences by employing a bootstrap of 1000 replications on MEGA 11 generated a well-supported clade grouped with the species M. phaseolina. To verify the pathogenicity of the fungus, 10-day-old Lagenaria siceraria plants at vegetative stage were inoculated using the toothpick method. Sterile toothpicks were previously placed in Petri dishes containing each of the three M. phaseolina isolates until full colonization was achieved. Subsequently, small wounds were made at the stem base of ten plants per isolate using the colonized toothpicks. For the control treatment, sterile (non-colonized) toothpicks were used. Pathogenicity assays were conducted in two independent trials, each including 10 replicates per M. phaseolina isolate. All plants were maintained in a growth chamber under a 12-hour photoperiod, at 70 ± 5% relative humidity and 28 ± 2 °C. After two weeks, 100% of the plants still undergoing vegetative growth stage displayed symptoms similar to those observed in the commercial fields, whereas no symptoms were observed in the control plants. Macrophomina phaseolina was successful reisolated and identified from symptomatic root tissues, fulfilling Koch’s postulates. To date, M. phaseolina has been previously reported infecting L. siceraria in India (Richardson 1990) and South Korea (Kim et al. 2021). To the best of our knowledge, this is the first report of M. phaseolina causing disease in L. siceraria in Mexico. This finding provides a foundation for future studies on charcoal rot in this host and supports the development of improved management strategies for the disease in the country.