First report of charcoal rot caused by Macrophomina phaseolina on dry beans in New York, United States.

Charcoal rot, a disease caused by the soilborne fungus Macrophomina phaseolina, affects a broad range of host plants globally. The disease has been reported on soybean in New York (NY) (Cummings et al. 2013), however, there have been no previous reports of M. phaseolina infecting dry bean (Phaseolus vulgaris) in NY. In August 2024, dry bean roots were collected from a research plot at Musgrave Research Farm in Aurora, NY (42.73422° N, 76.65898° W) to identify potential root rot pathogens. Affected roots showed characteristic symptoms of charcoal rot, including dark lesions on the lower stem and root tissue and a dry rot of the cortex. Signs of fungal growth were also present on the diseased roots including numerous black microsclerotia. Symptomatic root tissues were surface sterilized (10% sodium hypochlorite for one minute), rinsed three times with sterile distilled water and placed on Petri plates with 2% water agar with ampicillin (1 mg/l), then incubated at 18°C. After three to four days, hyphal tips were transferred to potato dextrose agar (PDA) and incubated at room temperature in the dark for 10 days. Colonies were initially hyaline, later developing a gray to black pigmentation. Microsclerotia were abundant, globose to oblong, consistent with morphological descriptions of M. phaseolina (Smith and Wyllie, 1999). Three isolates (2024_34, 2024_38, and 2024_40) were selected for further characterization. The internal transcribed spacer (ITS) region was amplified using primers ITS1/ITS4 (White et al. 1990), and calmodulin (CAL) and translation elongation factor (TEF1-α) genes were amplified using M. phaseolina-specific primers MpCalF/MpCalR and MpTefF/MpTefR, respectively (Santos et al. 2020). BLASTn analysis revealed 100% identity with reference M. phaseolina sequences in the NCBI GenBank database. The sequences of the three isolates were deposited in GenBank under the following accession numbers: Isolate 2024_34 - PV605619 (ITS), PV626632 (CAL), PV613809 (TEF1-α); Isolate 2024_38 - PV605620 (ITS), PV640550 (CAL), PV613810 (TEF1-α); Isolate 2024_40 - PV605624 (ITS), PV640551 (CAL), PV613811 (TEF1-α). A maximum likelihood tree based on concatenated ITS, CAL, and TEF1-α sequences constructed using RAxML v.8.2.11 with the GTR + gamma substitution model (Stamatakis, 2014) placed the three isolates within the M. phaseolina clade. Pathogenicity tests were conducted using dry bean cv. 'Zoro'. Four seeds per 15-20 cm pot were planted, with five replicates for each treatment: inoculated and non-inoculated control. Inoculum consisted of 10 g of a macerated, 12-day-old M. phaseolina culture grown on PDA, applied using an inoculum layering technique. The non-inoculated control plots received the same amount of sterile and noncolonized PDA. Plants were maintained in a greenhouse at 28/17°C (day/night), with a 13-h light/11-h dark cycle and 70% relative humidity. Inoculated plants showed symptoms (root browning, dry rot) within five to six weeks post-inoculation, while non-inoculated controls remained asymptomatic. The pathogen was reisolated and confirmed as M. phaseolina based on morphology, completing Koch's postulates. Pathogenicity tests were repeated twice for all three isolates. This report confirms the occurrence of charcoal rot caused by M. phaseolina on dry bean in NY and highlights the need for surveillance and integrated disease management to mitigate its potential impact in the northeastern region of the United States.