First report of Xanthomonas campestris pv. campestris as the causal agent of necrotic leaf spot in Phaseolus vulgaris at Puebla, Mexico.

Beans are the most cultivated legume in the world. In Mexico, it is the second most important crop after corn (FAO 2020; SIAP 2020). Bean plants "Flor de Mayo M38" variety were affected by a foliar disease during the agricultural cycle 2019 in Puebla-Mexico (19°02'46.6" LN and 98°05'15.6" LO). Necrotic V- shaped lesions were observed on the margins of the leaves surrounded by yellow halos followed by foliar necrosis, affecting 40% of the crop. In Mexico this variety of cultivars is in great demand for local consumption and generates income in foreign currency (Castellanos et al. 1997). Sampling was carried out on 50 plants "Flor de Mayo M38" variety, with necrotic leaf symptoms from ten plots of one hectare. Samples were cut into pieces (5 mm), disinfested with 1% hypochlorite 3 min, and washed with sterile distilled water. Subsequently, samples were dried on sterile paper and placed on Petri plates containing yeast extract calcium carbonate dextrose agar (YDC) medium and kept at 36°C for 3 days. Colonies of ten typical bacteria isolated from all symptomatic plants were Gram (-), small and uniform in size with rounded edges, yellow, convex with entire borders and mucoid appearance on YDC. Bacteria did not grow on 0.1% triphenyl tetrazolium chloride amended casamino acid, peptone, and glucose medium (CPG). Biochemical tests showed that isolates did not reduce nitrate to nitrites, had positive catalase and starch hydrolysis, while the Kovac oxidase test was negative (Schaad and White 1974). Genus identity of the representative isolate Xcf1-APJR, was confirmed by 16S rRNA encoding gene partial sequencing, using universal primers 518F (5'-CCAGCAGCCGCGGTAATACG-3') and 800R (5'-TACCAGGGTATCTAATCC-3') (Halim et al. 2020). BLASTn alignments against the nucleotide collection were 100% identical to Xanthomonas sequences including Xanthomonas campestris pv. campestris strains NZ_AP019684.1, CP025750.1, and MN108237.1. The 1,418 bp sequence was deposited in the GenBank database under accession number MT645246. The identification of species/pathovar was accomplished by serological methods using a polyclonal antiserum specific for X. campestris pv. campestris (Popovic ́ et al. 2013) with the DAS-ELISA commercial kit (catalog number 07122C/096, LOEWE Biochemica GmbH, Germany). The pathogenicity test was carried out on 50 healthy bean plants from the "Flor de Mayo M38" variety. Bacterial culture incubated at 28°C for 48 h in YDC medium was used to prepare the bacterial suspension (108 CFU mL-1). The first two lower leaves of 30-day-old plants were inoculated by sprinkling. Ten plants sprayed with sterile distilled water were used as negative control. All plants were kept for 20 days in greenhouse at 18-26°C and relative humidity of 60%. After seven days, chlorotic lesions developed on all inoculated plants that became necrotic from 14 days after inoculation (dai). Necrotic leaf spots merged at 14 dai to form necrotic areas of more than 20 mm in diameter, reaching total necrosis of the leaf tissue at 20 dai and were similar to the symptoms observed in the field. Koch's postulates were confirmed by the reisolation of Xcf1-APJR strain, which presented the same colony morphology, partial sequence, and polyclonal specific detection. This is the first report of this pathogen causing necrotic leaf spot in beans from the "Flor de Mayo M38" variety in Puebla-Mexico. The author(s) declare no conflict of interest. References: FAO. 2020. FAOSTAT. Food and Agriculture Data. http://www.fao.org/faostat/en/#home/. SIAP. 2020. Atlas Agroalimentario. https://www.gob.mx/siap/. Castellanos, J. Z., et al. 1997. Arch. Latinoam. Nutr. 47:163. Schaad, N. W., and White, W. C. 1974. Phytopathology. 64:876. https://doi.org/10.1094/Phyto-64-876 Halim, R. A., et al. 2020. HAYATI J. Biosciences. 27:215. https://doi.org/10.4308/hjb.27.3.215 Popovic ́, T., et al. 2013. Plant Dis. 97:418. https://doi.org/10.1094/PDIS-05-12-0506-PDN.