First report of root-knot nematode Meloidogyne javanica infecting Cannabis sativa in Brazil

Cannabis sativa L. has gained significant attention globally, driven by the increasing recognition of its multiple therapeutic properties. However, the crop is susceptible to a wide range of pathogens, including root-knot nematodes, which can significantly damage Cannabis spp. (Song et al., 2017; Brito et al., 2023). In July 2024, few plants of C. sativa cv. ‘Easy sativa’ in a symptomatic spot were found in a field from Caçapava do Sul, RS, Brazil (30°30’43” S, 53°29’2” W). The plants exhibited foliar chlorosis and intense root galling, indicating the presence of Meloidogyne sp. Roots and soil from five symptomatic plants were sampled and pooled to form a single composite sample. The average extracted population was 1,000 second-stage juveniles (J2s) and eggs per gram of root. Morphological identification of the nematode was carried out using females from galls (n=10), J2s (n=20) and adult males (n=10) from soil and roots. The perineal patterns of females exhibited thick striae smooth to slightly wavy, along with a low dorsal arch featuring two lines in the lateral field, corroborating with the original description of Meloidogyne javanica (Chitwood, 1949). The J2s had the following morphometric characters: body length = 445.7 (402.6 – 503.4) μm, stylet = 14.7 (14.1 – 15.8) μm, dorsal esophageal gland orifice = 3.9 (3.1 – 4.4.) μm, tail length = 57.3 (52.6 – 60.5) μm, hyaline tail terminus = 13.8 (10.5 – 16.1) μm. The adult males had a labial region unseparated from the body, characterized by 2 or 3 incomplete rings and the stylet cone had the shape of a straight cone with a pointed tip, cylindrical shaft and the low, wide and separated bulbs, as described by Eisenback and Hirschmann (1981). The esterase phenotype was verified by the isoenzyme electrophoresis method (Esbenshade and Triantaphyllou, 1985; Ito et al., 2019). One young female was collected from the roots and placed into a microtube. Six tubes were prepared: four contained one female from C. sativa roots, and two contained ten reference standard specimens of M. javanica. The esterase phenotyping revealed the presence of M. javanica in all C. sativa tubes (Est: JV, Rm: 1.0, 1.12, 1.20). Polymerase chain reaction (PCR) was performed using DNA extracted individually from six females isolated from C. sativa roots (Castagnone-Sereno et al., 1991). Species-specific primers Fjav (5’-GGTGCGCGATTGAACTGAGC-3’) and Rjav (5’-CAGGCCCTTCAGTGGAACTATAC-3’), developed from RAPD fragments characteristic of M. javanica (SCAR), was used for amplification (Zijlstra et al., 2000). An expected fragment of approximately 670 bp was obtained, identical to M. javanica. The pathogenicity test was performed under controlled conditions (25°C, 12-hour photoperiod) in C. sativa cv. ‘Easy Sativa’. Thirty days after germination, roots of four plants were individually inoculated with 800 J2 extracted from the sampled plants. Four uninoculated plants served as controls. After 60 days, root galling and foliar chlorosis were observed in all inoculated plants, identical to field observations. The average reproductive factor (final population/initial population) in these plants was 5.21. Control plants showed no symptoms or signs. Consequently, this work confirmed M. javanica, making this, to our knowledge, the first report of the nematode infecting C. sativa in Brazil. This nematode represents a critical risk for the emerging C. sativa production across the country and grower instructions are necessary to prevent further spread.