First Report of Guava Root-Knot Nematode ( <i>Meloidogyne enterolobii</i> ) Infecting Tomato ( <i>Solanum lycopersicum</i> ) in Saudi Arabia

Guava root-knot nematode (Meloidogyne enterolobii Yang & Eisenback, 1983) is a highly polyphagous Meloidogyne species that causes significant damage to several economically important crops worldwide. The expansion of M. enterolobii is increasing with recent reports emerging from the Middle East and North Africa regions (Ibrahim et al. 2023). Its strains are highly pathogenic and have the capacity to break various sources of resistance against root-knot nematodes (Vashisth et al. 2024; Yang and Eisenback 1983). Due to its relevance, M. enterolobii has been recognized as an A2 pest by the European and Mediterranean Plant Protection Organization (EPPO 2021). In September 2023, tomato plants (Solanum lycopersicum L. cv. Castlerock) with stunting and yellowing symptoms were observed in open fields in both Najran (17°32'10.2"N 44°14'27.0" E) and Jazzan (17°23'50.1"N 42°32'55.0" E) regions, Saudi Arabia. Upon uprooting of these diseased plants, galled roots were observed. Soil and galled roots were taken to the nematology lab of King Saud University for proper diagnosis. Nematodes were extracted from soil using the modified sugar flotation method (Jenkins 1964) and from infected roots using the protocol described by Hussey and Barker (1973). Nematode population densities ranged from 2,200 to 2,500 individuals per 250 g of soil, while 9,864 and 11,388 eggs and second-stage juveniles (J2s) were recovered per root system in Najran and Jazzan, respectively. Nematodes were characterized morphologically and molecularly using DNA sequence analysis of the 28S D2D3 (Subbotin et al., 2006) and the internal transcribed spacer (ITS) (Subbotin et al. 2001) regions of the ribosomal DNA. The females were pearly white, globular, and had an annulated surface. The perineal pattern was typical of M. enterolobii, with an oval shape and moderately high dorsal arch. The average size of adult females (n = 32); was (L: 610 ± 51 µm; W: 435 ± 35 µm), stylet length (15.3 ± 1.4 µm), excretory pore to anterior end and stylet length ratio (4.3 ± 0.3 µm). The J2s presented a thin, bluntly pointed tail with a clearly defined hyaline terminus. The average J2 size (n = 32) body length was (472 ± 28 µm), tail length (41.6 ± 8.3 µm), and stylet length (13.8 ± 0.4 µm). The observations of the M. enterolobii populations from Saudi Arabia were consistent with those previously described (Subbotin et al. 2021; Yang and Eisenback 1983). For molecular characterization, juveniles hatched from a single egg mass were used for DNA extraction. DNA sequences were deposited in GenBank under accession numbers PV267741 (D2D3), PV267743 (D2D3), PV605585 (ITS), and PV605584 (ITS). The D2D3 sequences showed 99.86 % identity with M. enterolobii sequences from India (OP935640) and China (MT193449), while ITS sequences showed 98.28 - 99.86 % identity to M. enterolobii sequences from China (MT028373), India (MK940246), and Mexico (OM721664). To fulfill Koch’s postulates, 2-week-old seedlings of tomato cv. Marmande were transplanted into 25 cm-diameter pots containing 1.5 kg sterilized sandy loam soil and peat moss (3:1 v/v). A total of 7 pots were used. Each pot contained one tomato seedling and was infested with 3,000 J2s and eggs. Non-inoculated plants served as controls. After 62 days, infected plants showed root galls. No symptoms were observed in the controls. Adult females from galled roots were reidentified based on the rDNA regions as M. enterolobii. To our knowledge, this is the first report of M. enterolobii in Saudi Arabia.