Further observations on Meloidogyne enterolobii (Nematoda: Meloidogynidae) infecting guava (Psidium guajava) in India

The infestation of root-knot nematodes is rapidly expanding in guava growing areas in India; and more than one RKN species have been found to be severely damaging and resulting in decline of orchard with slow-wilting symptom of plants in the presence of other pathogens. A severe infestation of root-knot nematode in guava was reported in the Tamil Nadu state of India (Anonymous, 2016). The distribution of nematode infested planting materials of guava has possibly enabled dissemination and spread of the dreaded nematode species across the country.

Severe decline of guava with outright crop failure due to the infestation of M. enterolobii in commercial guava orchards has been experienced in Brazil (Gomes et al., 2012). Since then, the nematode species is widely known as guava root-knot nematode. The occurrence of M. enterolobii in guava has also been reported from other Asian countries like China (Hao et al., 2005) and Vietnam (Iwahori, 2009); but how the nematode species got introduced and established in India still remains elusive. Besides, decline of guava yield and quality has been reported in several parts of Malaysia to be caused by M. incognita (Razak and Lim, 1987). Khan et al. (2007) also reported the presence of M. incognita and M. javanica in the rhizosphere soil of guava in West Bengal state of India, but they were not found to be economically damaging for the crop. While investigating the association of the nematodes in the infested guava plants (Fig. 1A), we found massive deformation (root galling) in the root system (Fig. 1B). Based on the morphometric illustrations of the present population and their simultaneous comparison with earlier descriptions (Yang and Eisenback, 1983; Rammah and Hirschmann, 1988; Karssen et al., 2012) the specific status of M. enterolobii was confirmed. We measured the important taxonomic characters of the female anterior end and posterior cuticular patterns and found an overlap of the ranges of all morphometric variables in the Indian population with the reported morphometrics of M. enetrolobii. However, mean stylet length (13.4 µm) is slightly shorter than that of previous observations (14.7-15.4 µm). Measurements of vulva-slit (24.8 µm), inter-phasmidal distance (24.6 µm), vulva-anus (20.2 µm) in the perineal pattern are relatively less as compared to the published reports (Table 1). Similarly, in male, distance from the head end to secretor-excretory (SE) pore (130.4 µm) and spicule length (25.2 µm) is relatively less (Table 2). Morphometrics of second-stage juveniles (J2s) are almost similar to the previous descriptions except for the DGO position (2.8 µm), which is slightly closer to the stylet base in the Indian population (Table 3). Morphology of perineal pattern is typical for the species; oval shaped dorsal arch moderately high to high squarish and rounded, with smooth and coarse striae, dorsal and ventral arch forming a weak lateral line, ventral arch broad with smooth striae. However, perineal patterns (Fig. 2A-F) from Indian population of M. enterolobii are slightly different (consistently high squarish dorsal arch) from that of Yang and Eisenback (1983), Rammah and Hirschmann (1988), and Karssen et al. (2012). Female (Fig. 3A: anterior end), male (Fig. 3B-D), and J2 (Fig. 3E-H) are also similar for the species. The tail of J2s has a characteristic constriction that could be useful to distinguish it from that of M. incognita. The perineal patterns of M. enterolobii populations from India are slightly similar to M. incognita. Therefore, morphological separation of two closely related species based on the study of few perineal patterns is rather difficult. The difficulty of accurate identification of root-knot nematode species is often compounded by the occurrence of mixture of several species infecting guava. In the present study, we used isozyme and species-specific SCAR markers to confirm the morphological identification.

Figure 1:

Figure 2:

Figure 3:

Biochemically, the present RKN population showed species-specific esterase phenotype of M. enterolobii (Xu et al., 2004; Paes et al., 2012) (Fig. 4A). Taxonomic identification of M. enterolobii has proved to be very challenging based on only the traditional means and has resulted in incorrect identification and misreporting (Brito et al., 2004). Molecular characterization based on ITS rDNA sequences (NCBI GenBank accession number KT271569) and SCAR marker resulted in authentication of the species (Fig. 4B). In this line, use of molecular markers, viz., ribosomal D2D3 expansion segment, ITS rDNA, IGS rDNA, mitochondrial COI, viable region between COII and 16S rDNA, mitochondrial NADH5 and SCAR marker has been found to be more robust for specific confirmation of the species (Tigano et al., 2010; Onkendi and Moleleki, 2013; Rashidifard et al., 2018); and SCAR marker proved best in the situation of occurrence of mixed populations consisting of M. enterolobii, M. incognita, M. javanica, and M. hapla Chitwood, 1949 (Rashidifard et al., 2018).

Figure 4:

In conclusion, here we report detailed morphological and morphometric observations of the Indian M. enterolobii population infecting guava. While most of the observations are identical with the earlier descriptions of Yang and Eisenback (1983), Rammah and Hirschmann (1988), and Karssen et al. (2012), some minor variations were observed that could be attributed to differences in hosts, geographical isolates, methodology of specimen processing, artefacts or any microscopic observational bias. The description and illustration in the name of M. enterolobii previously reported from India gave rise to the confusion regarding its identification. This study is of cardinal importance for resolving the contention that all RKN infections in guava are M. enterolobii. This information would be useful for phenotypic comparisons of various M. enterolobii populations from across the world, and might help with the population genetics as well as comparative virulence studies.