Symptoms
The infected plants were found to be stunted in growth with yellowing, browning and bronzing of leaves with marginal necrosis (Fig 1a), defoliation and smaller fruits. The infected plants were uprooted and observed for nematode symptoms. The presence of simple and compound galls in the roots indicated the nematode infestation in guava plants (Fig 1b).
Morphological and morphometric characteristics
Mature females were pear- to globular-shaped with a long, prominent neck and no posterior protuberance, distinguishing them from other root-knot nematode species (Fig 2d). The head region was continuous, comprising the labial disc and medial lips. Annulations were distinct in the posterior region of the body. The stylet conus was slightly curved and tapered distally. The morphometric characteristics of
M.
enterolobii females isolated from guava was closely associated with the original description by
Yang and Eisenback (1983), with minor variations (Table 1). The mean body length (752.6 µm) and width (610.5 µm) were slightly higher than the original (735.0 µm and 606.8 µm), whereas neck length was slightly shorter (212.3 µm vs. 218.4 µm). Stylet measurements were similar, with minimal reductions in knob height and width. The excretory pore was positioned slightly further from the head (65.1 µm vs. 62.9 µm). Inter-phasmidial and vulval lengths showed small variations, while the vulva-anus distance remained similar. The body ratio (A = 1.23) was consistent with the original (1.2) and coefficients of variation (2.45-18.75%) indicated low morphological variability. Nematode egg masses were gelatinous, irregular to oval in shape, extruded on the root surface and contained numerous oval, thin-shelled eggs. (Fig 2a and 2b).
The perineal pattern of
M.
enterolobii females obtained from guava was predominantly oval, with a moderately high dorsal arch and a rounded to squarish outline in some individuals. Coarse and fine striae were clearly visible, while the perivulval region appeared smooth, lacking striations. Striations extended onto the lateral areas near the vulval tail terminus (Fig 2e). This confirmed the identity of the species as
M.
enterolobii based on its morphological characteristics.
The male was long, translucent white and vermiform with a rounded tail. The head was slightly elevated from the body and the cephalic framework was moderately developed. The stylet was robust with a straight conus, cylindrical shaft and large, rounded knobs detached from the shaft. Spicules were arcuate with a rounded base and a short, smooth tail (Fig 2f, g). The morphometric traits of
M.
enterolobii males from guava closely resembled those described by
Yang and Eisenback (1983), with minor quantitative differences (Table 2). The mean body length (1285.6 µm) and width (35.4 µm) were slightly lower than the original description (1599.8 µm and 42.3 µm, respectively). Stylet and spicule lengths were comparable, measuring 22.3 µm and 28.6 µm against 23.4 µm and 30.4 µm in the original, while the gubernaculum length was marginally higher (7.0 µm vs. 6.2 µm). The DEGO value (5.6 µm) and excretory pore position (146.2 µm) showed slight variations, indicating natural population variability. The tail was shorter (11.2 µm vs. 12.5 µm) and the testis length was average with 315 µm. Ratios A (37.8) and C (118.5) are closely aligned with the original description (37.9 and 131.6).
Infective second-stage juveniles were transparent, slender and tapering at both ends, with a slightly offset head region (Fig 2c). The stylet was fine, with a pointed conus, broader shaft and round knobs. The procorpus was distinct, the metacorpus was elliptical and the tail was hyaline, thin and contained few fat droplets and the phasmids were indistinct. The morphometric traits of
M.
enterolobii juveniles from guava were closely matched with the original description by
Yang and Eisenback (1983), showing only slight variations (Table 3). The present population had a determined mean body length of 427.5 µm and a width of 14.6 µm, which is slightly lower than the original values. Tail length (52.4 µm) and excretory pore position (82.1 µm) were marginally reduced. Stylet dimensions (12.3 µm; knobs 1.7 x 3.0 µm) and DOGO (3.0 µm) were comparable to the reference. The body ratios A (29.5) and C (8.1) remained consistent, confirming overall morphological similarity with minor variations induced by host or environment.
Molecular confirmation
PCR amplification produced a distinct band of approximately 720 bp for ITS and 450 bp for COI (Fig 3), confirming their species identity. The sequences of the isolates was submitted to the GenBank with an Accession Number PX 667703, PX667704 and PX667705 of ITS primer and PX 658232, PX658233 and PX658234 of COI primer. BLAST analysis of the sequences showed 99-100% similarity with
M.
enterolobii reference isolates from China (MN509752), Brazil (ON341109) and India (OK678123). Phylogenetic clustering placed the guava isolate within the
M.
enterolobii clade (Fig 4), clearly separated from
M.
incognita and
M.
javanica.
Morphological identification of
M.
enterolobii is challenging due to similarities with
M.
incognita and
M.
javanica. However, the high dorsal arch and smooth perineal striae observed in this study are consistent with earlier descriptions by
Kiewnick et al., (2019) and
Ashokkumar et al., (2021). The morphometric ranges of J2 and females corroborate previous findings on
M.
enterolobii populations from tropical fruit crops. The characterization of root knot nematode species based on morphological features is time consuming and requires highly skilled technical personnel
(Blok et al., 2002). Identification of
Meloidogyne species is important to design effective management practices such as crop rotation and use of plant resistance
(Zijlstra et al., 2000). Studies on varietal susceptibility have demonstrated differential host responses to
Meloidogyne incognita under controlled conditions, highlighting host resistance as a critical component of nematode management strategies
(Ikram et al., 2025). The root knot nematode,
M.
enterolobii was morphologically identified based on infective second stage juveniles, male, female and posterior cuticular pattern.
The bodies of infective second-stage juveniles were long, clear white, vermiform, tapered at both ends and had a slender tail. The head region was continuous and a little offset from the body and the tail tip was hyaline, very thin and had a few fat droplets. The description derived from the second-stage juveniles of the collected samples was similar to the original descriptions given by
Rammah and Hirschmann (1988).
Posterior cuticular pattern of
M.
enterolobii was rounded to oval with a medium to high dorsal arch and a lengthy vulval slit. The posterior cuticular patterns of
M.
enterolobii encountered during the current study showed round to dorsoventrally ovoid, long vulval slit with medium to high dorsal arch, as observed from multiple populations of collected samples and their characters were similar to the original descriptions given by
Rammah and Hirschmann (1988) and
Yang and Eisenback (1983). However,
Brito et al., (2004) determined that the perineal patterns of
M.
enterolobii isolates in 70% of the populations studied were round to dorsoventrally ovoid, while the remaining 30% had trapezoidal dorsal arches comparable to those of
M.
incognita in Florida.
Brito et al., (2004) found that the morphometrics of
M.
mayaguensis females were identical to those reported in the original description in West Africa.
Molecular approaches for identifying
Meloidogyne species using DNA are being developed by utilizing polymerase chain reaction (PCR), which permits the investigation of a single nematode (
Akyazi and Felek, 2013). In the current study, PCR analyses were used to amplify the internal transcribed spacer using ITS and COI primers. Isolates from various guava orchards were identified as
M.
enterolobii. The sequencing results revealed 90-100% similarity with the relevant
M.
enterolobi strains. For
Meloidogyne spp. ITS regions of nematodes were site-specific and the size of the amplicon was amplified at 620 base pairs. Molecular identification using ITS and COI markers proved conclusive, aligning with global reports highlighting their diagnostic reliability
(Dong et al., 2021). The detection of
M.
enterolobii in multiple guava-growing regions indicates its widespread prevalence and potential to exacerbate the guava wilt-nematode complex. The integrative diagnostic approach employed here and provides a reliable basis for surveillance, quarantine and development of host-resistance screening programs.
