The guava root-knot nematode Meloidogyne enterolobii was first recognised as a species in 1983, from roots of pacara earpod tree, Enterolobium contortisiliquum (Vell.) Morong, on Hainan Island in China (Yang and Eisenback 1983). Previously, morphologically similar root knot nematodes were classified as Meloidogyne incognita. Meloidogyne enterolobii is a highly aggressive parasite of a wide range of economically important crops such as sweet potato, potato, peppers, tomato, cucumber, pumpkin, and guava. Early detection of M. enterolobii is essential for managing the disease, as crops often show symptoms only when the nematode is well-established, and roots are already significantly damaged (Fig. 1). Meloidogyne enterolobii is distinguished from other described species of the genus by the form of perineal pattern, stylet morphology, and position of the excretory pore in the female; morphology of the head in the male; and morphology of the head and tail in the second-stage juvenile (Yang and Eisenback 1983).

Fig. 1
figure 1

Plant roots showing significant galling due to infection with Meloidogyne enterolobii. a cucumber Cucumis sativus; b butterfly pea Centrosema molle. Scale bars: a 2 cm; b 4 cm

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The first confirmed case of M. enterolobii in Australia was made from a chilli plant grown in a suburban garden in Darwin, Northern Territory. The plant, which displayed typical root knot nematode galling symptoms, was submitted for diagnosis to the Northern Territory Government Plant Health Laboratory in October 2021, (sample number 36773; see Table 1). After the initial detection described above, samples from additional plants displaying significant root-galling symptoms (Fig. 1) were obtained and material was sent to the CSIRO Australian National Insect in Canberra, Australian Capital Territory, for secondary confirmation. Roots were examined independently in both laboratories and sedentary female nematodes, second stage juveniles, and males were extracted from roots/galls under dissecting microscopes (Fig. 2). Genomic DNA was extracted from individual adult females extracted from plant roots using Qiagen DNeasy Blood and Tissue kits, following the manufacturer’s instructions.

Table 1 Collection data for M. enterolobii from the Northern Territory, Australia
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Fig. 2
figure 2

Light microphotographs of various life cycle stages of Meloidogyne enterolobii collected from infested plants from the Northern Territory between 2021–2023. a J2 anterior; b J2 tail; c male anterior; d male tail; e–g female perineal patterns; h females isolated from roots of eggplant Solanum melongena; i female excised from roots of pepper Capsicum annuum. J2s and males were isolated from the same eggplant roots as the females shown in (h). Scale bars: a-d = 20 µm; e–g = 10 µm; h, i = 500 µm

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A multi-gene approach was taken to amplify extracted DNA: the COI gene region using JB3/JB5 primers (Bowles et al. 1992); COII region using C2F3/ #1108 primers (Powers and Harris 1993); M. enterolobii species specific IGS2 primers MeF/MeR (Long et al. 2006); the D2-D3 region of the 28S rRNA using the primers D2A/D3B (Nunn 1992) and a SCAR species-specific primer set MK7-F/MK7-R (Tigano et al. 2005). Cycling conditions for PCR followed those from the original source of the respective primer sets. Amplicons were visualised on 1% m/v agarose gels supplemented with 0.01% v/v SYBR™ Safe (Invitrogen). Amplified PCR products of the COI, COII, and 28S genes were Sanger sequenced bi-directionally at the Australian Genome Research Facility, Brisbane and the Biomolecular Resource Facility, Australian National University, Canberra. Raw sequences were analysed and consensus sequences were obtained using the Geneious bioinformatics platform (http://www.geneious.com). Species identification was confirmed by matching gene sequences from the current study with those of M. enterolobii publicly available on GenBank using the megablast mode of the BLASTN database from the National Centre for Biotechnology Information (https://www.ncbi.nlm.nih.gov/).

Amplification using the IGS2 and/or SCAR species specific primers sets was successful for eleven samples from eleven separate plants collected between 2021–2023, indicating the root knot nematodes found represented M. enterolobii (Table 1). An additional sample (38452) was collected in 2023 and nematodes were confirmed as M. enterolobii using sanger sequencing and morphological analyses. BLAST results of the COI, COII and 28S sequences indicated 100% similarity with sequences of M. enterolobii previously deposited on GenBank. The newly generated COI sequences matched over 60 sequences on GenBank at 100% query cover (e.g., MH128521), the COII sequences matched over 70 sequences at 100% query cover (e.g., MN269935), and the 28S sequences matched over 40 sequences at 100% query cover (e.g., MT193450). There was no intraspecific variation among the 44 COI, two COII and two 28S sequences generated across the various samples and crop types thus only a single representative sequence was submitted to GenBank for each gene region (COI: PP514374; COII: PP526177; 28S: PP515714).

Molecular identification was validated with morphological data (Table 2; Fig. 2). Perineal patterns were prepared following the lactic acid method as described by Hartman and Sasser (1985) and mounted on slides using the wax-ring method. Photographs were taken using a ZEISS Axiocam 506 mono camera mounted on a ZEISS Axioscope light microscope. Perineal pattern images were stacked using Zerene stacker image stacking software version 1.04. A subset of J2s and males were mounted on temporary slides in water and photographed as above and measurements were made using ZEISS Blue imaging software (ZEISS, Germany). These and additional J2s and males recovered were then killed in hot water, preserved in 4% formalin, processed to glycerol using the slow method (Hooper 1986) and mounted in glycerol on wax-ring slides. Voucher specimens of perineal patterns, J2s and males are lodged in the nematology section of the Australian National Insect Collection, CSIRO, Canberra, under the accession numbers: 8802–8837. Morphology of perineal patterns, J2s and males were consistent with previous reports (see Subbotin et al. 2021).

Table 2 Morphometrics of second stage juveniles (J2s) and males of Meloidogyne enterolobii from eggplant, the Northern Territory, Australia. Measurements are presented in the form: mean ± standard deviation (range). Abbreviations follow Subbotin et al. (2021)
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Meloidogyne enterolobii was confirmed on a variety of market-relevant crops in the Northern Territory including Butternut Pumpkin (Cucurbita moschata), Capsicum and Chilli peppers (Capsicum annum), Cucumber (Cucumis sativus), Zucchini (Cucurbita pepo), Sweet potato (Ipomoea batatas), Snake bean (Vigna unguiculata ssp. sesquipedalis), Tomato (Solanum lycopersicum), and Eggplant (Solanum melongena). The nematode was also confirmed on some naturalised plants in the Darwin region: Alysicarpus vaginalis and Centrosema molle. The ability of M. enterolobii to parasitise many crops, weeds, and native Australian plants challenges in-field management of this nematode. Presently, the route of introduction of this nematode into Australia is unknown, however, this is under investigation.