Meloidogyne paramali n. sp. (Nematoda: Meloidogyninae) and First Report of M. marylandi in maple and yacca tree from Japan

Rhizospheric soil and medium soil samples of maple and Podocarpus trees from Japan were obtained by making two to three holes, each having a depth of about 20 cm to 30 cm, and about 1 kg of soil mixed with medium soil and some roots were collected. Nematodes were extracted with a Baermann funnel for 24 hr to 48 hr. After extraction of J2, the roots were washed and observed with a dissecting microscope, and females and males were recovered. For preparation of light microscopy (LM), males and J2s were relaxed with gentle heat, fixed in a solution of 4% formaldehyde, and processed using the glycerin–ethanol method. Perineal patterns of mature females were prepared as described previously (Hartman et al., 1985). The perineal pattern was trimmed and transferred to a drop of glycerin for observation. Nematodes were measured and photographed with a Zeiss Imager Z1 microscope equipped with a Zeiss AxioCam MRm CCD camera (Oberkochen, Germany).

DNA samples were obtained from freshly extracted nematodes. Briefly, two repeats of each single J2 individual, one male and one female, were transferred separately to a PCR tube containing 8 μL ddH₂O and 1 μL 10× PCR buffer. The tubes were stored at −70°C for at least 2 hr, and then pipetted 1 μL proteinase K (100 μg/mL) was introduced into the solution containing the melted remains of the nematodes. Subsequently, the tubes were incubated at 65°C for 1 hr to 2 hr and the proteinase K was denatured at 95°C for 10 min. Four sets of primers (synthesized by Invitrogen [Shanghai, China]) were used in the PCR analyses to amplify partial 18S, full length ITS, and partial 28S D2-D3 regions of rDNA. The 18S region was amplified with one pair of primers (forward primer 988F, reverse primer 1912R) and then another pair (1813F and 2646R) (Holterman et al., 2006), the ITS region was amplified with the forward primer TW81 (Ferris et al., 1993) and the reverse primer AB28 (Vrain, 1993), and the 28S D2-D3 region was amplified with the forward primer D2A and the reverse primer D3B (De Ley et al., 1999). The intergenic region between the cytochrome oxidase subunit II (CO II) and 16S rRNA (lrRNA) genes was amplified with forward primer C2F3 and reverse primer MRH106 (Stanton et al., 1997). For M. marylandi, only full length ITS and partial 28S D2–D3 regions of rDNA were amplified. PCR products were separated on 1% agarose gels and visualized by staining with ethidium bromide. Positive PCR products were cloned with the pMD 18-T Vector Cloning Kit (Promega, Beijing, China) using TOP10 competent cells following the manufacturer’s instructions before sequencing. PCR products were sequenced in both directions with PCR primers in Invitrogen.

The sequences of partial 18S, full length ITS, 28S D2-D3 region, and intergenic region between CO II and 16S rDNA of M. paramali n. sp. were compared with other relevant Meloidogyne sequences available with GenBank. The selected sequences were aligned by MAFFT v.7.205 (Katoh and Standley 2013) with default parameters. The alignments of sequences were manually edited and assembled in one dataset by using AliView (Larsson, 2014). The best-fitted model of DNA evolution was obtained using jModelTest2 (Darriba et al., 2012) with the Akaike information criterion (AIC). The SYM + I + G model was selected for 18S region partition, the TVM + I + G model was selected for 28S D2–D3 region partitions, and the GTR + G model was selected for full length ITS and intergenic region between CO II and 16S rDNA.

The Bayesian concatenated tree was inferred using MrBayes 3.2.3 (Ronquist and Huelsenbeck, 2003) with four chains (three heated and one cold). Model parameters were unlinked and the overall rate was allowed to vary across partitions. The number of generations for the total analysis was set to 3 × 107, with the chains sampled every 1,000 generations and the burn-in value set to 25%. The Markov chain Monte Carlo (MCMC) method within a Bayesian framework was used to estimate the posterior probabilities of the phylogenetic trees using the 50% majority rule (Larget and Simon, 1999). The consensus tree was selected to represent the phylogenetic relationships with branch length and support level and visualized using TreeGraph 2 (Stöver and Müller, 2010).

(Figs. 1–4)

Figure 1

Measurements of J2 are available in Table 1.

Female (n = 5)

Females were rare, and only 5 could be measured. The measured parameters are the following – length (including neck): 820.6 ± 82.0 (710–962) μm; length of neck: 166.0 ± 19.4 (135–190) μm; body width: 737.2 ± 72.8 (630–849) μm; DGO from stylet base: 5.6 ± 0.8 (4.7–6.5) μm; and stylet length: 14.5 ± 1.5 (12.5–16.9) μm.

Females were completely enclosed by gall tissue. Their bodies were translucent-white, variable in size, and ovoid to pear-shaped. The neck was sometimes prominent. The body cuticle was distinctly annulated, and annuli were smaller in the anterior neck region. The head region was set off from body and the cephalic framework was weakly sclerotized, with distinct head caps and indistinct annulations. The stylet cone was slender and slightly curved; the shaft was observed to have a gradually wider shape posteriorly near the junction, with knobs. The knobs were well developed, round to transversely ovoid, and slightly concave anteriorly. The excretory pore was located posterior to stylet base. The distance of dorsal pharyngeal gland orifice to stylet base was approximately 5 μm to 7 μm. Perineal patterns were oval or irregular, with distinct lateral lines; dorsal arches were often round, and dorsal and ventral striae smooth. A dorsal arch with shoulders was sometimes present, formed by a slight indentation of the dorsal striae near the lateral lines. The anus was within a cuticlar fold, and the phasmids were conspicuous. Inter-phasmidal distance (22.2 [18.9–28.7] μm) was wider than the length of vulval slit (17.3 [16.1– 18.3] μm), the distance of the vulva from an imaginary line joining the phasmids was 25.3 (24.0–26.9) μm, and the distance between the vulva and anus was 16.3 (12.4–19.1) μm.

Males are rare, and only two were recovered. Their characteristics are the following – body length: 1,545 μm and 1,819 μm; body width: 32.1 μm and

* Specific epithet is derived from its similarity to M. mali. 47.0 μm; a: 48.1 and 38.7; stylet length: 18.9 μm and 20.1 μm; DGO: 6.5 μm and 7.0 μm; spicules’ length: 31 μm and 33 μm; head to the middle of valve plate: 93 μm and 99 μm; and tail length: 9.8 μm and 11.4 μm.

Figure 2

The bodies were vermiform, tapering anteriorly, bluntly rounded posteriorly twisting through 90°, and the tails were bluntly rounded. Body annuli were large, distinct, and 2.4 μm to 2.9 μm in width. The lateral field was with four lines in the mid body region, beginning as two lines at about the stylet base and ending as two or three lines at the tail tip. The cephalic framework was moderately developed, and vestibule extensions were distinct. The head was set off from the body, without labial annulus. The stylet was robust, with rounded basal knobs. The cone was slightly longer than the shaft. The shaft was cylindrical, widening slightly near the junction, with knobs. The dorsal pharyngeal gland orifice branched into three channels, with distinct ampulla. The procorpus was distinct, while the median bulb was ovoid. Pharyngo-intestinal junction occurred at the level of nerve ring, and was indistinct. Three nuclei were present in gland lobe. The testis was outstretched or anteriorly reflexed. Spicules were arcuate, and gubernaculum distinct. The tail was short with broadly rounded terminus, with indistinct phasmids.

Figure 3

Figure 4

About 50 J2s were observed, and 20 of them were measured. The bodies were observed to be vermiform, clearly annulated, and straight or slightly ventrally bent near the tail region. The anterior ends were truncated, with the head region only slightly set off from the body. The lateral region was slightly larger than one-third of the body width, with four lines. The stylet was delicate, and the cone was straight, narrow, and sharply pointed; the shaft was observed to become slightly wider posteriorly, while the knobs were small and rounded. The procorpus was faint, while the metacorpus was oval-shaped with an enlarged lumen lining. The isthmus was clearly defined, while the pharyngo-intestinal junction was difficult to observe. The gland lobe variable in length, and it ventrally overlapped the intestine. The excretory pore was distinct, and was located 64 μm to 81 μm from the head, while the hemizonid was three to five annuli posterior to the excretory pore. The tail was conoid, with finely rounded to broad pointed terminus, but never sharply pointed. The hyaline tail terminus was very short (4.3 [3.0–4.9] μm), and indistinct. The phasmids were small, difficult to observe, and located posterior to the anus.

The eggs had a morphology that is typical for this genus. The egg masses were usually protruding from root tissues on the roots.

The nematodes in question were isolated from Japanese maple (Acer palmatum Thunb.) imported from Chiba, Japan and inspected in Ningbo Port, P.R. China during May, 2018.

To earmark the necessary holotype female, five females, two males, and 26 second-stage juvenile paratypes (slide number 5445-1~5445-10) have been deposited at the nematode collection of Ningbo Customs Technology Center, China. Additionally, five paratype J2s (slide numbers T620) are deposited in the Canadian National Collection of Nematodes, Ottawa, Canada.

Meloidogyne paramali n. sp. is characterized by J2 with a short tail length of 32.2 (24–36.8) μm, finely rounded to broad pointed tail terminus, and an extremely short hyaline tail terminus of 4.3 (3.0–4.9) μm. The perineal pattern of females was oval or irregular, with round dorsal arch, distinct lateral lines, and fine and smooth striae. The polytomous key codes of the new species according to Subbotin et al. (2021) are: Female: A21, B2, C32, D4; Male: A21, B3, C2, D1, E2, F2; J2: A2, B23, C43, D34, E12, F34.

Based on the perineal pattern of females (A4) and the similar J2 tail length (C43), we may infer that Meloidogyne paramali n. sp. is very similar to M. mali and M. nataliei Golden et al., 1981. The new species can be distinguished from M. mali by the perineal pattern, which has a distinct lateral field; additionally, the hyaline terminus of the J2 tail is shorter (4.3 [3.0– 4.9] μm vs. 8.2 [4.8–12.7] μm), and the tail is finely rounded to broadly pointed in M. paramali n. sp., but it is never sharply pointed as it is in M. mali.

It differs from M. nataliei by shorter J2 body length (433 [402–455] μm vs. 599 [539–641] μm), shorter J2 stylet length (11.7 [10.8–12.5] μm vs. 22.4 [21.9–22.8] μm), shorter tail hyaline portion (4.3 [3.0–4.9] μm vs. 8.4 [7.0–11.0] μm), and much smaller c value (13.6 [11.7–18.9] vs. 22 [19–26]).

M. caraganae Shagalina et al., 1985, M. coffeicola Lordello and Zamith, 1960, M. kikuyensis De Grisse, 1961, M. nataliei, M. suginamiensis Toida and Yaegashi, 1984, M. turkestanica Shagalina et al., 1985, and M. vandervegtei Kleynhans, 1988 have similar J2 tail length (C4 or 43), but the perineal patterns are different: D3 for M. caraganae, M. suginamiensis, M. turkestanica and M. vandervegtei, D2 for M. coffeicola, and D5 for M. kikuyensis.

In the phylogenetic trees (Figs. 6–9), M. vitis, M. paramali n. sp., and M. mali formed a well-separated clade, which is named molecular group VIII (Subbotin et al., 2021). Meloidogyne paramali n. sp. differs from M. vitis in that the J2 tail is shorter (32.2 [24–36.8] μm vs. 57.43 [47.01–63.77] μm), and the J2 c value is bigger (13.6 [11.7–18.9] vs. 6.95 [6.15–8.77]).

Figure 5

Figure 6

(Fig. 5)

Fourteen individuals were measured and observed. Their bodies were vermiform, almost straight, and tapering at both extremities, but tapered more posteriorly. The head was not offset, and was observed with labial disk, while the lip region was without annulation. The stylet was delicate (11.5 μm to 13.4 μm long). The cone was straight, narrow, and sharply pointed; the shaft was observed to become slightly wider posteriorly, with a small rounded knob.

The cuticular annulation was fine and distinct. The lateral field was prominent, with four incisures. The excretory pore was three to four annuli anterior to the hemizonid. The rectum was inflated, while the phasmids were small and indistinct. The tail tapered gradually, and was 49.5 μm to 54.0 μm long. The hyaline tail terminus was 11.2 (9.5–13.5) μm long, and the terminus was bluntly rounded.

Males and females were not found.

The partial 18S region, full length ITS, 28S D2-D3 region, and the cytochrome oxidase subunit II (CO II) sequences of M. paramali n. sp. were deposited in GenBank with the accession numbers MW342769-MW342772, MW342760-MW342768, MW342773-MW342775, and ON430646-ON430649, respectively. The full length ITS and 28S D2-D3 region sequences of M. marylandi were deposited in GenBank with the accession numbers ON453657-ON453659 and ON453660-ON453661.

The 18S phylogenetic tree demonstrated that M. paramali n. sp. clustered with M. mali as an independent clade and close to the basal branch of Meloidogyne with high support value (posterior probability = 100). The intraspecific sequence divergence value of the four acquired 18S sequences of M. paramali n. sp. is 0.23% to 1.45% (5– 22/1,724 bp). M. paramali n. sp. shares the smallest sequence divergence value of 2.58% to 2.64% (41– 45/1707 bp) with M. mali.

The ITS phylogenetic tree demonstrated the new species clusters with M. mali and M. vitis as an independent clade with fully supported value (posterior probability = 100). The new species showed a sister phylogenetic relationship with M. mali with low supported value (posterior probability = 60).

The intraspecific sequence divergence value of the nine acquired M. paramali n. sp. ITS sequences is 0.15% to 7.35% (1–48/653 bp). The new species also shares the smallest sequence divergence value of 11.86% to 18.80% (81/683–129/686 bp) and 17.85% to 19.70% (108/605–120/609 bp) with M. mali and M. vitis, respectively.

The 28S D2-D3 phylogenetic tree also displayed a similar topology with the ITS tree. The new species clustered with M. mali, an unidentified species Meloidogyne sp. GKL-2016, and M. vitis as a clade with fully supported value (posterior probability = 100). However, the new species is sistered to the clade that is comprised of M. mali, M. vitis, and Meloidogyne sp. GKL-2016. The analysis of the three acquired sequences of M. paramali n. sp. showed the intraspecific sequence divergence value of 3.24% to 6.35% (25–49/772 bp). M. paramali n. sp. shares the smallest sequence divergence value of 5.96% to 9.46% (46/772–74/782 bp) with M. mali. Further, the new species shared the sequence divergence value of 8.42% to 10.49% (65–71/772 bp) and 8.17% to 9.33% (63/771–72/772 bp) with Meloidogyne sp. GKL-2016 and M. vitis, respectively.

Figure 7

Figure 8

Figure 9

In the COII phylogenetic tree, M. paramali n. sp. clustered with M. mali and M. vitis into a fully supported clade (posterior probability = 100). However, the phylogenetic relationship between these three species has remained unresolved. The analysis of the four acquired M. paramali n. sp. intergenic regions between CO II and 16S rDNA sequences showed the intraspecific sequence divergence value of 0.14% to 0.44% (1/687–3/688 bp). M. paramali n. sp. shares the sequence divergence values of 13.19% to 13.37% (72–73/546 bp) and 18.92% to 19.22% (102/539– 104/541 bp) with M. mali and M. vitis, respectively. This molecular phylogenetic analysis supports the status of M. paramali n. sp. as a new species.

The sequence analysis of ribosomal rDNA 28S and ITS regions showed a 98% to 100% sequence similarity between the M. marylandi population from Japan and the registered M. marylandi, but it was quite different from other similar species. Therefore, the nematode was identified as M. marylandi.