Characterization of root-knot nematodes infecting mulberry in Southern China

Samples were collected from rhizosphere soil and mulberry root-knots in six locations in South China, including Huadu City of Guangdong province, Guangzhou City of Guangdong province, Zhanjiang City of Guangdong province, Shaoguan City of Guangdong province, Nanning city of Guangxi province, and Changsha City of Hunan province. Samples were placed in sealed plastic bags, which were then placed in sample boxes and stored at 4°C before further analysis, in order to minimize changes in nematode populations.

Male nematode was extracted from soil samples using the method described in Zeng (2012) study. Using a dissecting microscope, females and egg masses were isolated from infested roots with a scalpel and a nematological needle.

Individual nematodes were picked and heat-killed, then fixed in FG solution (containing 1 mL glycerol, 10 mL formalin, and 89 mL distilled water). The specimens were added slowly into glycerol and mounted on microscope slides. Measurements were made with a stage micrometer of Nikon microscope. Morphometric data were processed using Excel software. Images of key morphological features were taken using a Nikon DS-Fi1 attached to a Nikon ECLIPSE 80i microscope and processed using Photoshop CS5.

A female was identified and separately placed in 5 μL of worm lysis buffer (WLB) containing proteinase K for DNA extraction (Williams et al., 1992). DNA samples were stored at −20°C.

To amplify the ITS region, we used primers 5367 (5′-TTGATTACGTCCCTGCCCTTT-3′) and 26s (5′-TTTCACTCGCCGTTACTAAGG-3′) described by Vrain et al. (1992). PCR reactions contained 12.5 μL 2× PCR buffer for KOD FX, 5 μL 2 mM dNTPs, 1 μL of each primer, 2 μL of isolated DNA, and distilled water up to 25 μL. The amplification was carried out in a lab cycler (Applied Biosystems) using the following program: initial denaturation at 94°C for 4 min; 35 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, and elongation at 72 °C for 2 min; followed by a final extension at 72 °C for 10 min.

The D2-D3 region of the 28S gene was amplified with primer D2A (5′-ACAAGTACCGTGAGGAAAGTTG-3′) and D3B (5′-TCGGAAGGAACCAGCTACTA-3′) described by Sturhan et al. (2006). PCR reaction conditions were the same as those described for the ITS region.

The sequences obtained were submitted for a search in GenBank using the BLAST algorithm. Sequences for each gene were then aligned with corresponding published gene sequences using ClustalX 1.83 with default parameters. BI (Bayesian inference) analysis under the GTR + I + G model was initiated with a random starting tree and was run with four chains for 1.0 × 10⁶ generations. The Markov chains were sampled at intervals of 100 generations. Two runs were performed for each analysis. The log-likelihood values of the sample points stabilized after approximately 10⁴ generations. The topologies were used to generate a 50% majority rule consensus tree. Posterior probabilities (PPs) are given on appropriate clades. Sequence differences between samples were calculated with PAUP* 4b 10 (Cummings, 2004) as an absolute distance matrix and the percentage was adjusted for missing data.

The morphology of the root-knot nematode population isolated from mulberry trees is shown in Figure 1. The morphometric measurements are shown in Tables 1-3.

Figure 1:

The female body is white, pear-shaped to globular, with a prominent neck of variable size, and no posterior protuberance (Fig. 1A). The head region was not distinctly set off from the neck. Position of excretory pore is variable, and often found near the metacorpus (Fig. 1B). Cuticular body annulations become progressively finer posteriorly. Stylet is slender and its conical portion slightly curved dorsally, tapering toward tip. Contains cylindrical shaft, posterior end often enlarged (Fig. 1B). The conical portion is slightly curved dorsally, tapering toward the tip, with a cylindrical shaft that is posterior and often enlarged (Fig. 1C). Perineal pattern usually oval shaped, with coarse and smooth striae. The dorsal arch is moderately high to high, often rounded, with no lateral lines on perineal pattern for most females, or only one or two unclear lateral lines for a few samples (Fig. 1D,E).

The body is translucent white, vermiform, and tapered at both ends. It is small in size with a clear body ring (Fig. 1F). The head cap is highly rounded and slightly shrinking, with no ring pattern. Cephalic framework is moderately developed, with a distinct vestibule and extension (Fig. 1G). The stylet is robust with a straight cone. The boundary between the pole and the Base ball is clear (Fig. 1G). The Base ball of the stylet is large and the middle esophagus ball is fusiform. The tail is short and rounded, with a round base (Fig. 1G). The posture is slightly curved, while the base is round (Fig. 1H).

It has a body with translucent white and vermiform. The body ring is small and clear. The head slightly shrinks with fold marks. The labial disc is rounded and raised slightly above medial lips (Fig. 1I). The stylet is slender, with a straight cone, that is narrow and sharply pointed. The boundary with the rod is obvious. The distance of the DGO to the base of the stylet is long (3-5 μm). The mid-esophageal is bulbous shaped, with a clear valve (Fig. 1J). The Base ball of the stylet is clear, large, and oblong (Fig. 1I). There is a narrow tail, and the Hyaline tail terminus is clearly defined. The tail tip is obtuse, and has a 1 to 3 notches, with a dilated rectum (Fig. 1K).

The main morphological features are consistent with the description of the morphology of the ear worms of Yang and Eisenback (1983), Liao et al. (2001) and Long et al. (2015). This morphological similarity indicates that all six of the root-knot nematode populations are Meloidogyne enterolobii, but confirmation through molecular phenotyping is required to be certain.

The primer pairs D2A/D3B and 26S/V5367 were used to amplify the D2-D3 region of the 28S and rDNA-ITS gene sequences of root-knot nematodes in the Guangdong, Guangxi, and Hunan province of China. The amplified products were 765 bp and 715 bp, respectively. The sequence of the amplified product was submitted to GenBank for a BLAST search, and the results revealed the highest similarity (99-100%) to sequences of Meloidogyne enterolobii. The phylogenetic tree inferred from rDNA-ITS (Fig. 2) suggests that MK850135, MN102393, and Meloidogyne enterolobii (KX823381.1 and KX823382.1) are in the monophyletic clade, while MK850136, MK850137, MK850138, and MK116406 are in another monophyletic clade in relation to a population of Meloidogyne enterolobii (KX823381.1 and KX823382.1). The phylogenetic tree inferred from the D2-D3 region of the 28S rRNA (Fig. 3) indicated that MN01721, MN01724, MN017127, and Meloidogyne enterolobii (MF467276.1) are in the monophyletic clade, while MN01722, MN01723, MN01725, and Meloidogyne enterolobii (KX823404.1) are in another monophyletic clade.

Figure 2:

Figure 3: