Characterization of Meloidogyne indica (Nematoda: Meloidogynidae) Parasitizing Neem in India, with a Molecular Phylogeny of the Species

Victor Phani; Satyapal Bishnoi; Amita Sharma; Keith G. Davies; Uma Rao

doi:10.21307/jofnem-2018-015

Figures & Tables

Plant symptoms (A–C) and morphology (D–K) of Meloidogyne indica infecting neem. A, Healthy neem seedlings; B, Infected neem seedlings devoid of lateral roots; C, Root gall with eggmass; D, Adult females; E, Anterior region of adult female; F and G, Perineal pattern morphology; H, Anterior region of male; I, Male tail; J, Anterior end of second-stage juvenile (J2); K, Second-stage juvenile tail (scale bar = D: 550 µm; E: 100 µm; H,I: 10 µm; J,K: 20 µm).

Scanning electron microscopy photomicrographs of Meloidogyne indica. A, Female lip region; B, Male lip region; C, Male tail; D, Lateral field with lateral lines; E, Second-stage juvenile tail (scale bar in µm).

Citrus roots showing development of galls and eggmasses upon inoculation with the neem population of Meloidogyne indica. A, Citrus plant inoculated with neem population of M. indica; B, Healthy roots of citrus; C, Infected roots of citrus with galls and eggmasses.

Evolutionary relationship of Meloidogyne indica using ITS rRNA sequence. The evolutionary history was inferred by using the maximum likelihood method based on Kimura 2-parameter model. The bootstrap consensus tree inferred from 1,000 replicates is taken to represent the evolutionary history of the analyzed taxa. Branches corresponding to partitions reproduced in less than 30% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying neighbour-joining and BioNJ algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood approach and then selecting the topology with superior log likelihood value (–1869.0466). A discrete Gamma distribution was used to model evolutionary rate differences among sites (five categories [+G, parameter = 1.0929]). The analysis involved 29 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 170 positions in the final dataset. Evolutionary analyses were conducted in MEGA6.

Evolutionary relationship of Meloidogyne indica using D2D3 expansion segment of 28S rRNA sequence. The evolutionary history was inferred by using the maximum likelihood method based on General Time Reversible model. The bootstrap consensus tree inferred from 1,000 replicates is taken to represent the evolutionary history of the analyzed taxa. Branches corresponding to partitions reproduced in less than 30% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying neighbour-joining and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood approach and then selecting the topology with superior log likelihood value (−4112.4125). A discrete Gamma distribution was used to model evolutionary rate differences among sites (five categories [+G, parameter = 0.4693]). The analysis involved 34 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 525 positions in the final dataset. Evolutionary analyses were conducted in MEGA6.

Evolutionary relationship of Meloidogyne indica using mitochondrial COI sequences. The evolutionary history was inferred by using the maximum likelihood method based on General Time Reversible model. The bootstrap consensus tree inferred from 1,000 replicates is taken to represent the evolutionary history of the analyzed taxa. Branches corresponding to partitions reproduced in less than 30% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying neighbour-joining and BioNJ algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood approach and then selecting the topology with superior log likelihood value (−2471.1920). A discrete Gamma distribution was used to model evolutionary rate differences among sites (five categories [+G, parameter = 0.5728]). The analysis involved 21 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 309 positions in the final dataset. Evolutionary analyses were conducted in MEGA6.

Morphometrics for Meloidogyne indica infecting neem and citrus_ All linear measurements are in micrometer and in the form of mean ± SD_

	Neem population			Citrus population (After Whitehead, 1968)
Character	J2	Male	Female	J2	Female
n	20	15	30	25	8
L	484 ± 31.5 (430–520)	1253 ± 80 (1180–1380)	653 ± 92.2 (450–790)	414 ± 4.5 (381–448)	–
Body width	18 ± 1.5 (16.77–21.15)	28 ± 4 (24.55–34.66)	408 ± 75 (325–550)	–	–
A	26.68 ± 1.9 (24.20–29.87)	44.89 ± 3.5 (39.81–48.06)	1.60 ± 0.3 (1.38–2.10)	–	–
Stylet length	13.8 ± 0.1 (13.57–14.21)	16.3 ± 0.4 (15.90–17.08)	13.7 ± 0.4 (13.32–14.18)	12 ± 0.9 (10–14)	14 (12–16)
DGO	2.8 ± 0.2 (2.45–3.25)	3.1 ± 0.1 (2.92–3.30)	2.9 ± 0.3 (2.49–3.67)	–	3 (2–4)
Head-metacorpus	50 ± 2.3 (46.45–53.02)	73 ± 4.1 (68.70–78.14)	–	–	–
Head-oesophageal gland	138 ± 4.8 (129.97–144.65)	–	–	–	–
b′	3.5 ± 0.1 (3.10–3.65)	–	–	–	–
c	26.2 ± 1.2 (24.15–27.65)	–	–	24.9 ± 1.36 (21.2–31)	–
c′	1.6 ± 0.1 (1.52–1.91)	–	–	1.57 ± 0.012 (1.06–1.78)	–
Tail length	18 ± 0.6 (17.50–19.50)	–	–	16.8 ± 1.88 (13–20.1)	–
Anal body width	11.1 ± 1.0 (9.85–12.45)	–	–	–	–
Spicule	–	26 ± 0.6 (25.90–27.50)	–	–	–

List of primers used for polymerase chain reaction amplification in this study_

Primer name	Gene	Sequence	References
V5367	ITS	5′-TTGATTACGTCCCTGCCCTTT-3′	Vrain et al. (1992)
26S	ITS	5′-TTTCACTCGCCGTTACTAAGG-3′	Vrain et al. (1992)
D2A	LSU	5′-ACAAGTACCGTGAGGGAAAGTTG-3′	Castillo et al. (2003)
D3B	LSU	5′-TCGGAAGGAACCAGCTACTA-3′	Castillo et al. (2003)
JB3	COI	5′-TTTTTTGGGCATCCTGAGGTTTAT-3′	Bowles et al. (1992)
JB5	COI	5′-AGCACCTAAACTTAAAACATAATGAAAATG-3′	Derycke et al. (2005)

List of GenBank accession numbers used in phylogenetic analyses (** Not found in NCBI database)_

Species	ITS rRNA	D2D3 28S rRNA	COI mtDNA
Meloidogyne arabicida	**	KF993624	**
Meloidogyne africana	**	**	KY433441
Meloidogyne arenaria	AF387092	JX987332	JX683705
Meloidogyne artiellia	KC545880	AY150369	KU517173
Meloidogyne baetica	AY150366	AY150367	**
Meloidogyne camelliae	JX912885	KF542869	KM887148
Meloidogyne chitwoodi	AY281852	AF435802	KU517168
Meloidogyne christiei	KR082319	KR082317	**
Meloidogyne dunensis	EF612711	EF612712	**
Meloidogyne duytsi	**	**	KU517177
Meloidogyne enterolobii	KM046989	KJ146862	KT936633
Meloidogyne ethiopica	KF482366	KF482372	**
Meloidogyne exigua	**	AF435795	**
Meloidogyne fallax	AY281853	KC241969	KU517182
Meloidogyne graminicola	KM111531	KJ728847	KY250093
Meloidogyne graminis	JN157866	JN019326	**
Meloidogyne hapla	EU908052	DQ145641	JX683719
Meloidogyne haplanaria	**	**	KU174206
Meloidogyne hispanica	EU443613	EU443607	JX683712
Meloidogyne ichinohei	**	EF029862	KY433448
Meloidogyne incognita	KJ739707	JX100425	JX683696
Meloidogyne indica	KC311146	MF680038	MF662179
Meloidogyne inornata	KF482368	KF482374	**
Meloidogyne izalcoensis	**	KF993621	**
Meloidogyne javanica	KJ739709	KC953092	JX683711
Meloidogyne konaensis	**	AF435797	**
Meloidogyne lopezi	**	KF993616	**
Meloidogyne luci	KF482365	KF482371	**
Meloidogyne mali	JX978228	KF880398	KU517175
Meloidogyne marylandi	JN157854	JN019333
Meloidogyne minor	KC241953	JN628436	KU517178
Meloidogyne naasi	KJ934132	KC241979	KU517170
Meloidogyne panyuensis	**	**	**
Meloidogyne paranaensis	**	AF435799	**
Meloidogyne silvestris	EU570216	EU570214	**
Meloidogyne spartelensis	KP896294	KP895293	KP997301
Meloidogyne thailandica	AY858795	EU364890	**
Meloidogyne trifoliophila	JX465593	AF435801	**
Pratylenchus vulnus	FJ713011	EU130885	KX349427
Hirschmanniella oryzae	DQ309588	JX291142	**
Tylenchorhynchus leviterminalis	EF030984	KJ475548	**
Heterodera glycines	HM370421	GU595446	**
Radopholus similis	KJ845638	JN091964	KX349430
Heterotheca mucronata	**	**	KR819278
Tylenchorhynchus sp	**	**	KY639376

Characterization of Meloidogyne indica (Nematoda: Meloidogynidae) Parasitizing Neem in India, with a Molecular Phylogeny of the Species

Figures & Tables

Fig. 1

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Fig. 6

Morphometrics for Meloidogyne indica infecting neem and citrus_ All linear measurements are in micrometer and in the form of mean ± SD_

List of primers used for polymerase chain reaction amplification in this study_

List of GenBank accession numbers used in phylogenetic analyses (** Not found in NCBI database)_

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