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Int.J.Curr.Microbiol.App.Sci (2018) 7(7): 1701-1708 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 7 Number 07 (2018) Journal homepage: http://www.ijcmas.com Case Study https://doi.org/10.20546/ijcmas.2018.707.201 Morphological and Morphometric Variations among the Population of Root-Knot Nematode(s) of Assam Kshetrimayum Sumita*, Debanand Das and Bhupendra Nath Choudhury Directorate of Research, Central Agricultural University, Manipur, India Department of Nematology, Assam Agricultural University, Jorhat-785013, India * Correspo ndingauthor ABSTRACT Keywords Morphological variations, Morphometric variations, Populations, Meloidogyne incognita Article Info Accepted: 15 June 2018 Available Online: 10 July 2018 Study on the morphological variations among the populations of Meloidogyne incognita from nine districts of Assam exhibited variations only in the tail shape, while other morphological characters showed similarities among the populations.Tail shape of second stage juveniles (J2) of M.incognita collected from Golaghat, Kokrajhar, Nagaon and Sibsagar district was similar in shape. Likewise, J2 tail shape of Dibrugarh, Jorhat and Lakhimpur population showed similarity but, Dhubri and Kamrup population showed variation from other populations. No variation was observed in size and shape of perineal pattern of M. incognita collected from nine districts of Assam. Studies on morphometric variations (body length, stylet length, lip height, lip width, MB, a, b, b´, c and c´) among the populations of M. incognita from nine districts of Assam showed no distinct variations in the major characters, except in MB value. MB value of Kamrup population was higher (61.79) than other populations. Introduction Nematodes are highly diversified, ubiquitous group of invertebrates that occurs in all possible kind of climatic conditions and habitats. Until middle of the last century, plant parasitic nematodes did not get worldwide recognition as important causal agents of plant diseases, but are now realized as a great menace in successful cultivation of crops, limiting production, productivity and quality as well. Plant parasitic nematodes are responsible for 12.3 per cent average annual loss of the world’s major crops. Their threat being more severe in underdeveloped and developing countries with warm and humid climate than developed countries that lie in the temperate zones. It is evident from the fact that an average loss of 14.6 per cent was estimated due to plant parasitic nematodes in developing countries as compared to 8.8 per cent in developed countries (Sasser and Freckman, 1987). In India, the loss on crops due to plant parasitic nematodes goes up to 10 per cent (Dasgupta, 1998). Among the important economic plant parasitic nematodes, Meloidogyne spp. ranks first, causing a great loss to the crops ranging upto 90.9 per cent in okra (Bhatti and Jain, 1977).Due to many biotic and abiotic influences, morphological 1701 Int.J.Curr.Microbiol.App.Sci (2018) 7(7): 1701-1708 and morphometric characters have been found to be altered among this nematode. Geographical variation is also considered as one of the major cause for morphological and morphometric variations. Considering the importance of accurate identification of rootknot nematode for proper management strategies the following work has been carried out. Soil along with root samples were collected from around the rhizosphere of vegetable crops from nine districts of Assam viz., Golaghat, Kokrajhar, Nagaon, Sibsagar, Dibrugarh, Jorhat, Lakhimpur, Dhubri and Kamrup. Root-knot nematode population collected from these nine districts were extracted from soil as well as from root using Cobb’s sieving and decanting method and Baermann funnel technique, respectively. Nematode suspension, concentrated in fewml of water were killed and fixed simultaneously by pouring equal amount of warm 8 per cent formalin (8ml formaldehyde solution + 92 ml distilled water). Clearing of nematodes were done using Seinhorst’s method (Seinhorst, 1962). Paraffin waxes were used for mounting and sealing of nematodes for permanent slide preparation. For preparation of perineal pattern, entire knotted root system was cut into a small pieces and stained by NaOCl-Acid Fuchsin method (Byrd et al., 1983). Ten mature females from each district were taken for preparation of perineal pattern. Measurements of the mounted nematode were taken with the help of an ocular micrometer, caliberated with a stage micrometer. For denoting the dimensions of nematodes, de Man’s (1880) formulae were used. cylindrical procorpus and rounded metacarpus. Excretory pore just behind the level of stylet knobs. Perineal pattern wavy dorsally. Dorsal arch high and squarish. Perineal pattern of Meloidogyne spp. collected from nine districts of Assam were found to be similar with the perineal pattern of M. incognita with high dorsal arch, distinct lateral lines absent. Vulva, anus and phasmid present in perineum. No variation on perineal pattern was observed in size and shape of perineal pattern of M. incognita collected from nine districts of Assam (Fig. 1). J2(n=15): Body slender, lip region continuous with the body contour. Cephalic framework well developed. Stylet slender with distinct round small basal knobs. Metacorpus distinct with basal bulb extension overlapped ventrally, genital primodia developed, rectum and anus clearly visible. Tail with hyaline terminus conoid shaped. Morphological characters of J2 M. incognita population collected from nine districts of Assam exhibited similarity in head portion (Fig. 2) with variations in tail shape (Fig. 3). Similar tail shape of J2M. incognita collected from Golaghat, Kokrajhar, Nagaon and Sibsagar district was recorded. Likewise, tail shape of J2of Dibrugarh, Jorhat and Lakhimpur; Dhubriwith Kamrup population showed similarity. Morphometric Morphology J2(n=15): Populations of M. incognita from nine districts of Assam showed no distinct variations in the major characters, except in MB value. MB value of Kamrup population was higher (61.79) than other populations. Mature female (n=10): Body pearly white, globular to pear shaped, with projecting neck. Cuticle translucent and glistering. Annulations visible in neck and vulva-anus region. Lip setoff. Stylet slender with distinct rounded knobs. Esophagus well developed with large Table 1 and 2 showed that from all the nine populations of M. incognita J2collected, longestbody length were found in Kamrup (0.478 mm) while, shortest being in Golaghat (0.360 mm). Lip height, lip width and stylet length were in same range with 2-3 µm, 4-5 1702 Int.J.Curr.Microbiol.App.Sci (2018) 7(7): 1701-1708 µm and 10-12 µm, respectively. Length from the anterior end to the centre of median bulb in relation to esophagus length (MB) showed variation. Highest value of MB was recorded in Kamrup(61.79) followed by Jorhat (54.63) while, lowest being recorded in Sibsagar (36.36). Highest a and b ratio was recorded in Sibsagar (33.38) and Golaghat (5.61) while, lowest was recorded in Golaghat(26.07) and Sibsagar (3.81), respectively. Highest b’ ratio was recorded in Lakhimpur (4.48) while, lowest being found in Jorhat (3.01). Highest c and c´ ratio was recorded in Kokrajhar (10.66) and Lakhimpur (4.90), while lowest being found in Lakhimpur (7.73) and Kokrajhar (3.00), respectively. Table.1 Comparison of morphometric data of Meloidogyne incognita (J2) from nine districts of Assam Characters Jorhat Golaghat Sibsagar Lakhimpur Dhubri Nagaon Dibrugarh Kokrajhar Kamrup J2 n=15 n=15 n=15 n=15 n=15 n=15 n=15 n=15 n=15 L 0.3660.434mm (0.398±0. 225) 0.3600.440mm (0.410±0. 234) 0.3820.473mm (0.390±0.03 0) 0.3720.459mm (0.426± 0.291) 0.3730.451mm (0.411± 0.023) 0.3640.449mm (0.405±.02 4) 0.3820.422mm (0.400± 0.013) 0.3840.448mm (0.410± 0.023) 0.4170.478mm (0.440± 0.020) Stylet Length 10.012.0µm (11.5±0.6) 10.012.0µm (10.8±0.7) 10.0-12.0µm (11.0±0.6) 10.0-12.0µm (11.0±0.5) 10.0-12.0µm (11.2±0.7) 10.0-12.0 µm (10.8± 0.05) 10.0-12.0µm (11.5±0.06) 10.0-12.0 µm (11.2±0.79) 10.0 - 12.0 µm (11.2±0.72) Lip height 2.0µm 2.0µm 2.0µm 2.0-3.0µm 2.0µm 2.0µm 2.0µm 2.0µm 2.0 µm 4.0-5.0µm (4.6± 0.05) 4.0µm 4.0 µm 4.0 -5.0µm (2.0±0.02) Lip width 4.0µm 4.0-5.0µm (4.2±0.04) 4.0-5.0µm (4.3±0.04) 4.0-5.0µm 4.0 -5.0µm (4.6±0.04) (4.5±0.51) 27.71-30.53 (29.13±0.89) 28.0030.71 (29.45± 0.69) 27.64-30.23 (29.02±0.93) 27.92-31.07 (29.74±0.85) 29.33-31.86 (30.33± 0.81) (4.6±0.04) a 26.1433.30 (29.96±1. 17) 26.0732.53 (30.71±1. 61) 27.78-33.38 (30.80±2.74) 28.61-30.64 (29.87±0.67) b 4.11-5.41 3.95-5.61 (4.69± 0.42) 3.81-4.91 (4.80± 0.67) 4.59-5.20 4.82 -4.96 (5.01±0.19) (4.88±0.04) 4.78-4.97 (4.89± 0.04) 4.22-4.66 (4.41±0.15) 4.82-5.30 (5.02±0.14) 4.96-5.27 (5.05± 0.13) 3.39-3.63 (3.53±0.08) 3.47-3.50 (3.48±0.02) 3.46-4.35 (4.87± 0.36) b´ c c´ MB 3.01-3.89 3.08-3.79 3.05-4.01 3.17-4.48 3.46 -4.12 3.15-4.10 (3.53±0.4 1) (3.44±0.1 8) (3.49± 0.67) (3.59±0.37) (3.71±0.33) (3.52± 0.32) 8.77-9.64 (9.16±0.3 0) 8.29-9.84 (9.28± 0.47) 8.64-10.50 (9.23±0.59) 7.73-9.2 (8.47±0.48) 8.22-9.43 (8.82±0.27) 8.66-9.55 (9.09±0.31) 8.88-9.28 (8.98±0.13) 9.00-10.66 (9.90±0.55) 9.00-10.15 3.75-4.40 (3.95± 0.17) 3.75-4.50 (4.10± 0.22) 3.50-4.18 (3.96±0.27) 4.00-4.90 (4.39±0.28) 3.66 - 4.16 (3.83±0.18) 3.58-4.09 (3.89±0.18) 3.58-4.00 (3.77±0.11) 3.00-4.08 (3.57±0.35) 3.46-4.00 41.0454.63 42.1853.09 36.36-49.07 46.15-53.71 44.00-54.16 (50.42±2.62) (48.98±4.46) 42.2753.92 38.63-49.53 (45.77±4.56) 40.75-45.16 (43.83±1.85) 39.06-61.79 (46.42± 5.84) (46.99± 4.79) (46.61± 4.59) (48.90± 4.17) *data in parenthesis is mean ± SD 1703 (3.87± 0.31) (9.43 ± 0.36) (3.76± 0.14) (51.87± 6.39) Int.J.Curr.Microbiol.App.Sci (2018) 7(7): 1701-1708 Table.2 Comparison of morphometric data of Meloidogyne incognita (J2) with Kofoid and White (1919), Whitehead (1968), Khan and Murmu (2006) and Das (1993) Characters Kofoid and White (1919) Whitehead (1968) Khan and Murmu (2006) works on West Bengal Das (1993) works on Assam Present work on Assam populations n=10 n=15 Second stage juvenile L 0.36- 0.393 mm 0.337-0.403 mm 0.327-0.471 mm (0.404) 0.40-0.43 mm 0.360-0.478 mm (0.414±0.01) Stylet Length 10 µm 9.6-11.7 µm (10.5) 11.88-13.86 (13.32) 10.0-12.0 µm 10.0-12.0 µm (11.0±0.6) Lip height - - - - 2.0 µm Lip width - - - - 4.0-5.0 µm (4.31±0.26) A 29.0-33.0 24.9-31.5 (28.5) 23.60-34.76 (28.42) 24.0-29.0 (25.5) 26.07-33.38 (29.89±0.63) B 5.6-6.4 2.02-3.14 (2.36) 3.94-5.08 (4.49) 2.5-3.0 (2.7) 3.81-5.61 (4.84±0.19) b´ - - - - 3.01-3.48 (3.57±0.135) c 8.0-9.4 6.9-10.6 (8.1) 6.16-8.70 8.12-9.2 (8.52) 7.73-10.66 (9.15±0.39) c´ - - - - 3.00-4.90 (3.91±0.23) MB - - - - 36.36-61.79 (47.54) *data in parenthesis is mean ±SD Fig.1 Perineal pattern of M. incognita 1704 Int.J.Curr.Microbiol.App.Sci (2018) 7(7): 1701-1708 Fig.2 Head portion of M. incognita J2 from nine districts of Assam (100X) Dhubri Dibrugarh Golaghat Jorhat Kamrup Kokrajhar Lakhimpur Nagaon Sibsagar 1705 Int.J.Curr.Microbiol.App.Sci (2018) 7(7): 1701-1708 Fig.3 Tail of M. incognita J2 from nine districts of Assam (40X) Dhubri Dibrugarh Golaghat Jorhat Kamrup Kokrajhar Lakhimpur Nagaon Sibsagar 1706 Int.J.Curr.Microbiol.App.Sci (2018) 7(7): 1701-1708 Observations on perineal pattern of M. incognita collected from nine district of Assam exhibited same shape. Perineal patterns with high dorsal arch. Anus anteriorly located at 14-17 µm from the vulva. Striae distinct and wavy. Dorsal striae smooth and closely spaced. Similar finding were also observed by Chitwood (1949) and considered the perineal pattern as the most striking and distinguishing character for identificationof root-knot nematode species. Kaur and Attri (2013) also opined that perineal pattern is the most stable characters of root-knot nematodes. Ley et al., (1997) considered stylet length and tail length as the most stable characters in J2 of M. incognita. Jepson (1987) also assessed the characters of different life stages like J2, female and male and concluded that tail length in J2, stylet length in female and male were most differential character whereas stylet length in juvenile was considered as supplementary differential character for identification of Meloidogyne species. Kaur and Attri (2013) stated that most of the morphometric characters of J2of M. incognita like body length, stylet length, head to median bulb length, distance from median bulb to excretory pore, tail length, anal body width, c and c´ are highly variable character. References Bhatti, D.S. and Jain, R.K. (1977). Estimation of loss in okra, tomato and brinjal yield due to Meloidogyne javanica. J. Nematol.7:37-41 Byrd, D.W.; Kirkpatrick, T. and Barker, K.R. (1983). An improved technique for clearing and staining plant tissues for detection of nematodes. J. Nematol. 29: 16-22. Chitwood, B.G. (1949). Root-knot nematodes. Part-1, A Revision of the genus Meloidogyne Goeldi, 1887. Proc. Helminth. Soc. Wash. 16: 90-104. Das, D. (1993). Occurrence and distribution of plant parasitic nematodes at Assam Agricultural University, Jorhat campus. M. Sc. (Agri.) Thesis, Assam Agricultural University, Jorhat, pp. 84. Dasgupta, M.K. (1998). Phytonematol. Naya Prakash, Calcutta. pp. 840 de Man, J.G. (1880). diefrei in der reinenerd und in sussenwasserleb. End enNematoden der neiderlandischen fauna Leiden, Brill. pp. 206. Jepson, S.B. (1987). Identification of rootknot nematodes (Meloidogyne spp.) CAB International, Wallingford, Oxon, UK. pp-265. Kaur, H. and Attri, R. (2013). Morphological and morphometric characterization of Meloidogyne incognita from different host plants in four districts of Punjab. Indian J. Nematol. 45(2): 122-127. Khan, M.R. and Murmu, B. (2006). Morphometric and Morphological variations in a nematode Meloidogyne incognita from West Bengal, India. Proc. Zool.Soc. Calcutta. 59(2): 15-22. Kofoid, C.A. and White, W.A. (1919). A new nematode infection of man. J. Amer. Med. Assoc. 72: 567-569. Ley,I.T de; Machon, J.E.; Hunt, D.J. and DeLey,I.T. (1997). Morphometrical and morphological variability of six populations of Meloidogyne species from rice ecosystem in Phillipines. Mededelingen Faculteit Landbouwkundige-en T oegpaste Biologische Wetenschappen Gent. 62(3a): 675-689 Sasser, J.N. and Freckman, D.W. (1987). A world perspective on nematology: the role of the society. In: Vistas of nematology. Veech, J.A. and Dickerson, D.W. (Eds.). Society of Nematologists, Hyattville, Maryland, 1707 Int.J.Curr.Microbiol.App.Sci (2018) 7(7): 1701-1708 pp. 7-14. Seinhorst, J.W. (1962). On the killing, fixation and transferring to glycerine of nematodes. Nematologica8: 2932. Whitehead, A.G. (1968). Taxonomy of Meloidogyne (Nematoda: Heteroderidae) with description of four new species. Trans. Zool. Soc.London31: 263-401. How to cite this article: Kshetrimayum Sumita, Debanand Das and Bhupendra Nath Choudhury. 2018. Morphological and Morphometric Variations among the Population of Root-Knot Nematode (S) of Assam. Int.J.Curr.Microbiol.App.Sci. 7(07): 1701-1708. doi: https://doi.org/10.20546/ijcmas.2018.707.201 1708