Mulberry, Root-knot nematode, Root incubation, Perineal pattern, Meloidogyne incognita.

The rhizosphere soil is more intensified zone of microbial activity where the population of microorganisms is comparatively high (Katznelson, 1965). The association of microorganisms with the plant root system may be mostly beneficial and sometimes become detrimental. Among the rhizosphere organisms of mulberry, fungi and nematodes contribute to a markedly higher population (Nandi et al., 2004). In the global front, forty-two nematode species belonging to 24 genera were reported to be associated with mulberry as a pest (Swamy and Govindu, 1965). Among them, nematodes species belonging to genera Meloidogyne, Rotylenchulus, Helicotylenchus, Hoplolaimus and Xiphinema were majorly reported from India.

The root-knot nematode (RKN) are parasites of underground roots, which is difficult to recognize and hence the damage symptoms very often go unnoticed (Sengupta and Govindaiah 1991). The affected plants show stunted growth, marginal necrosis and yellowing of leaves. The characteristic knots or galls appear on the roots and affect the utilization of water and nutrients resulting in poor plant growth (Govindaiah et al., 1991). Arunakumar et al. (2018) delineated that Meloidogyne incognita infesting mulberry caused 20-50 per cent leaf yield loss apart from deteriorated leaf quality. This connotes the importance of characterising the nematode species appertaining to mulberry ecosystem. 

In this purview, an experiment was conducted to isolate and characterise the RKN associated with mulberry roots.

A. Experiment site details

The experiment was carried out in the RKN infested mulberry garden in Kalyapura Village, Shidlaghatta Taluk, Chikkaballapura District i.e., in the Eastern Dry Zone (Zone-5) of Karnataka, India at 13°14'20"N latitude and 77°52'17"E longitude, at an altitude of 904m above mean sea level.

The mulberry plantation selected for isolation of nematodes had red sandy loam type of soil with six years old V1 variety plants, planted at the spacing of 90 × 90cm; bottom pruning (Kolar method) was followed; field was irrigated in two-days interval with drip irrigation facility i.e., based on soil moisture conditions; organic manures and inorganic fertilizers were applied in accordance with the recommended package of practice. Except for the management of RKN, the selected field was well maintained.

B. Isolation and identification of mulberry RKN 

To isolate the RKN, five grams of infested root sample was processed following the root incubation method (Ayoub, 1977). After incubation for 48 hours, the suspension column was made up to 200 ml, out of which 10 ml was pipetted out and used for the isolation of root-knot nematode infesting the mulberry plants in the experimental site. The severely infested plants were tagged based on the above ground visual symptoms before collecting the root samples (Fig. 1).

The RKN infested roots were washed free of soil. Female nematodes were isolated from the well-developed galls under a stereo-zoom binocular microscope and transferred to a petri plate containing water. The posterior portion was cut with a perineal pattern knife (Taylor and Sasser 1978) and the body contents were cleared. The cleaned posterior portion of the female was further trimmed and transferred to a drop of glycerin on a clean microscopic glass slide. Bits of glass wool were placed around the trimmed portion to avoid damage. A cover slip was placed over the trimmed portion and sealed, then observed under the stereo binocular microscope. The species identification and characterisation were done based on the perineal patterns as described by Chitwood (1949).

C. Taxonomical status of RKN

D. Differentiation between the RKN species based on the perennial pattern

A. Stunted growth; B. Yellowing and marginal necrosis; C. Root-knots

Fig. 1. Symptoms of RKN infestation in mulberry.

The RKN juveniles isolated from the root samples collected from the experimental site possessed the following characters – body was typically saccate and spheroid like with a distinct neck; stylet was found to be slender with strong basal knobs; oesophageal median bulb was observed and the excretory pore was found to be situated between stylet knob and the median bulb; perineal patterns were found surrounding the anus and vulva similar to that of human fingerprint pattern, at the side exactly opposite to the neck; perineal pattern striae were smooth and wavy without any lateral lines, and the dorsal arch was high having a distinct whorl around the tail terminus (Fig. 2). The key observations on perineal pattern using scanning electron microscope images were in line to those described for Meloidogyne incognita by Chitwood (1949). The described morphological characteristics confirmed that the nematode species associated with the mulberry plants was M. incognita (Kofoid and White). 

Fig. 2. SEM image of the perineal pattern in Meloidogyne incognita female larva.

The morphological characteristics observed were in consonance with that of those described by Narasimhamurthy (2010). Nalini Singh and Sunita Chahar (2021) reported on morphological description of M. incognita infesting cucumber roots which were also on similar lines. The perennial patterns of female juveniles, morphological characteristics and the DNA-specific markers confirmed the pathogenicity of nematode species, M. enterolobii in the mulberry trees in Hainan Province of China (Sun et al., 2019), M. arenaria on white mulberry in Europe (Castillo et al., 2001) and M. enterolobii in Morus nigra L. seedlings (Paes-Takahashi et al., 2015). Stella et al. (2022) revealed that the species of root-knot nematode associated with papaya plants were M. javanica based on the perineal pattern of female nematodes, specifically the lateral lines.

A roving survey may be conducted in different zones of Karnataka practising sericulture, to study the diversity of RKN infesting mulberry. Molecular characterisation could also be emphasized along with morphological characterisation.

Bharath K.B., Vinoda K.S., Kavitha T.R., Banuprakash K.G. and Shashidhar K.R. (2024). Isolation, Identification and Characterisation of Root-knot Nematode in Mulberry (Morus alba L.). Biological Forum – An International Journal, 16(6): 15-17.