The wild jute (Corchorus aestuans) belongs to the genus Corchorus, comprising about 40–100 species in the family Tiliaceae, and is native to tropical and subtropical regions of the world. The cultivated jute species (Corchorus olitorius and C. capsularis) are commonly grown as one of the most important fibres in Asian, African, and European countries. Its tender leaves at early stages are also consumed as green leafy vegetables (Choudhary et al. 2013). The wild species of jute (C. aestuans) known locally as Nalte saag is cultivated primarily as a popular minor leafy vegetable in some parts of the West Bengal, India. The edible species of Corchorus are good sources of proteins and vitamins (A, C, and E). They are also rich in mineral nutrients such as calcium and iron (Steyn et al. 2001; Dansi et al. 2008). The cultivation of this species has been adopted by the poor farmers of West Bengal during the summer season as a leafy vegetable for economic and nutritional benefit from April to June. Traditionally, it is grown in the bed of a shallow pond that gets dry during the summer months. Nowadays, it has been successfully grown in open fields with adequate nutrient supplements. Root-knot nematodes (Meloidogyne spp.) are well-recognized polyphagous pests of various crops, including vegetables grown in tropical and sub-tropical parts of the world. The root-knot nematodes (RKNs) have been documented as one of the most damaging nematode pests of crops in India (Khan et al. 2014). The infection of RKN species on the wild jute (Corchorus aestuans) remained unnoticed when its cultivation was limited to the bed of the shallow pond. In a preliminary survey of RKNs infecting vegetables, we noticed a heavy infection on wild jute (Corchorus spp.) from the Brahmani river-bank areas of Nalhati, in the Birbhum district of West Bengal. Therefore, the present investigation focused on the identification and establishment of the pathogenic potential of the RKN species infecting wild jute species.

Twelve RKN-infected root samples were collected from Nalhati (24.595 N, 88.05E), Birbhum (West Bengal), India, and processed in the Plant Health Clinic laboratory, Directorate of Research, Bidhan Chandra Krishi Vishwavidyalaya, West Bengal (India). Nematode species identification was initially performed using morphology and morphometric of special characts of females, males and second-stage juveniles (J2s), and molecular analysis was used to confirm the species identification. The J2s and males from the fresh root samples, were recovered by Cobb’s decanting and sieving method (Cobb 1918), followed by modified Baermann’s technique (Schindler 1961). A part of the infected root sample was stained with Acid Fuchsin method (Byrd et al. 1983). The females were dissected from stained roots and used to take the measurements, while the males and J2s were processed using glycerol-ethanol method (Seinhorst 1959). The nematode specimens were mounted in anhydrous glycerine on the glass slide and sealed by the wax-ring method (de Maeseneer and d’Herde 1963). Measurements of special characters of the female were taken, and the posterior end was used for the preparation of the perineal patterns (Taylor and Netscher 1974). Individual egg masses were hand-picked from the stained roots and placed on a glass slide with glycerine for observation under a compound microscope at X200 magnification. The measurement was taken using ocular micrometres at suitable magnification. The photomicrograph of the specimens was taken under a compound light microscope (ZEISS-Axioskop 40) fitted with a digital camera (Canon PowerShot S3 IS) for recording morphological variations. To confirm the nematode species identification, different SCAR primer sets, including one M. graminicola species-specific (SCAR) were used.

Genomic DNA was extracted from a single J2 using the worm lysis buffer method following Carvalho et al. (2019). The crude DNA extracted was amplified using the following SCAR primer sets: SCAR-MgFW/SCAR-MgRev for M. graminicola; Finc/Rinc for M. incognita; Fjav/Rjav for M. javanica; and MnSCARF/MnSCARev for M. naasi (Fanelli et al. 2017). Amplifications of extracted DNA with SCAR markers was performed using a thermal cycler (SureCycler 8800, Agilent) under following conditions: initial denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 60 °C for the 30s, and extension at 72 °C for 7 min. A 100 bp (Takara) ladder was used to determine the product size of amplified DNA following electrophoresis of 10 µl of DNA on a 1.2% agarose gel (Fanelli et al. 2017).

A single egg-mass progeny of M. graminicola collected d from the natural RKN population infecting wild jute and maintained on a susceptible rice cultivar (IET-4094) was used for the pathogenicity test. The test was carried out on an earthen pot (10 cm dia.) under net-house conditions. The jute seed (cv Local, procured from the sampling areas) is directly sown in the cemented pot containing 1000cm3 of sterile soil with the required amount of organic manure for crop growth. Two weeks after sowing, the seedling was inoculated with 0 (control), 10, 100, 500, 1000, and 5000 freshly hatched J2 suspensions of M. graminicola around the root zone of each plant by making three holes. Each treatment was replicated six times and suitably randomized. The plant growth parameters, viz., shoot length, fresh shoot weight, dry shoot weight, root length, fresh root weight, dry root weight, and nematode infection included number of galls/plant and galling severity (on a 1–5 scale where 1 = no galls/plant, 2 = 1–10 galls/plant, 3 = 11–30 galls/plant, 4 = 31–100 galls/plant, and 5 = more than 100 galls/plant per root system) (Khan et al. 2014) were recorded. The final populations (J2, female and male) were recovered from the root system (5 g) and soil (100 cm3) by modified Baermann’s technique (Schindler 1961) and Cobb’s decanting and sieving method (Cobb 1918). Then the final nematode population (root and soil) was used to determine reproduction factors (R) = Pf/Pi, where Pf represents the final and Pi represents the initial population of the nematode (Oostenbrink 1966). All statistical analysis and figures were prepared using ‘ggstatsplot’ package in R (version R-4.2.1, R Core Team, 2021; statistical software.

The root galls induced by the root-knot nematode species wild jute species are quite distinctive; they are typically terminal, forming bead-like structures almost like those on rice. However, the egg masses are clearly visible outside the root (Fig. 1F). Morphological and morphometric studies carried out using the RKN infecting wild jute (C. aestuans) at various life stages (Fig. 1; Table 1) were compared with that available from the original description of M. graminicola (Golden and Birchfield 1965; Mulk 1976; Jepson 1987; Handoo et al. 2003). However, there were little deviations in the measurements from the previous observations. As with other root-knot nematodes, the population from the different hosts, geographic locations, and cropping systems may contribute to induced variations in morphometrics and often make it difficult to differentiate M. graminicola from closely related species such as M. oryzae, M. graminis, and M. triticoryzae infecting rice and wheat crops.

Fig. 1
figure 1

Corchorus aestuans infected by Meloidogyne graminicola: A- Male, B- Second-stage juvenile (J2), C- Anterior and posterior ends of J2, D- Anterior and posterior ends of male, E- Flowering stage of Corchorus aestuans, F- infected root with eggmasses (arrowhead showing eggmass), G- Perineal pattern, H- Females, and I- Infected root system of C. aestuans. Bar length: A-100 μm, B-30 μm, C-D- 10 μm, F-2 mm, G-20 μm, H-200 μm

Table 1 Morphological and morphometric parameters of different life stages of rice root-knot nematode (Meloidogyne graminicola) population infecting wild jute in West-Bengal, India. Note: Measurements were taken from 20 individuals of each life stage. Each body parameter (in µm) has been represented as mean± SE with their respective range

Our findings are in agreement with those reported previously for M. graminicola. The identified slides of four second-stage juveniles (No. NNCI-2022/1), three males (No. NNCI-2022/2) and four perineal patterns (No. NNCI-2022/3) are deposited in the National Nematode Collection of India, New Delhi. Further, the DNA amplification using the species-specific SCAR primer set for M. graminicola yielded a single fragment of 640 bp l (Fig. 2). No amplifications were found on other species-specific SCAR primer sets except for M. graminicola. Thus, the species is further confirmed as M. graminicola.

Fig. 2
figure 2

Species-specific amplification by using SCAR primers. Mg: Meloidogyne graminicola, Mi: M. incognita, Mj: M. javanica and Mn: M. naasi

The pathogenicity results are presented in Table 2. Root galling severity measured based on a root-knot index (RKI) on a scale of 1–5 (Khan et al. 2014) was found to increase with inoculum density. RKI varied significantly (Kruskall-Wallis χ2 = 20.36, p < 0.01) across inoculum levels. No significant difference in RKI was observed between inoculum density of 500 and 1000 J2s/ 1000 cc soil; however, it decreased at 5000 J2s level (Table 2). At the higher inoculation level, the root system of the crop is damaged badly. Reproduction factor varied significantly (Kruskall-Wallis χ2 = 23.09, p < 0.01) across different levels of inoculum. Highest RF of 145 was observed at an inoculum density of 10 J2s/1000 cm3 soil. With increased inoculum density, RF decreased significantly.

Table 2 Effect of different inoculum densities of Meloidogyne graminicola on nematode reproduction and plant growth wild jute in West Bengal, India

A significant reduction of plant growth parameters was observed with increased inoculum density of J2s. Control plants were healthier than inoculated ones. While the average shoot length in the control plant was found to be 40.5 cm, at an inoculum density of 5000 J2s, it was 21.13 cm. Significant (Kruskall-Wallis χ2 = 27.42, p < 0.01) variation in shoot length was found across the treatments. Both fresh (Kruskall-Wallis χ2 = 27.94, p < 0.01) and dry (Kruskall-Wallis χ2 = 23.14, p < 0.01) shoot weight reduced significantly with an increased inoculum density. A similar pattern was observed for root parameters. Both fresh and dry root weights didn’t show significant differences up to an inoculum level of 500 J2s/ 1000 cc soil. However, a sharp decline in weight was observed when inoculum density reached afterwards 1000 J2s per 1000 cc soil (Table 2).

The rice root-knot nematode (Meloidogyne graminicola) is a global nematode pest of rice. More than 100 plant and weed species have been reported as hosts (MacGowan and Langdon 1989; Jain et al. 2012). In India, this nematode species is widely distributed (Khan et al. 2010) and is a serious pest of rice (Jain et al. 2012). M. graminicola was found in rice from West Bengal (Pal and Jayapraksh 1983; Khan et al. 2010) and many other weed species, mostly belonging to different families, including Gramineae (Khan et al. 2004). The two jute species (Corchorus olitorius and C. capsularis) used in this study, are commercially cultivated as fibre crops in West Bengal, India and Bangladesh. In West Bengal, rice cultivation coincides with the growing season of popular jute cultivars. However, M. graminicola has never been reported to infect jute in a rice-jute crop sequence. This is the first report of a RKN natural infection of wild jute species in the field. The galls induced by M. graminicola on wild jute species are a little different from the typical terminal root gall on rice, where the egg mass remains concealed in the cortex tissue, but on wild jute, the egg masses are visible from outside the root.

More than 100 hosts, including fodder, fruit, weeds, etc. (MacGowan and Langdon 1989; Jain et al. 2012), 77 weed hosts (Roy 1977) from Assam, and 17 weed hosts from West Bengal (Khan et al. 2004) have been documented. Bridge et al. (2005) reported several hosts of M. graminicola, including rice and an isolate infecting Corchorus capsularis. Thus, the susceptibility of hosts to M. graminicola varies with crops and populations. There were differences in M. graminicola isolates for infectivity on different crop hosts and varieties (Pokharel et al. 2010). For example, the Nepalese isolates reproduced on jute cv Deshi but not on cv Tosa (Pokharel et al. 2007). Pathogenic variability in M. graminicola populations from India was observed in six resistant rice genotypes, with the Jorhat isolate from Assam being considered highly virulent to rice (Singh et al. 2013). To our knowledge this is the first report of M. graminicola infecting wild jute in West Bengal, India.

The present study identified M. graminicola infecting a wild species of jute (C. aestuans) and conclusively proved that C. aestuans is a good host of M. graminicola. This finding also indicated that the pathogenic population variability might occur in M. graminicola in West Bengal. Taking into consideration that this wild jute species is a popular and nutritious underutilized vegetable crop that is being cultivated in open-field conditions in this region the infection of M. graminicola in this crop could be a potential threat to its profitable cultivation. Further investigation is essential for understanding pathogenic variability in M. graminicola on many crops, including cultivated jute species, which could also suffer yield losses.