Occurrence and distribution of cyst nematodes (Heterodera spp.) in India

Introduction

Cyst nematodes (Heterodera; Globodera) are the most economically important pests of crops, second only to root-knot nematodes (Jones et al., 2013) [9] . The cyst nematode, upon infecting host plants, induces characteristic histological changes termed the syncytium. Further, the nematode is well adapted to transform the body cuticle into a persistent tanned structure after the death of the female body (termed a cyst) to protect its eggs. Earlier, the genus Heterodera included 12 species (Sharma, 1998) [24] , but currently the number of described species stands at 87 nominal species (Yang et al., 2024; Wen et al., 2024) [28,27] . They are distributed in eight genera: Heterodera (87 species), Globodera (14 species), Cactodera (14 species), Dolichodera (1 species), Paradolichodera (1 species), Betulodera (1 species), Punctodera (4 species), and Vittatidera (1 species), totalling 121 valid species (Handoo et al., 2018; Perry et al., 2018) [6 18] , . The most economically important one is the cereal cyst nematode (Heterodera avenae), which severely impacts cereal crops, particularly wheat. Infected fields can suffer yield losses ranging from 30% to 100%, with a reported loss of up to 28.5% in India (Kumar et al., 2020) [16] . Since 1958, 22 species of Heterodera have been identified in India based on the structures of the vulval cone top (Kaushal et al., 2007; Kaushal, 2013) [10,11] . Among these, H. avenae causes 'molya' disease in wheat and barley, especially in northern regions, leading to potential losses of up to 97.28 million rupees in wheat (Jain et al., 2007) [8] . Another significant species is the pigeon pea cyst nematode (Heterodera cajani), which was first reported in Delhi (Koshy, 1967) [14] . Other economically important Heterodera species infecting maize, sorghum, rice, and banana are H. zeae, H. sorghi, and H. oryzicola, respectively. Over the past several years, there has been no systematic survey conducted on the occurrence and distribution of cyst nematodes in India. Therefore, this study aims to identify Heterodera species based on morphological observations and to understand their presence and distribution across different states in India.

Materials and Methods

Soil sampling and nematode extraction

Soil samples were collected from the fields of various crops grown in 14 states of India. Altogether 67 samples were collected across India. Nematode extraction from soil samples was performed using Cobb's decanting and sieving method (Cobb, 1918) [3] followed by the modified Baermann funnel technique (Schindler, 1961) [21] . The collected nematode suspension was then observed under a stereoscopic binocular microscope to identify the presence of juveniles and males. Cysts were isolated from residue retained on the 60-mesh sieve and further examined using OLYMPUS, Japan, stereo-zoom microscopes (SZ-PT and SZ-9). Cysts were carefully picked using forceps and segregated into different Petri plates based on their types (shape, size and colour) for identification. Second-stage juveniles (J 2s ) that hatched from these cysts were subsequently used for detailed morphological studies.

Morphological observations of Heterodera spp

For species identification, vulval cone tops were prepared from 10 cysts from each population following the methods described by Cooper (1955) [4] and Perry et al. (2021) [17] . The prepared vulval cones were mounted in modified Kaiser's glycerine jelly (Dioni, 2003) [5] . Identification was done based on the cyst's size, colour, shape, vulval cone structures, and J 2s (Handoo et al., 2018) [6] . J 2s collected from the sample were heat-killed and preserved in TAF solution (7 ml of 40% formaldehyde, 2 ml of triethanolamine, and 91 ml of distilled water), and were subsequently placed in glycerol (Seinhorst, 1959) [22] . Permanent and semi-permanent slides were prepared (Handoo, 2002) [7] , and photomicrography was taken under stereoscopic (Leica, Germany) and compound microscopes (Leica DMLB, Germany) using an image-analysing camera.

Results and Discussion

A total of 67 soil and root samples were collected from 14 states across various agroecological zones in India. The sampling included a diverse range of crops, such as cereals, millets, pulses, oilseeds, fruits, vegetables, cash crops, and one weed (Cyperus rotundus). Table 1 and 2 provides details on crops, locations, and states and the identified Heterodera species, as well as crops and locations where no cyst nematodes were recorded. The data represent presence or absence records only; cyst counts and population densities are not included due to the variability in sampling times throughout the year. The analysis of soil samples revealed the presence of four species of Heterodera: Heterodera avenae, H. zeae, H. cajani, and H. mothi (as detailed in Tables 1 and2; Figure 1). Populations were identified morphologically based on diagnostic features including cyst shape, size, colours, vulval cone structures and J 2s head and tail shapes, etc. Among the 67 samples collected from different agroecological zones, H. avenae, commonly known as the cereal cyst nematode, was found in Delhi, Uttar Pradesh, and Rajasthan. H. cajani, the pigeonpea cyst nematode, was identified in Delhi, Uttar Pradesh, Rajasthan and Assam. H. zeae was found in Delhi and Uttar Pradesh, while H. mothi, known as the Nutgrass cyst nematode, was identified only in Delhi. The presence of Heterodera was found in 15 out of 67 samples (22.4%). Fifty-two samples (77.6%) were negative. The identified species included H. avenae in 12 samples, H. zeae in eight, H. cajani in five, and H. mothi in two. H. avenae was the most widespread species, found in wheat (5 records), pigeonpea (Cobb, 1918) [3] , maize (Bhatti et al., 1981) [2] , and soybean (Bhatti et al., 1981) [2] across Rajasthan, Uttar Pradesh, and Delhi. H. zeae was recorded in Uttar Pradesh and Delhi, affecting wheat, maize, soybean, and pigeonpea (with 2 records each). H. cajani exhibited the broadest geographic distribution, occurring in Uttar Pradesh, Delhi, Assam, and Rajasthan, specifically on wheat, pigeonpea, and soybean. This study first recorded the occurrence of H. cajani from Assam in a soybean sample. H. mothi was found associated with motha (C. rotundus L.), a common weed, with two samples collected at IARI in Delhi. Among the crop field samples, the occurrence of cysts was found in soybean (3 of 6 samples positive), wheat (5 of 11), pigeonpea (3 of 8), maize (2 of 14), and motha (2 of 2). No cyst nematodes were found in chickpea, mungbean, rice, sorghum, finger millet, pearl millet, cowpea, other beans, vegetables, litchi, mango, taro, sugarcane, or cotton. Samples with cysts were primarily found in four regions. Delhi (IARI) had the highest prevalence, with 6 positives out of 11 samples, showing the presence of all four species. All four samples from Uttar Pradesh and all four samples from Rajasthan were positive. In Assam, one out of four samples were positive. All samples from the other ten states-Telangana (13 samples), Chhattisgarh (Handoo et al., 2018) [6] , Punjab (Dioni, 2003) [5] , Bihar (Cooper, 1955) [4] , Jharkhand (Cooper, 1955) [4] , Arunachal Pradesh (Cobb, 1918) [3] , Meghalaya (Cobb, 1918) [3] , West Bengal (Cobb, 1918) [3] , Tamil Nadu (Bhatti et al., 1981) [2] , and Odisha (Ali et al., 2005) [1] -had no cyst nematodes detected in the samples. More than one species of cyst nematodes was common. Nine out of the 15 samples (60%) contained two or three species, while six samples had a single species. H. avenae was present in all nine mixed samples, most often alongside H. zeae. Three-species mixtures occurred in wheat from Bijnor, UP and in pigeonpea from IARI, Delhi and Meerut, UP. The presence of multiple species in the crop field may be attributed to the residual populations from previous crops in the rotation or to weed hosts that support these populations. The cysts of H. avenae, which complete only one generation during the wheat-growing season, were observed in maize fields. In contrast, H. cajani, a multivoltine species with a host range limited to leguminous crops, was commonly found in both maize and wheat fields. Two species of Heterodera viz., Heterodera avenae infecting graminaceous hosts and H. cajani infecting leguminous crops, are considered India's most frequently encountered and economically important species. Cereal cyst nematode (H. avenae and H. filipjevi) has a very limited host range, mostly infecting wheat, barley, and oat, all belonging to the graminae family. H. avenae was identified by its cyst shape, which is predominantly lemon-shaped, varying in colour from light brown to black. The individual cyst sizes were variable (Figure 2A). The cone-top structure was ambifenestrate, with a short vulval slit (9-10 µm) in the fenestral bridge and pronounced bullae in the vulval cone structure (Figure 3A). Its first occurrence was reported from Sikhar district in Rajasthan by Vasudeva (1958) [26] . Currently, it is widespread in the northern wheat-growing regions of India and causes the characteristic 'molya' disease in wheat and barley. Among cyst nematodes reported in India, H. avenae is the most economically significant species and a considerable concern for wheat cultivation in northern India. In India, wheat is a major crop affected by H. avenae, causing yield losses of 49% (Bhatti et al., 1981) [2] . dark brown to black, with a narrow posterior end and a short protuberance (Figure 2B). Its cone-top structure is ambifenestrate, featur ing finger-like bullae, a long vulval slit, and a thick but short underbridge (Figure 3B). This species is the second most important cyst nematode affecting leguminous crops such as pigeonpea and cowpea; however, its small cysts and lack of above-ground symptoms make detection challenging. H. cajani females are very different from females of other species. A large egg sac, almost twice as big as the cyst itself, is a distinguishing feature of the nematode (Ali et al., 2005) [1] . H. cajani has been found primarily in sandy-loam and black cotton soils in India (Reddy et al., 1993; Varaprasad et al., 1997) [20,25] . The 'pearly root' of pigeonpeas (Sharma, 1991) [23] is a confirmatory symptom of H. cajani infection. The occurrence of H. cajani has been reported earlier in Delhi (Rao et al., 2011; Kumar et al., 2022) [19,15] . H. zeae was identified by having a light brown, thin-walled cyst that is lemon-shaped, with a prominent vulval cone (Figure 2D). The vulval cone has an ambifenestrate structure and features finger-like cross-bullae located beneath the underbridge, often leading to confusion with H. cajani. It also possesses a long vulval slit and a short vulval bridge, with an indistinct anus (Figure 3B). H. zeae was first identified in Chapli, Udaipur district, Rajasthan, India by Koshy et al. (1970) [13] . Subsequently, it was found in numerous maize-growing regions across northern, western, central, and southern India (Kaushal et al., 2007) [10] . H. mothi cysts are light to dark brown, lemon-shaped, and transparent, allowing the eggs to be visible from outside. They also feature a protruding vulval cone (Figure 2C). The cone top is ambifenestrate with prominent bullae, and the lengths of the vulval bridge and slit are equal (Figure 3C). The anus is distinct. A notable characteristic of the J 2s is its long tail with an acute terminus and an elongated hyaline part, with the phasmid located far from the anus (Figure 4C). The J 2 is distinct from the other three species of Heterodera (Figure 4). This nematode species was first described in Uttar Pradesh, India by Khan and Husain (1965) [12] in nut grass (Cyperus rotundus). Although it has since been found in northwestern India, its economic significance to crops remains unclear.

Conclusion

Cyst nematodes (Heterodera spp.) were detected in approximately one in five samples, primarily in wheat, maize, soybean, and arhar in Delhi, Uttar Pradesh, and Rajasthan. Four species of Heterodera: H. avenae, H. cajani, H. zeae and H. mothi were identified based on cyst, vulval cone and second-stage juvenile morphology. H. avenae was identified as the dominant species, and mixed-species infestations were common. Motha (Cyperus rotundus) was found to harbour H. mothi. Samples collected from southern, eastern, and northeastern states were uniformly negative; however, the limited sampling in these regions indicates a need for more extensive studies with population counts to confirm and get the distribution pattern in India. The study confirms the occurrence of H. cajani in soybean from Assam. It is believed to be the first report in Assam, India.

Acknowledgement

AcknowledgementThe authors thank Post Graduate School, ICAR-Indian Agricultural Research Institute, New Delhi and Division of Nematology for supporting this study.

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