Morpho-molecular characterization of cereal cyst nematode, Heterodera avenae from Hisar region of Haryana

developing effective control measures, particularly when identifying sources of host-plant resistance against Heterodera species (Dababat et al. 2015) [2] . Rapid and precise identification is also essential for quarantine and regulatory purposes. To address these needs, molecular methods have been widely explored for identifying species within this group. The internal transcribed spacer (ITS) regions of ribosomal DNA (ITS-rDNA) have proven to be highly effective for differentiating species within the H. avenae group (Zheng et al. 2015) [22] . Comparing ITS-rDNA sequences of unidentified species with published sequences in GenBank has further facilitated the rapid and accurate identification of most cyst-forming nematodes. The present study focuses on the morpho-molecular characterization of cereal cyst nematodes from Hisar, Haryana region.

Plate 1:

Heterodera avenae cysts and their symptoms on wheat roots

Materials and Methods

Collection of nematode populations

The infested soil was collected from farmer's field of Hisar district of Haryana (India). Cysts were extracted from this soil by Cobb's sieving and decanting technique (Cobb, 1918) [1] ; on 60 mesh sieves (pore size 250 ηm). Cysts retained on 250 µm sieve were then hand-picked under a stereomicroscope and stored at 4 °C for further use. Identification of cysts was done on the basis of cone top structure, and separate pure culture of H. avenae was maintained on wheat susceptible variety (WH 1105) in screen house in 5 kg earthen pots filled with steam sterilized sandy soil for further use in experimentations.

Vulval cone preparation

From pure culture, the vulval cone part of the ten similar looking cysts were cut and mounted in glycerine jelly for some morphological features like semi fenestra shape, underbridge and bullae development. Depending on these structures, the identifications of cysts were done (Handoo, 2002) [5] . Among the selected cysts, five cysts were observed for morphological characters and measured using Zeiss Upright Axio Imager M2 Fluorescence Microscope in Dept. of Plant Pathology, CCSHAU, Hisar. For the preparation of vulval cone, cysts were soaked in water for 24 h. After placing the cyst on a Perspex plate and cut off the posterior end with the help of a small surgical blade and trimmed to small squarish shape to keep fenestral area is in the centre of the piece. After removing the adhering body contents, the portions were transferred to melted glycerine jelly for providing support, on to a glass slide.

Killing, fixing of nematodes and preparation of slides

Juveniles were collected in 100 ml beaker containing a known quantity of water. An equal amount of double strength 8% boiling formalin as fixative, was added to the suspension followed by putting the suspension in a vial and closing the lid tightly. Processing of the cyst nematodes for the permanent mount was done after 24 hours using the glycerol-ethanol method of Seinhorst (1959) [16] . The morphological characters of cyst and juvenile of H. avenae were studied.

DNA extraction

Genomic DNA was extracted from cyst nematodes using a method based on the protocol established by Reid and Pickup (2005) [15] . Eggs and second-stage juveniles (J2s) were extracted by puncturing the cyst and transferred into a sterile 200 ηL PCR tube. Next, 20 ηL of deionized water (ddH2O) and 2 ηL of 10× PCR buffer containing Mg2+ were added. The tube was stored overnight at -80 °C to ensure stabilization. After thawing, 2 ηL of protease K (20 mg/mL) was introduced to the mixture, which was incubated at 65 °C for 90 minutes to promote cell lysis. This step was followed by an incubation at 95 °C for 30 minutes to deactivate the protease K enzyme. To remove debris, the sample was centrifuged at 14,000 rpm for 3 minutes. The resulting genomic DNA was stored at -20 °C for future use in polymerase chain reaction (PCR) analysis.

PCR amplification

PCR amplification of the COX 1 region was done with following JB3-JB5 primers: 1. Forward primer: JB3 (5′-TTT TTT GGG CAT CCT GAG GTT TAT-3′)

2. Reverse primer: JB5 (5′-AGC ACC TAA ACT TAA AAC ATA ATG AAA ATG-3′)

The PCR thermal cycler program consisted of following steps:

• 5 min at 95 °C • 40 cycles of 94 °C for 30 sec • 48 °C for 45 sec, and 72 °C for 45 sec • Final extension for 8 min at 72 °C After PCR amplification, 5 µL of each PCR product was mixed with 1 µL of 6X loading buffer (Fermentas Life Sciences) and loaded on a 1.5% standard TAE buffered agarose gel. After electrophoresis at 100 V for 40 min, the gel was stained with ethidium bromide (0.1 µg/mL) for 15 min and visualized and photographed under UV light. The remaining PCR product was stored at -20 °C.

PCR product purification

PCR products were purified using a PCR purification kit to remove any residual primers, nucleotides, and other impurities. The purified products, along with appropriate sequencing primers, were then sent to SOLS sequencing facility, Cheeka, Kaithal (Haryana) for Sanger sequencing.

The obtained sequences were compared to known sequences in public databases to identify the closest matching pieces by performing a BLAST (Basic Local Alignment Search Tool) search using the GenBank NCBI database.

Results and discussion

Cysts were predominantly lemon-shaped, dark brown with a protruding neck and posterior protuberance (Plate 2). Earlier the cyst colour was white which changes to brown. The cyst wall was dark brown to black in colour. Vulval cone of cysts was bifenestrate with a vulval slit smaller than the fenestral width. Numerous dark brown bullae were prominent (Plate 3). Total length without neck was 653.115±30.597 ηm, neck length as 72.439±2.592 ηm, 512.410±14.449 ηm width.

Fenestral length was 50.032±1.372 ηm, fenestral width 23.017±1.087 ηm, while vulval slit length 10.444±0.278 ηm and vulval bridge width as 9.960±0.216 ηm (Table 1).

Based on the morphological characters, the second-stage juveniles (J2s) were vermiform in shape, slender shaped strong stylet with large anteriorly flattened to concave basal knobs and hemispherical head region, sharply pointed tail (Plate 4). Tail was conoid with hyaline portion. Body length was 620.626±8.481 ηm, max. body width 20.510±1.063ηm, stylet length 24.80±0.47ηm, robust, exhibiting moderately concave stylet knobs. Hyaline length and tail length were 39.45±1.23 ηm and 62.26±2.84 ηm, respectively (Table 1). The method was validated by inspecting underbridge structures of cyst vulval cones. The molecular analysis proved to be a robust tool for species identification and differentiation. The COX 1 sequences obtained from Haryana populations were compared with existing sequences in GenBank, confirming their identity as H. avenae. The results align with previous studies that highlighted the utility of conserved regions for resolving taxonomic ambiguities within cyst-forming nematodes (Subbotin et al. 2000) [18] . The molecular data also facilitated the detection of genetic variability, which could have implications for understanding the adaptability and virulence of H. avenae in different agro-climatic zones.

Conclusion

The present study provides the morpho-molecular characterization of Heterodera avenae populations in Haryana, addressing a significant knowledge gap in understanding this economically important nematode in the region. The combination of morphological and molecular approaches enabled precise identification of the cyst nematode species, which is crucial for developing effective management strategies. Morphologically, the populations studied showed typical characteristics consistent with the H. avenae group, including minor morphological variations that are commonly observed among geographically distinct populations. However, the reliance on morphological features alone can be challenging due to the subtle differences within the group and the influence of environmental factors on nematode morphology.

Conflict of Interest

The authors declare no conflicts of interest.

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