Page | 158 2. PCR-based detection of Xanthomonas campestris pv. campestris: A set of rpf region- based primers namely rpfH_F and rpfH_R were developed for Xanthomonas campestris pv.
campestris. The annealing temperature for primers were optimized at 60oC and specific bands of 304 bp for X. campestris pv. campestris were obtained in PCR (Figure 7). The detection sensitivity of the primer pairs was performed by dilution of genomic DNA and results revealed that it could detect up to 0.1 ng μl-1 of template DNA of the pathogen (Kiran et al., 2019; Kumar et al., 2019). This primer is specific to X. c. pv. campestris and there is no cross amplification with other related fungal and bacterial pathogens.
Figure 7: PCR amplification for X. campestris pv. campestris isolates using specific primers rpfH_F and rpfH_R. Lane M- 1 Kb plus DNA ladder; Lanes 1 to 11- X. campestris pv. campestris isolates, Lane C- Negative control.
In addition to conventional diagnostic tools, LAMP assay and PCR-based molecular are substantial in detecting and intercepting large number of pathogens of quarantine significance on a wide range of crops from different sources/ countries which emphasizes the pivotal role of plant quarantine in minimizing risk of introduction of exotic pathogens associated with PGR importing into the country, otherwise huge losses would have occurred due to exotic pathogens/ diseases.
SECTION III: Detection and Identification of Plant Parasitic
Page | 159 The nematode spreads from one place to other by active or passive means. The active movement through its own locomotory system is important for plant-to-plant or field to field spread. The passive spread is a long distance movement of nematodes by various agents like wind, water, field equipments, planting material etc. Dispersal through wind, rain and floodwater respects no national boundaries. This kind of spread is important at regional as well as national scale. Many nematodes of quarantine significance as well as of economic importance spread into the new, clean or nematode free areas along with these passive agents, particularly with planting material (Ferris et al., 2003). Seed-borne nematodes like A. tritici, Aphelenchoides arachidis, A. besseyi, D. dipsaci) have ability to withstand desiccation, which helps in their survival and dispersal. There are other few species, which use insects as vectors for their transmission (Rhadinaphelenchus cocophilus and Bursaphelenchus xylophilus.
Besides these natural means of dispersal, anthropogenic factors are very common for long distance dispersal of nematodes. Increased trade through land, sea and air tremendously increased over the decades and played very important role in dispersal of devastating nematodes across the boundaries. Exotic nematodes spread through plants, imported grain, plant debris, soil attached to machinery, tyres of vehicles, tools and even shoes. The nematodes like Pratylenchus penetrans, Hoplolaimus spp., Tylenchorhynchus spp.
Rotylenchus minutus, Meloidogyne spp. were intercepted from rooted saplings of various plants during processing of exotic germplasm at ICAR-NBPGR, New Delhi. Besides, seeds of paddy are regularly found infested with A. besseyi.
In India, examples of introduction of quarantine nematode are golden nematode of potato, Globodera rostochinesis and Globodera pallida that spread from Andean mountains to Europe to the India. Burrowing nematode of banana, Radopholus similis is another example, the nematode was introduced in India from Sri Lanka, now present in every banana growing region of India and causing heavy losses (Khan, 1999). Similarly, root-knot nematode, Meloidogyne enterolobii that was reported recently from Tamil Nadu is causing heavy damage in guava plantations. Therefore, quarantine processing of germplasm for nematode detection and its proper management is utmost important to prevent entry and establishment of new nematodes pests into country. It involves careful observation of germplasm, extraction of nematodes stages, their proper identification and control. Germplasm material received in the quarantine laboratory is subjected to detailed examination by employing various nematode detection techniques depending upon the kind of plant material, size of the sample and availability of time. Some commonly used techniques are described below:
Nematode extraction from suspected germplasm
Seeds / grains: Seeds known or suspected to carry seed-borne nematodes are soaked in water which help in softening of seeds. The hard seeds are cut open / teased / crushed which enables the nematodes present in seed to come out in water. The suspension is observed for presence of nematode and if present processed for taxonomic identification.
Page | 160 Plant debris / soil clods: The plant debris or associated soil clods are soaked in water, sieved through nematological sieves of 100, 200, 325 and 400 mesh. The finest sieve (400 mesh per linear inch) allows nematode juveniles and eggs extraction. These are recovered and examined under the compound microscope for identification. Critical examination of accompanying soil is necessary as it carries different stages of nematodes such as eggs, eggmasses, cysts.
Vegetative propagules / roots: The part of the plant tissue especially roots, bulbs, corms, rhizomes etc. is used as a planting material. Migratory endoparasitic nematodes live within root tissue can be extracted using a modified Baermann method. Roots are rinsed, cut into small pieces of 2-3 cm long, macerated using blender with small quantity of water. The incubation for 24 h allows migratory stages to come into water suspension which can be observed under a light microscope. Staining technique is used for quick detection of nematodes are sedentary in nature. The plant tissue is boiled in acid fuchsin lacto-phenol solution for a few minutes and de-stained in clear lacto-phenol. The nematodes, if present, retain the red stain more deeply than the plant tissue and can easily be detected under a stereo microscope.
Figure 1: Quarantine processing for nematode detection and identification
Some important nematodes of quarantine importance include Aphelenchoides besseyi, A.
arachidis, Anguina tritici, A. agrostis, Bursaphelenchus cocophilus, B. xylophilus, Ditylenchus africanus, D. destructor, D. dipsaci and Heterodera goettingiana.
Identification of nematodes
The nematodes detected during quarantine processing of samples are identified using morphological, biochemical and molecular methods. The universal standard keys are used for
Page | 161 morphological identification of nematodes. Morphological identification needs careful processing, fixing and mounting nematodes. The extracted nematodes are concentrated in small quantity of water and killed by pouring hot water (65° to 85°C) over them and then fixed in 4% formaldehyde solution. For taxonomic studies, nematode are dehydrated by adopting the method of Seinhorst (1959) and subsequently mounted on glass slides in anhydrous glycerine for light microscopic studies. Nematodes are later observed under microscope, measurements are taken using an ocular micrometer and de Man’s formula is used to determine pertinent values of measurements.
Morphological identification is now days supplemented with bio-chemical and molecular methods. Biochemical approaches include the separation of proteins, enzyme analysis, esterase profiling etc. Molecular methods are more useful for detection, characterization and study of inter- and intra-specific variability in nematodes. The techniques such as the polymerase chain reaction, restriction fragment length polymorphism, randomly amplified polymorphic DNA, amplified fragment length polymorphism satellite-DNA probes, sequence-characterized-amplified regions (SCAR), high-resolution-melting-curve analysis, real-time-PCR assays and DNA sequencing are being used for nematode characterization and identification.
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