ImportAll import consignments are subjected to the following seed health testing procedures.

Routine testsVisual examination: Each consignment is examined under illuminated magnifying lens (2 X) and cleaned to eliminate the storage pests and pathogens in the form of sclerotia, nematode galls, weed seeds and soil clods. In specific cases, samples are also examined under low power stereo-binocular microscope to detect the presence of seed borne rust, anthracnose and downy mildew spores.

Blotter test: After visual examination each seed sample is plated for pathological examination as recommended by International Seed Testing Association (ISTA) for observation of seed as-sociated pathogens (fungi/bacteria/nematodes). Petri-dishes containing seeds are incubated for seven days at 20 + 20C under alternate light and darkness.

Microscopic examination: After seven days of incubation, samples are examined under a ste-reo-binocular microscope for the presence of pathogens. Seeds showing poor germination or infection by pathogenic fungi, bacteria or nematodes, are rejected. A detailed record of the seed samples and pathogens detected is maintained in a register.

Specific testsRadiography: Chickpea, pigeonpea, groundnut and other legume seeds are X-rayed for detec-tion of latent infestation by insect pests, particularly bruchids and chalcids and their developing instars. The procedure involves exposure of seed samples to soft X-rays emitted by Softex X-ray equipment (Fig. 1) specially designed for the purpose. The seed sample is placed on a Polaroid film and exposed to X-rays. Images of adult insects, instars and eggs can be seen in the print as dark patches on the seed (Fig. 2). Apparently healthy seeds showing internal infestation are rejected for release.

Figure 1. Figure 2.

Agar plate method: The principle of recording in the agar plate test is macroscopical examina-tion of fungal and bacterial colonies. The method is reliable and quick, but one has to be well acquainted with colony characters of different fungi and bacteria on agar media. Seeds are gener-ally pre-treated with mild disinfectant such as sodium hypochlorite (1% aqueous solution) before plating on agar to prevent the growth of saprophytic pathogens. Potato dextrose agar, Potato car-rot agar, Malt extract agar and Oat meal agar for fungi and Nutrient agar and Tetrazolium chloride agar for bacteria are the commonly used media.

Sedimentation: Seed samples of sorghum and pearl millet are picked up at random and washed with distilled and sterilized water, then centrifuged (Fig. 3). The suspension is examined micro-scopically for oospores of downy mildew, smut spores, nematodes and Striga seeds.

Figure 3.

Enzyme-linked immunosorbent assay (ELISA): The test is used to detect seedborne viruses in groundnut seed as well as in plants that are subjected to grow-out test. Generally, 10 seeds per accession are used in the test for economic reasons. A piece of tissue (of about 50 mg) from cotyledon opposite to embryo is removed using a sterilized razor blade. Ten such seed samples of each germplasm line are pooled and processed by ELISA. The cut seeds are placed in a seed storage wooden tray having the same configuration as in ELISA plate (Fig. 4) and hold until the test is completed. The pooled tissues are ground in a mortor at 1:50 w/v dilution of 0.01 M car-bonate buffer, pH 9.6 containing 0.01 M sodium diethyl dithio-carbonate. Appropriate positive and

negative controls are used. Antisera are cross-adsorbed with healthy groundnut seed extracts at 1 g tissue/20 ml of antibody buffer for 1 h at 370C. The antisera to Peanut mottle virus (PeMoV) and Peanut stripe virus (PStV) are used at 1:5000 dilutions. Goat antirabbit Fc specific Ig G conjugated with alkaline phosphatase enzyme is used at 1:5000 dilutions. The substrate used is P-nitro phenyl phosphate @ 0.25 mg/ml. The plates are incubated for 30 min at room tempera-ture. The color developed is recorded visually. Absorbance values are recorded at 405 nm in an ELISA reader. Samples showing absorbance values, 3-times more than that of healthy seeds are regarded as positive.

The ten seeds corresponding to the well showing positive reaction (Fig. 4) to PeMoV and PStV are taken out from the seed storage tray and cotyledon tissue obtained from each seed is tested again individually by ELISA to identify the particular seed(s) infected with the viruses.

Figure 4.

Grow-out: Seeds of groundnut germplasm found negative in ELISA tests are grown after appro-priate fungicidal treatment in plastic pots containing sterilized soil mixed with fertilizer in a green-house. The plants are observed for virus symptoms up to 4 weeks and those that show virus or virus-like symptoms are kept separately.

All healthy looking plants are ELISA tested by pooling the leaf tissues of 10 or <10 plants of each accession. Four-week-old seedlings that are found free from viruses are transplanted in the post-entry quarantine isolation field for further observations until harvest (Fig. 5).

Salvaging procedures and treatment schedules

NBPGR conducts seed health tests, gives mandatory seed treatments as per the ICAR guidelines and releases the consignment for growing in the post-entry quarantine isolation area (PEQIA) or greenhouse at ICRISAT, Patancheru. In case of groundnut the six-week-old seedlings are re-leased to PQL, ICRISAT.

Seed treatment

Seed should be treated with appropriate chemicals prior to planting to avoid the spread of patho-gens. The chemical treatment schedules followed for import of ICRISAT mandate crops are pro-vided in Table 6.

Table 6. Mandatory treatment schedules for import crop germplasm Crop Against (disease) TreatmentChickpea Ascochyta blight Thiabendazole 3g kg-1 seedGroundnut Seedborne fungal pathogens Thiram 2g kg-1 seedPigeonpea Wilt Benomyl+ thiram 2g kg-1 seed eachSorghum Seed rots and molds Benomyl+ thiram 2g kg-1 seed eachMillets Downy mildew Hot water treatment at 550C for 15 min followed by dry seed treatment

with metalaxyl 2g kg-1 seed

Figure 5.

All imported accessions, except healthy chickpea germplasm are grown in the PEQIA and kept under surveillance from sowing until harvest to detect and avoid the introduction of any exotic pests and pathogens. This ensures the release of healthy first-generation seed from imported consignments.

Post-entry quarantine inspection

Plant quarantine, which acts with the principle that prevention is better than cure has the re-sponsibility of preventing entry, spread and multiplication of hazardous pests. Pathogens, which may not be routinely detected in seed examination using blotter test are likely to cause disease on field-grown crops. Therefore, all exotic ICRISAT mandate crop germplasm accessions are grown for one season in PEQIA (Fig. 6). Weekly inspections are undertaken to detect ex-otic pests associated with growing plants. Interceptions made during 1986-2004 in PEQIA are provided in Table 7.

Figure 6.

Table 7. Post-entry quarantine field interceptions of pathogens in crop germplasm accessions at ICRISAT, Patancheru (1986-2004)

Crop/Diseases Causal organism Country of origin

SorghumBacterial leaf streak Xanthomonas vasicola pv.

holcicolaYemen Arab Republic

Bacterial leaf stripe Ralstonia andropogoniDowny mildew Peronosclerospora sorghi Kenya, Mali, Mexico, Namibia, Niger,

Rwanda and USAPearl millet

Downy mildew Sclerospora graminicola USA and Togo

Pennisetum violaceum

Downy mildew Sclerospora sp Niger

Pigeonpea

Wilt Fusarium udum Kenya and Indonesia

Groundnut

Bacterial wilt Ralstonia solanacearum Australia, Brazil and Malawi

Small millet

Blast Pyricularia setariae Zimbabwe