SEED HEALTH TECHNOOGY H.S.

Prakash and Jan Torp*

Professor, Department of Applied Botany & Biotechnology, University of Mysore, Mysore-570006 India. *Director, Danish Seed Health Center for Developing Countries & Associate Professor, Department of Plant Biology, University of Copenhagen, Denmark. 1. INTRODUCTION Since the scope for expansion of cultivable area is very limited, we have to look for maximizing the productivity of available area through improved utilization of resources, cultivation practices and inputs. In this context the supply of quality seed is strategically important. Quality seed should ensure, genetic identity and physical homogeneity, high standards of vigor and germination, and the seed should be free from weeds and other crop seeds, impurities and free from seed-borne diseases that may constitute a risk to crop establishment. Seed health refers to presence or absence of disease causing organisms like fungi, bacteria and viruses, animal pests such as eelworms and insects, as well physiological conditions such as trace element deficiency (ISTA, 2003). In the past, seed health aspects were not always an essential component of seed quality. However, incidence of more and more pathogens in seeds and their transmission resulting in epidemics in crop production is raising awareness about seed health issues among seed producing agencies, researchers, seed quality control agencies as well as among extensionists, farmers and other seed consumers. This resulted in increased emphasis upon occurrence and importance of seedborne pathogens in seeds, seed-borne nature, seed-transmission of the pathogen, reestablishment of pathogens in seeds, epidemiology of seed-borne diseases and management of seed-borne diseases in agricultural and horticultural crops. Reliable, repeatable and economical diagnostic procedure is an important pre-requisite in managing seed-borne diseases. Serological tests especially Enzyme linked immunosorbent assay (ELISA) and dot-blot techniques are being used in seed health testing. Modern molecular biology tools especially methods based upon Polymerase Chain Reaction (PCR) is being assessed for suitability in seed health testing. Many high yielding varieties have shown susceptibility to different diseases and many of these diseases are seed-borne. The seed primordium or the maturing seed may be infected either (i) directly from the infected plant through the flower or fruit stalk and the seed stalk or directly from the seed surface, or (ii) infection from outside may be introduced through stigma or ovary wall or pericarp, and the fruit stalk, and later through the seed coat. A pathogen may penetrate into several other parts of the seed and in turn infect them. The infection/contamination of the seed may occur during harvesting, threshing and processing. The pathogen may thus be carried with the seeds in three ways. (i) Admixture: Pathogens are independent of seeds but accompany them, eg. Ergot sclerotia mixed with healthy seeds during threshing. (ii) External: The pathogen may be present on seed surface as spores, oospores and chlamydospores as in case of Karnal bunt of wheat, covered smut of barley, downy mildew of pearl millet etc.

(iii) Internal: Pathogens establish within the seed with definite relationship with seed parts as in Ustilago tritici (Loose smut in wheat). The seed-borne pathogens may result in (i) reduced germination/crop establishment (ii) discoloration and shriveling of seeds (iii) development of plant diseases leading to epidemics (iv) loss of yield and quality (v) distribution of pathogens to new areas (vi) introduction of new strains or physiologic races of the pathogen along with new germplasm from other countries (vii) toxin production in infected seed. Healthy seeds are harvested from a healthy crop. Production of healthy crop starts from selection of healthy seeds for raising the seed crop. Good seed health can be ensured through appropriate practices during crop cultivation, during harvest and storage. For a transplanted crop the nursery plants should be free from any diseases. Even while transplanting, infected seedlings shall be rejected. All prophylactic steps should be followed to grow a healthy crop. Ultimately the seed shall be collected from healthy plants/plots followed by proper cleaning to remove soil debris and other plant parts, and by proper drying. 2. SEED-BORNE DISEASES/PATHOGENS Seed borne diseases are generally caused by fungi, bacteria, virus and nematode. The major group of pathogens belong to fungi, bacteria, virus. 2.1 Brief description of seed-borne fungal pathogens Seed-borne fungal pathogens have been reported from almost all agricultural and horticultural crops. The description of important seed-borne fungal diseases has been given in table 1. A majority of the seed-borne fungi belong to deuteromycetes and easily sporulated on seeds upon incubation and they can be identified based on colony characteristics considering the pattern of arrangement of conidia on the conidiophore, conidial morphology or fruiting bodies. Several downy mildew pathogens belonging to oomycetes, ergot pathogens of ascomycetes and smuts of basidiomycetes are also seed borne. Field symptoms of some important seed-borne fungal diseases are shown in plate 1 and 2. Table 1: Brief description of important seed-borne fungal diseases of cereals
Disease
Rice Brown spot Bipolaris oryzae

Pathogen

Description
Disease on the leaves are small, circular, dark brown or purplish dots. Fully developed lesions are oval, brown, with grey or whitish centres and are up to 1 cm long in susceptible cultivars. Symptoms mainly appear on the seedlings, leaves and the grains. When seedlings are infected, necrotic spots develop in the roots and coleoptiles. Heavily infected seedlings eventually die. This disease can infect the rice plants at any stage of the growth, but it is more frequent in the nursery and flowering stages. Spots or lesions are produced on leaves, nodes, different parts of the panicle and the grains. Usually seen on mature leaves of older plants. Symptoms are zonate lesions starting in the form of oblong or diamondshaped water soaked blotches, eventually developing into

Stack burn

Alternaria padwikii (Trichoconiella padwickii)

Blast

Pyricularia oryzae

Leaf scald

Rhynchosporium oryzae

large areas encircled by dark brown bands with light brown haloes.

Disease
Udbatta disease

Pathogen
Ephelis oryzae

Description
Disease seen only at the time of panicle emergence. An erect grayish white cylindrical rod much like Agarbatti (incense rod) emerges from the boot leaf sheath instead of a normal ear head, covered with white mycelium, no grains will be formed. Smut sori are produced within the ovaries, which remain covered by the glumes. They may burst at maturity. Spore mass granular and black, sterile cells intermixed with the spores, few to many, usually globose, hyaline to yellowish. Symptoms mainly seen on maturing panicles. Usually few, occasionally several gains are affected in each panicle, on the surface of which spherical smut-spore-like, olive green to blackish-brown chlamydospres are formed. Due to infection by several microorganisms on the glumes or kernels or both, infection may occur before or after harvest.

Kernel smut

Neovossia indica (Tilletia barclayana)

False smut

Ustilaginoidea virens

Grain discoloration

Alternaria sp., Fusarium spp., Curvualria lunata, Bipolaris oryzae, Phoma sorghina & Nigrospora oryzae Claviceps fusiformis

Pearl millet Ergot

Disease becomes evident at the time of flowering. It is easily recognized by small droplets of creamy to pink, mucilaginous fluid exuding from the infected florets in different parts of the ear. Systemic infected plants are chlorotic stunted and occasionally have white stripe leaves and abnormal seed set.

Downy mildew

Sclerospora graminicola

Wheat Loose smut

Ustilago tritici

Mainly visible at the time of ear emergence. In infected plants, a few or all the ears are transformed into a black powdery mass. In rare cases, an infected ear may be partially diseased. Lesions on the leaves caused by Race O are elongated between the veins, tan, 2-6 x 3-22 mm long, with limited parallel margins and buff to brown borders. Rotting commonly affects the roots, plant base, and lower internodes. Rot normally begins soon after pollination and becomes more severe as the plant matures. A whitish pink to salmon discoloration of the pith, stalk breakage and premature ripening are the same as for Giberella stalk rot. A salmon pink to reddish-brown discoloration first appears on the caps of individual kernels or groups of kernels scattered over the ear. Grains severely infected appear almost normal except for slight pink or black discoloration on a small portion of the surface; internally the grain appears normal.

Maize Southern corn leaf blight Fusarium stalk rot

Drechslera maydis

Fusarium semitectum Fusarium verticillioides Fusarium graminearum

Ear/kernel rot

Fusarium verticillioides

Sorghum Grain mould

Fusarium semitectum Fusarium moniliforme Curvularia lunata

2.2 General description for seed-borne bacteria Several plant pathogenic bacteria are also seed-borne. The seed-borne bacteria are of special significance in rice, french bean, tomato, crucifers, cotton and soybean. The seed-borne bacteria belong to Pseudomonas, Ralstonia, Burkholderia, Acidovorax, Xanthomonas, Clavibacter and Curtobacteria. The description of important species of seed-borne bacteria is given in table 2. Field symptoms of few seed-borne bacterial diseases are shown in plate 1 and 2. Table 2: Description of important seed-borne bacterial diseases
Disease
Phaseolus vulgaris Halo blight

Pathogen
Pseudomonas savastanoi pv. phaseolicola (Burkholder) (Syn. Pseudomonas syringae pv. phaseolicola) Xanthomonas axonopodis pv. phaseoli (Smith) (Syn. Xanthomonas campestris pv. phaseoli) Curtobacterium flaccumfaciens pv. flaccumfaciens (Hedges) (Syn. Corynebacterium flaccumfaciens pv. flaccumfaciens) Pseudomonas savastanoi pv. glycinea (Coerper) (Syn. Pseudomonas syringae pv. glycinea) Xanthomonas axonopodis pv. glycines (Nakano) (Syn. Xanthomonas campestris pv. glycines) Clavibacter michiganensis subsp. michiganensis (Smith) (Syn. C. michiganense subsp. michiganense) Xanthomonas vesicatoria (Doidge) (Syn. X. campestris pv. vesicatoria)

Description
Gram-negative rod, occurring singly or in pairs, motile with 1-6 polar or bipolar flagella. Small water soaked spots on cotyledons are first symptoms. The spots become red-brown, necrotic & are often, not always, surrounded by a light green halo, due to action of phaseolotoxin. Aerobic, Gram-negative rod, motile with a single polar flagellum. Small water soaked spots on leaves, petioles, pods and stems are the first symptoms. On leaves these spots become reddish-brown and necrotic, surrounded by a bright yellow halo. Aerobic, Gram-positive bacterium, slightly curved rods with some straight rods and wedge shaped forms, single or in pairs, non-capsulated motile with 1-3 polar or lateral flagella. The bacteria can be located internally and externally on bean seed. The bacterium is a motile, Gram-negative rod with rounded ends, with one to several polar flagella. The bacterium is mainly located in the seed coat infected during harvest but it is also found internally in the cotyledons The bacterium is a motile by single polar flagellum, single, straight rods. Small, light green to red-brown spots with a raised centre that develop first at the lower side of the leaf are the first symptom. In later stages the raised spots shrivel and develop into irregular spots with a yellow halo. The bacterium is Gram positive, aerobic, curved rods, singly or in pairs. Initially vascular infection unilateral wilting of leaflets. Later, severe wilting occurs and fruits show characteristic marbling, cankers are formed on stems and petioles during later stages of pathogenesis. Aerobic, Gram-negative rod, motile with a single polar flagellum. Irregular green water soaked spots that become brow black and necrotic in later stages on cotyledons. On fruits small light green to brown-black, oval to irregular blisters with a water soaked margin develop, show a sunken necrotic centre surrounded by a cracked epidermis. Gram-negative rods, aerobic, motile with a tuft of polar flagella. Rapid wilting of leaves and stems occurs. Sometimes brown steaks on the stem may be observed above the soil line. Grey-white bacterial slime exudes from vascular bundles, which are broken or cut.

Common and fuscous blights

Bacterial wilt

Glycine max Bacterial wilt

Bacterial pustule

Lycopersicon esculentum Capsicum sp. Bacterial Canker

Bacterial spot

Lycopersicon esculentum Capsicum sp. Solanum melongena Bacterial wilt

Ralstonia solanacearum (Smith) (Syn. Burkholderia solanacearum)

Conti.. 5

Disease
Brassica spp.

Pathogen
Xanthomonas campestris pv. campestris (Pammel) Dowson (Syn. Xanthomonas campestris ) Pseudomonas syringae pv. lachrymans (Smith and Brian) (Syn. Pseudomonas lachrymans) Xanthomonas axonopodis pv. malvacearum (Smith) (Syn. Xanthomonas campestris pv. malvacearum) Pantoea stewartii subsp. stewartii (Smith) (Syn. Erwinia stewartii)

Description
An aerobic, Gram-negative rod, occurring singly or in pairs, motile with a single polar flagellum. Along the margin of leaves V shaped yellow sectors with blackened veins develop. These sectors enlarge, become brown and necrotic, papery and may cover the whole leaf. An aerobic, Gram-negative, non-spore forming rod, capsulated, 1-5 polar flagella. Water soaked spots on leaves and petioles often surrounded by a shallow white-yellow to brown halo. These spots become angular, because larger veins will hamper extension of the diseased tissue. The bacterium is an aerobic, Gram-negative rod, motile by one polar flagellum. Green, water soaked spots on the underside of the leaves. These spots become angular and turn brown to black and are visible on the upper surface. Spots can also occur on stems and bolls. A facultative anaerobe, Gram-negative, non-motile rod, in pairs or in short chains. Short to long pale-green to whiteyellow streaks on the leaves are formed. Streaks turn brown on later stages of the disease. The entire vascular tissue may be infected and bacteria can be found in roots, stalks, leaves, tassels, cobs, husks and kernels. The pathogen is a Gram-negative bacterium, rods with one and rarely two polar flagella. No fluorescent pigment is produced. Plants become grey-green and then yellow; in later stages they wilt and die. The vascular bundles show yellow to brown discoloration and roots are destroyed. Cells are Gram-negative, non-spore forming rods, one or two polar flagella. Water soaked leaf spots are first symptom of the disease. Spots enlarge and elongate, become reddish-brown with a pale brown centre and may cover a larger part of the leaf. The bacterium is a Gram-negative, aerobic, capsulate rod, occurring singly, rarely in pairs, but not in chains, motile with a single polar flagellum. On the leaf blade pale green to yellowish, water-soaked stripes develop, often near to the top of the leaf. Sometimes milky to yellowish bacterial slime can be observed on young stripes under humid conditions. The bacterium is a Gram-negative rod, occurring singly or in pairs, single polar flagellum, no capsules, nor endospores. Symptoms first appear on the lower part of the leaf sheath, where water soaked dark green longitudinal stripes are formed. They soon turn dark brown. Aerobic Gram-negative rods with 1-3 polar flagella, encapsulated, without spores. Initial, symptoms include discoloration or pale yellowing at the basal part of the husk, which rapidly advances over the entire husk, becoming grayish-white to yellowish-brown or reddish brown.

Cucumis spp. Angular leaf spot

Gosypium sp. Bacterial blight

Zea mays Bacterial wilt

Sorghum vulgare Bacterial leaf stripe

Burkholderia andropogonis (Smith) (Syn. Pseudomonas andropogonis) Xanthomonas vasicola pv. holcicola (Elliot) (Syn. Xanthomonas campestris pv. holcicola) Xanthomonas oryzae pv. oryvae (Ishiyama) (Syn. Xanthomonas campestris pv. oryzae)

Bacterial leaf streak

Oryza sativa Bacterial leaf blight

Bacterial stripe

Acidovorax avenae subsp. avenae (Manns) (Syn. Pseudomonas avenae)

Bacterial Grain Rot

Burkholderia glumae (Kurita & Tabei) (Syn. Pseudomonas glumae)

2.3 Seed-borne viruses Plant viruses cause systemic infection. Except for the meristem, all other tissues are infected. However, all the plants viruses are not seed transmitted. The developing seed may escapes viral infection due to limited vascular and plasmodesmatal connection, even if the virus get an entry into embryo, the embryonic tissue may not support the replication of the virus. Of the 13 families and 71 groups, only five families and 18 groups have examples of seed transmitted viruses (Table 3). A small proportion of plant viruses are seed transmitted. Though about 110 viruses are reported as seed borne out of estimated 650 viruses, only about 30 are important (Table 4). Viruses are seed borne in about 350 hostvirus combinations. In addition, 31 cryptic viruses are reported that are of no economic relevance. Among viroids, 12 are reported to be seed borne (Albrechtsen, 2006) Typically embryo-infection leads to seed transmission. However viruses like tobamoviruses are located only on or in the seed coat and gain entry into the young seedlings through small wounds while transplantation. Tobamoviruses are highly stable and contagious. The embryo-borne viruses are generally unstable, for instance CMV and the seed coat infection in such cases will not contribute to seed transmission. The rate of seed transmission of viruses depends on host-virus combination. For instance the transmission rate of Lettuce mosaic virus in lettuce is low but sufficient to cause epidemics, whereas the seed transmission is common in barley stripe mosaic hordeivirus and nepoviruses. Once introduced into the field through the seed, the viruses can spread horizontally by vectors, especially aphids. Potyviruses, cucumoviruses and many others are typically transmitted by aphids in non-persistent way. Comoviruses are transmitted by beetles, furoviruses by soil fungi and nepoviruses and tobraviruses by nematodes. However, tobamovirus and hordeiviruses are transmitted only by mechanical means. Many of the seed-borne viruses are having limited host range emphasizing the role of seed as primary source of infection. In general, the viruses in seed survive as long as the seed is viable. General characteristics of seed-borne viruses All the seed-borne viruses are having ssRNA, they generally cause mosaic or mottle symptoms. These viruses are easily mechanically transmitted. Most of these viruses are also transmitted through aphids in a non-persistent manner. Field symptoms of few seed-borne virus diseases are shown in plate 1 and 2.

Table 3. Description of important seed-borne virus groups

Family/Genus 1. Bromoviridae Example Description (shape, size, mode of transmission) Isometric or bacilliform, 25-35 nm; sub genomic & m-RNA from genomic RNA-3, multicomponent, For some groups vectors are not known, tripartite virions without conical end, not stable in CsCl. Broad bean mottle virus (BBMV) Cucumber mosaic virus (CMV), Peanut stunt virus (PSV) Alfalfa mosaic virus (AMV) Prune dwarf virus (PDV), Prunus necrotic ringspot virus (PNRSV) Four RNAs, coat protein c.20k 4 RNAs, spherical, 30 nm, aphid Bacilliform, aphid Pleomorphic, not by aphids Bipartite, isometric Cowpea mosaic virus (CPMV), Cowpea severe mosaic virus (CPSMV) Tomato black ring virus (TBRV), Tobacco ringspot virus (TRSV) Broad bean wilt virus-2 (BBWV-2) Larger RNA less than 7 kb, beetles

1.1. Bromovirus 1.2. Cucumovirus 1.3. Alfamovirus 1.4. Ilarvirus 2. Comoviridae 2.1. Comovirus

2.2. Nepovirus 2.3. Fabavirus 3. Potyviridae 3.1.Potyvirus

More than 7 kb, 30 nm, nematodes Less than 7 kb, beetles Polyprotein, Polymerase is picorna-like, filamentous

Bean common mosaic virus (BCMV), Cowpea aphid borne mosaic virus (CABMV), Soybean mosaic virus (SMV) Wheat streak mosaic virus (WSMV)

Genome monopartite, more than 700 nm, Aphid

3.2 Tritimovirus 4. Tombusviridae 4.1. Tombusvirus Tobamovirus Furovirus Hordeivirus Tobravirus Tymovirus Carlavirus

Genome monopartite, more than 700 nm, Aceria mites Largest RNA, Lacks VPg, polymerase carmo-like

Tomato bushy stunt virus (TBSV) Tobacco mosaic virus (TMV), Tomato mosaic virus (ToMV) Peanut clump virus (PCV) Barley stripe mosaic virus (BSMV), Lychnis ringspot virus (LRSV) Tobacco rattle virus (TRV) Pepper ringspot virus (PepRSV) Dulcamara mottle virus (DuMV), Eggplant mosaic virus (EMV) Cowpea mild mottle virus (CPMMV), Hop mosaic virus (HpMV) White clover mosaic virus (WClMV), Foxtail mosaic virus (FoMV) Southern mosaic virus (SBMV), Southern cowpea mosaic virus (SCPMV)

RNA more that 4.6 kb Rigid rod, genome monopartite, 300 nm Rigid rod, 300 nm, lack triple gene block, Polymyxa graminis Rod, largest RNA less than 5 kb, sap Rod, virions >20 nm in diameter, bipartite, nematode Isometric or bacilliform, genome RNA about 6 kb, no vector known Filamentous, more than 700 nm, 6 ORFs, white flies

Potexvirus

Filamentous, less than 700 nm, genome less than 10 kb in one segment, triple gene block present, genome has 5 ORFs, soil fungi Isometric, genome or largest RNA more that 5 kb, 28 nm, beetles

Sobemovirus

Table 4

Description of important seed-borne virus diseases

Virus BCMV CMV CPMV CPSMV CABMV Bean common mosaic virus strain blackeye cowpea mosaic (BCMV-BlCM) CPMMV Description (symptoms, rate of seed transmission) Mottling or mosaic, stunting and rolling of leaves, abnormal flowers & pods, Upto 83%, normally 1-10% Vein banding, mosaic, mottling, up to 30% Mosaic, distortion of leaf and flower, necrosis, 1-5% Severe mosaic, up to 10% Vein banding, interveinal chlorosis, leaf distortion, stunting, Up to 40% Vein clearing, vein banding, mottle, up to 80%, do not infect French bean Mild systemic chlorotic mottling to severe chlorosis and necrosis Mosaic and mottling, 3-40% Mottling or mosaic, stunting and rolling of leaves, abnormal flowers & pods Expansion of lamina and pale green discoloration of the leaves leading to crinkling, up to 17% in black gram, up to 90% in green gram Mosaic or rugosity and stunted growth, pod malformation, reduction in size and number of seed, mottles seed; up to 30% Browning and drying of apical shoot and bud with downward curvature, dwarfing, reduced or no seed setting, underdeveloped, poorly filled pods with dark blotch, up to 100%, also transmitted in Petunia, N. glutinosa, Gomphrena globosa, Taraxacum officinale Up to 90% Prominent severe yellow-colored mosaic, c.2.5% Mottling and mosaic, irregular necrosis of seed testa No typical necrotic flecks on the seed coat Mottling, leaf distortion due to depression of interveinal tissue, necrosis, kernels may show discoloration and reduced size, 0.02 to 8%, smaller seeds show higher level of infection Stunting, malformation, abnormal pods, reduced seed size often with split seed coat, less than 1% Stunting, ring spots and line pattern, 6% Vein clearing, stunting, downward curling of the leaf margin, tight curling of tendrils, terminal rosettes, distorted flowers and pods, 10-90% Necrosis of leaves or stem, smaller and flat seeds, wrinkled seed coat with pits or greenish grey discoloration, up to 1-37% Not well described Only in green immature seeds, not seed transmitted Mosaic and malformation, transmission percentage is low Vein clearing, yellow vein banding, chlorotic spots, malformation and puckering; often present in seed coat Mottling, stunting and distortion, chlorotic to bright yellow streaks on the fruit, aucuba strain produce prominent bright yellow mottling; surface borne Mosaic, stunting , vein banding Mosaic, stunting and malformation, internal browning of fruits; located in or on the seed coat, highly infectious Not common, but reported for many cultivars Mosaic, mottling, stunted growth Mosaic Chlorotic stripes; often seed- borne

Crop French bean (Phaseolus vulgaris) Cowpea (Vigna unguiculata)

Urdbean or blackgram (Vigna mungo), Munbean or greengram (V. radiata) Soybean (Glycine max)

SBMV BCMV and CMV Urdbean leaf crinkle virus (ULCV) SMV TRSV

Broad bean (Vicia faba)

Peanut (Arachis hypogaea)

CPMMV Bean yellow mosaic virus (BYMV) Broad bean stain virus (BBSV) Broad bean true mosaic comovirus (BBTMV) Peanut mottle virus (PeMoV) PSV Peanut clump furovirus (PCV) Pea seed-borne mosaic potyvirus (PSbMV) Pea early browning tobravirus (PEBV) Pea enation mosaic virus (PEMV) Pea streak virus (PSV) CMV Squash mosaic comovirus (SqMV) in Cucurbita spp. Cucumber green mottle mosaic tobamovirus (CGMM) in cucumber Zucchini yellow mosaic virus (ZYMV) in squash ToMV Tomato black ring nepovirus (TBRV) TMV AMV BSMV

Pea (Pisum sativum)

Cucurbits (Cucumis spp., Cucurbita spp., Citrullus spp.)

Tomato (Lycopersicon esculentum) Chilli (Capsicum annuum) Barley (Hordeum vulgare)

3. Seed-borne Nematodes Nematodes are long thin roundworms measuring 0.3 to 1 mm (some may measure up to 10 mm) in length and 15 to 35 um in thickness. They have tapering and transparent body. Out of 10,000 species of nematodes 600 are reported to be parasitic. The nematodes require free moisture for their activity. They are abundant up to 30 cm depth in soil and mostly aggregated around roots. Nematodes cause root galls and knots, lesions, excessive branching, tip injury and root rot. The infection leads to restricted nutrient and water uptake, reduced vigor, yellowing, wilting, delayed flowering and low yield of poor quality. On the aerial part they can cause galls, necrotic lesions, twisting or distortion of leaves and stems and abnormal floral parts. The nematode life cycle is relatively simple, consisting of the egg, four juvenile stages and the adults. The length of the life cycle varies from a few days to nearly one year depending the genera and environmental conditions. Most of the nematodes are root parasites, however some of them are adapted to parasitism in nearly all plant tissues and organs. Plant parasites can be classified into four types based on the mode of parasitism. They are; (i) Ectoparasites: The nematodes generally remain on the surface of the plant tissues, feeding by inserting the stylet. They may be foliar ectoparasites feeding generally on epidermal plant cells of young leaves, stems, and flower primordia (Eg. Anguina, Aphelenchoides, Ditylenchus spp.) or root ectoparasites like Tylenchorhynchus, Trichodorus, Paratrichodorus, Longidorus, Paralongidorus, Xiphinema spp. (ii) Migratory endoparasites: All the stages of the nematodes can completely penetrate the plant tissues. The foliar endoparasites are found in stems, leaves, flower primordial, or seeds. (Eg. Aphelenchoides, Rhadinaphelenchus cocophilus, Bursaphelenchus xylophilus, Ditylenchus angustus, D. dipsaci). Below ground endoparasites are found in different tissues in roots, corms, bulbs, tubers and seeds (peanuts). (Eg. Ditylenchus, Hoplolaimus, Pratylenchoides) (iii) Sedentary endoparasites: The immature female or juvenile nematodes completely enter the plant tissues, develop a permanent feeding site, become immobile and swell into obese bodies. Eg. Globodera, Heterodera, Meloidogyne. In some cases the plant tissues can cover the nematodes resulting in galls. (iv) Semi-endoparasites: Immature female of juvenile larvae partially penetrate the roots. Eg. Totylenchulus, Tylenchulus spp. The important seed-borne nematode genera are: Anguina (seed-gall nematode) Ditylenchus (Stem and bulb nematode) Aphelenchoides (Bud and leaf nematode) Rhadinaphelenchus (Redi ring nematode) Heterodera (Cyst nematode) Meloidogyne (Gall nematode)

10

3.1 Mode of seed transmission The nematodes may be seed-borne as larvae harboured beneath the hull of cereals and grasses, in minor cavities of seed (hilum) or in lesions; carried with seed in infected plant debris or as galls and cysts mixed with seed. 3.2 Description of important seed-borne nematodes Anguina: These are commonly known as leaf and flower nematode. They become endoparatitic in seeds and transform the seeds into galls. The gall contains an adult male and female, and hundreds of Juvenile-2 (J-2) larvae. The larvae become active when water is available. The young ones are ectoparasitic and get attached to the growing point. The complete life cycle takes 5 months. Anguina tritici is known as ear cockle or seed gall nematode. It forms ear cockle in Avena, Secale and Triticum. This is the first plant parasitic nematode ever recognized. The adult females are large measuring 3.0-5.0 mm in length. The body tends to be thickened and curved ventrally. The stylet is delicate and the vulva is posterior. Earcockle is often associated with Corynebacterium tritici resulting in yellow gummy exudations. Other important species of Anguina are A. agropyronifloris, A. agrostis and S. spermophaga (plate 3a and b). Ditylenchus (Bulf and stem nematode): This nematode is polyphagus. The females have thin bodies, measuring up to 2 mm in length. The cuticle is finely striated with lateral line. They have four or more incisures. The vulva is posterior. The J-4 larvae (Dauerlarvae) are dormant. The life cycle is completed in 3 weeks. D. angustus causes Ufra disease in rice. This is an important pest in deepwater rice. The infected plants may show cottony masses beneath glumes. The leaves become distorted and discolored. The panicles get twisted, often fail to emerge and remain sterile. The nematode is an obligate ectoparasite. D. dipsaci is known as stem nematode. It infects onion and red clover. In onion the nematodes are found near the hilum, below the seed coat. It may also infect oat, beta, trifolium and vicia. Heterodera is known as cyst nematode. The adult female gets transformed to a cyst. The cyst is swollen, tough, white turning to brown or yellow at maturity, leathery, sac-like structure measuring 0.5 to 1 mm (plate 3c and f). It is spherical and lemon-shaped. The head of the female nematode is embedded in root cortex. Each female contains 10-600 embryonated eggs and J-2 larvae. They can survive for 5-10 years. The male nematodes are elongate, slow moving, 1-2 mm in length with short and blunt tail without caudal alae. The larvae measure 0.5 mm. They have thick spear. The life cycle is completed in one month. H. glycines (Soybean cyst) is endoparasitic causing premature yellowing, dwarfing and yellow seeds. H. Goettingiana causes pea cyst. It also infects Vicia and Lupinus. H. schachtii infects sugarbeet. It can also infect Beta and Brassica. The other important species of Heterodera are H. oryzae, H. avenae, H. rostochiensis, H. trifoliae, H. cajani and H. zeae.

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Reflexed testis Reflexed ovary

Posterior valva

Multiple rows of oocytes

a) Anguina tritici (Female coiled ventrally)

b) Anguina tritici (Male)

Delicate stylet, small knobs Large metacorpus Intestine joins directly to metacorpus

Esophageal glands in long lobe overlapping intestine Egg sac Long post-vacual sac One outstretched ovary

c) Heterodera spp.

d) Aphelenchoides

e) Adult females of Meloidogyne sp. dissected from within roots

f) Cysts of Heterodera spp.

Plate 3: Seed-borne nematodes

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Meloidogyne gall and Heterodera cyst have some resemblance. However, the Heterodera cyst is lemon-shaped, white or brown, with only neck embedded and posterior protuberance. The Heterodera is specific to monocots. The Meloidogyne gall is pear or flask-shaped, white and fully embedded in the host tissue (Plate 3e). It infects dicot hosts. Aphelenchoides (Bud and Leaf nematode): The mouthparts are weak having small stylets with tiny basal knobs. The metacarpous is long. The body is slender, measuring up to 1 mm. The tail is tapering often with mucrons. Males are morphologically similar to females. The life cycle is short taking only 10 days. Aphelenchoides besseyi (A. oryzae) is known as white tip nematode in rice. The nematode can survive beneath the hull for 2-3 years. Each seed may have 5-6 active nematodes. The nematodes are migratory ectoparasites of the foliage, feeding on stems, leaves and inflorescences. The female nematodes are thin, 0.6-0,9 mm in length, with a tapering tail ending with 3-4 pointed mucron. Vulva is posterior, approximately one-third of the body length from the tail tip (plate 3d). The other important Aphelenchoides species are A. ritzemabosi in Chrysanthemum and A. blastophorus in Aster. Rhadinaphelenchus cocophilus ( Bursaphelenchus cocophilus): This nematode is commonly known as red ring nematode in coconut. It is found only in West Indies and Latin America. The infected palm show orange or red ring in the cross-section of the stem. The leaves show yellowing. The nematode is carried by the palm weevil, Rhyncophorus palmarum. They oviposit in internode of crown and pupate. The nematodes are long, vermiform with long tapering tails. The vulva is positioned one-third of the body length from the tail tip. Styles are small and often obscure. 3.3 Management of seed-borne nematodes The nematodes can be managed through chemical and non-chemical means. Summer ploughing, fallowing, crop rotation, soil amendment, use of resistant cultivars and biological control are some of the approaches. The seed-borne nematodes could be effectively controlled by seed certification, physical cleaning and hot water treatment. The seed lots may be cleaned by flotation and sieving using fresh or slightly salted water. Seed health testing 1) Dry seed examination: The seed samples could be examined for galls (Anguina and Meloidogyne) and cysts (Meloidogyne). 2) Examination of water on soaking seeds: A. besseyi and D. angustus could be released from seeds by soaking in water at 28 oC. D. dipsaci in clover and onion seeds are released by placing the seeds in filter paper in a hopper closed at bottom and spraying with a mist of water. The cysts and galls can be floated and recovered with sieve. 3) Fenwicks small culture cells: The culture plates for D. dipsaci contain 50 cells of dimension 1 x 0.4 cm each arranged in rows on 15 cm places. Individual seed is soaked in each cell and examined for larvae. 4) Extraction from plant pieces and clods: The nematodes are extracted by using Baermann funnel and suspension is examined for presence of nematodes (plate 4a and b).

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4. SEED HEALTH TESTING 4.1 Sampling in seed health test The size of the working sample for seed health testing depends on the target pathogen, and the epidemic potential of seed-borne inoculum. It is suggested that the lot could be split into sub lots and each sub lot is sampled for seed health testing. The size of the sample should reflect even the low level of infection. The inoculum threshold refers to the level of infection on or in the seed or contamination that will significantly contribute to disease development and yield loss. Tolerance is the permissible or tolerable level of seed contamination or infection. 4.1.1 Working sample A working sample, which is a true representative of seed lot in composition and proportion of different components, is very essential to obtain reliable results. Hence standardized sampling methods are an integral part of any quality assessment. International Seed Testing Association (2003) has framed rules for drawing primary, submitted and working seed samples. Sampling for seed health especially by mechanical divider pose problem of contamination from one sample to another. Hence, it is very essential to clean the sampling devices between drawing successive samples. ISTA (2003) has given the maximum weight of seed lots, minimum weights of submitted samples and sample size of various categories of seeds. The sample for the purity analysis will be in terms of weight, which varies for different categories of seeds depending on the seed and density. The size is usually considered sufficient for seed health testing based on seed infection. However, in seed health testing, it may be essential to increase the sample size to determine the trace infection of 0.1% or less as minimum of 1000 seeds are required to detect one useful seed. This is of special significance for detection of seed-borne fungal infection like loose smut of wheat and barley and downy mildew from Sclerospora graminicola in pearl millet seeds. Further, it is necessary to test 2 3 sub samples of 1000 seeds. Seed health test for bacteria and viruses also need working/sub sample size. This again depends on the type of pathogen and threshold level. In some cases, it is very essential to prove that the working sample free from any infection. Larger working sample size is handled by taking replicates and level of rise is assessed based on number of infected replicate samples. In the detection of Tobamoviruses (TMV; ToMV; PMMV) on Pepper (Capsicum annum) the limit of detection is one infected seed in a sun-sample of 100 seeds-in case of low level of infection on the seeds or one infected seed in a subsample of 500 seeds-in case of high level of infection. Each sub-sample should be inoculated on two different plant species (preferable-Nicotiana tabacum cv. xanthi NN and Nicotiana glutinosa). The bioassay test is suitable tom identify all types of Tobamoviruses (TMV; ToMV; PMMV) (table 5). The percentage of Lettuce mosaic virus (LMV) can be estimated in a batch of 20 x 100 seedlings (table 6).

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Paper or cloth Nematodes in debris

Water level Rubber band

a) Inverted beaker in funnel to permit nematodes to migrate free of debris

Soil wrapped in cloth or paper towel Water level 2 cm or less

Soil Cloth or paper

Flexible tubing

Clamp

b) Standard Baermann funnel and a modification for increased efficiency

Plate 4

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Table 5: Number of sub-samples and seed per sub-sample to be tested in different percentage of infected seeds (0.1-0.5%) in detection of Tobamoviruses (TMV; ToMV; PMMV) on Pepper (Capsicum annum) (Hadas, 2000)
No. seeds/ sub-sample 0.1% No.sub sample 31 22 20 23 35 59 58 180 Total seed Percentage of infected seeds* 0.2% 0.3% No.sub -sample 16 11 10 11 15 18 17 27 26 13 Total seeds 1600 1650 2000 2750 4500 7200 8500 27000 39000 26000 No.sub sample 12 8 7 8 9 9 9 10 8 4 Total seeds 1200 1200 1400 2000 2700 3600 4500 10000 12000 8000 0.4% No.sub sample 8 6 5 6 6 6 6 5 4 2 Total seeds 800 900 1000 1500 1800 2400 3000 5000 6000 4000 0.5% No.sub sample 7 5 4 4 5 5 4 4 2 2 Total seeds 700 750 800 1000 1500 2000 2000 4000 3000 4000

3100 3300 4000 5750 10500 23600 29000 18000 0 1500 347 52050 0 2000 178 35600 0 * Probability of 95 (P> 0.95)

100 150 200 250 300 400 500 1000

Table 6: Estimation of percentage of LMV-infected seedlings as found with ELISA in which 20100 seedlings are tested (Oliver et al., 2001)
No. ELISA- positive sub samples 0 1 2 3 4 5 6 7 8 9 10 Estimated infection rate (%) of seed lot (P) 0.00% 0.05% 0.10% 0.16% 0.22% 0.29% 0.36% 0.43% 0.51% 0.60% 0.70% No. ELISA-positive sub samples 11 12 13 14 15 16 17 18 19 20 Estimated infection rate (%) of seed lot (P)* 0.80% 0.91% 1.04% 1.20% 1.38% 1.60% 1.88% 2.28% 2.95% >3%

*The estimated infection rate is calculated on the assumption that one LMV-infected seedling per subsample is detected in every assay.

The following formula is required: 1/n P=1-Y/N

(www.worldseed.org/ishi_manual_.htm)

4.2 Developments in seed health testing methods Seed health testing refers to detection and identification of pathogens associated with seeds. The methods depend on the type of pathogens. It is essential that the method for routine seed health testing shall be simple, inexpensive and repeatable. The method shall also reflect the actual load of live inoculum that contributed to disease development in the field. Of late, it is expected that the method shall handle bigger sample size and

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detect low level of infection. Earlier many seed health-testing methods were developed and standardized through comparative testing by plant disease committee of International Seed Testing Association (ISTA). The International Seed Health Initiative (ISHI) is also active in standardizing and publishing various methods. Accurate identification and early detection of pathogens is the backbone of disease management in crops. Seed-borne pathogens are difficult to identify using morphological key, which can be laborious and require extensive knowledge in taxonomy. Enzyme linked immunosorbent assay (ELISA) and DNA-based technologies such as the polymerase chain reaction have been the basis for molecular detection in modern seed pathology. Molecular detection techniques provide accurate results rapidly enough to be helpful for disease management. Technologies for the molecular detection of pathogens have already undergone two major breakthroughs over past 30 years. The first was the advent of antibody-based detection, in particular monoclonal antibodies (Kohler and Milstein, 1975) and enzyme-linked immunosorbent assay (Clark and Adams, 1977). This approach was an important turning point in virology and bacteriology because pathogens could be identified and detected much more rapidly (Hampton et al., 1990). Then came DNA-based technologies, such as the polymerase chain reaction (PCR) and its capability to amplify the original target DNA several million fold (Mullis and Faloona 1987). 4.3 Seed health testing for fungal pathogens Fungal propogules may be present as surface contaminants on seeds or as concominant contamination. In many cases, the seeds are infected and the mycelium of pathogen is present within the seed tissue. Accordingly, the seed health methods may vary based on presence of pathogen. 4.3.1 Dry seed examination Seed samples are subjected to dry seed examination and based on the visual examination of the seeds; they are classified into infected or healthy. This method is very useful in detecting the concontaminant contamination i.e., those seeds that are transformed as smut sori, bunt, gall or sclerotia (Table 7). The working sample, usually recommended for dry seed examination, are carefully observed under bright light on a purity board and represented as number of contaminants per gram or per 1000 units. Table 7: Examples of pathogens that could be detected by the dry seed examination
Disease/Pathogen Ergot Claviceps fusiformis Claviceps purpurea C. oryzae Softrot/white blight Sclerotia sclerotiorum Stem gall Protomyces macrosporus Aschochyta blight Aschochyta rabiei Leaf and pod blights Crop Pearl millet Barley, Wheat Rice Sunflower /Safflower Coriander Chick pea Soybean Propagule Sclerotia Sclerotia Sclerotia Sclerotia Chlamydospore Pycnidia Conidia Description Sclerotia get mixed with seeds while the crop is harvested Sclerotia get mixed with seeds while the crop is harvested Sclerotia get mixed with seeds while the crop is harvested Sclerotia act as initial source of infection. It is also present in the form of mycelium Tumor like swellings and hypertrophies on the seed surface Infected seeds are small, wrinkled and have dark brown lesions of various shapes and sizes Infected seed coat cracking, purple

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Cercospora kikuchi False smut Ustilaginoidea virens Bunt Karnal bunt

Rice Wheat

Chlamydospore Teliospore

spot/stain observed on the seed surface Seeds are covered by a mass of orangebrown to greenish-black spore balls Seeds are partially black, consisting a powdery mass of teliospore. The spore mass can be examined under a compound microscope

4.3.2 Washing test The sample is suspended in required amount of water to cover the seeds (10-100 ml) shaken thoroughly and spun in centrifuge at 1500-3000 rpm for 5 minutes. The suspension is mounted in glycerol and observed under the compound binocular microscope (40 x). The method is suitable to detect oospores or other fungal conidia present as contaminants. 4.3.3 Embryo extraction tests The test is suitable for mycelial/oospore infection in embryo (Aleurone layer) or endosperm. The seeds are soaked in 1 liter of a 5% freshly prepared solution of sodium hydroxide containing 0.15 g of trypan blue, at room temperature for 22-24 hours and the seed components are separated by repeated washing and passed through appropriate sieves. Examination of extracted embryos is done in an embryo examination plate containing lactic acid-glycerol mixture. The infected embryo may contain coenocytic mycelium as in S. graminicola infection or branched mycelium of Ustilago tritici causing loose smut in wheat. The kernel smut spores in rice can also be detected by clearing the seed tissue by water treatment and puncturing the seeds by fine needle, which release the spores. 4.3.4 Incubation tests The seeds are placed on suitable substrates and incubated under optimum light/temperature conditions for certain period. The sporulating colonies are identified based on morphology under stereomicroscope and conidia/spore morphology is detected by /compound binocular microscope. a) The standard blotter method test Four hundred seeds are subjected to the standard blotter paper method (SBM) by following the rules of ISTA (2003). Ten seeds per plate are plated on three layered moistened blotter discs in sterilized perspex plastic plates. These plates are incubated at 25 2 C under alternating cycles of 12/12h of near ultra violet (NUV) light and darkness for seven to thirteen days depending on the crop. The plated seeds are examined under a stereoscopic binocular microscope for fungal colonies. Subcultures are made on fresh PDA from fungi emerging on blotter and agar plates for proper identification and pathogenicity tests. Relative Percent Occurrence (RPO or Infection Incidence) is reported. RPO is calculated using the formula:
RPO = No. of samples having the fungus Total No. of samples screened X 100

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The RPO/Infection Incidence gives an idea for the relative occurrence of fungi using SBM. Similarly, Infection Percentage is used to designate frequency of infected seeds in a sample, while Infection Severity is often used to express the degree of infection of individual seeds in a sample. Habit character on seed and conidia of some important seed-borne fungi are presented in plate 5 and 6. b) 2,4-Dichlorophenoxyacetic acid (2,4-D method) The blotter discs are soaked in 2,4-D (0.1-0.2 %) and incubated on moist blotter as described above. This method is useful in preventing the germination of seeds and facilitating the observation of sprouting fungi. This method is widely used in testing seed of crucifers for Phoma lingam. The procedure should be adopted critically as 2,4-D may have a weakly fugistatic effect, e.g. Alternaria brassicola is delayed in development on cabbage seed by 1-2 days at the level of 0.2 per cent. c) Deep-freeze method: The petri dishes are incubated at 20 oC, This method has been used mainly to test commercial samples of cereals and grasses for Drechslera spp and Fusarium spp and various small seeded vegetables. This method is more suitable for testing maize and cabbage seeds for the incidence of fungi. d) Agar plate method Fungal contamination was also determined by agar plating method (Lacey, 1988). Two hundred seeds in four replicates of 50 seeds each are surface sterilized in 0.35 % sodium hypochlorite solution for 2 minutes and plated at 5 seeds per plate on PDA in an aseptic condition. The plates are incubated for 5 days at 25 2 C under alternative cycles of 12/12 h of NUV and darkness. They were observed using stereo-binocular microscope and the percent incidence of fungal contamination are recorded. 4.4 Seed health testing for bacteria The basic key for identification of bacteria is based on colony characters on specific media, gram reaction, biochemical and physiological criteria (Table 1). The identification of bacteria is relatively more cumbersome as it depends on successive culturing and several biochemical tests. Following are the important steps in identification of bacteria from seeds; Extraction of bacteria from the seeds. Isolation and purification. Identification of the bacterial isolates.

4.4.1 Detection of bacteria from seed samples Bacterial pathogens can be detected from seeds by following methods:

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Indirect detection: Bacteria are extracted from the seeds by soaking or by grinding using water or buffer solutions. Suspensions are then incubated on semi-selective or selective media at specified temperature, suspected colonies are isolated as pure colonies by staining and by using a set of other biochemical tests. The bacterial identity is further confirmed by infectivity test on indicator plant or pathogenicity test on susceptible natural host.

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Direct detection: Here the bacteria are detected without an extraction step. The seeds are directly plated on suitable medium with or without surface disinfection. The seeds can also be subjected to growing on tests, observed for specified symptoms and the isolated bacteria are subjected to other tests for confirmation. 4.4.1 Isolation and purification of bacteria The extracted bacteria are isolated on media of specified composition and purified as follows; Wash the suspected plant parts by dipping it in 95% ethanol followed by a dip in NaOCl (0.5-1.0%) for 2-5 min and rinsing in sterile water A small part of the tissue is cut in a drop of water and observed under a compound microscope after putting the cover slip for bacterial ooze Streak the water containing bacteria on a suitable media to get single colony of bacterium Incubate the streaked plates in an inverted position at 25-30 oC

The pure colonies are further identified by morphological and biochemical characteristics, pathogenicity, hypersensitivity, serological and molecular tests and electron microscopy. 4.4.2 Morphological characters The morphological characters include, cell shape, size, capsule, endospores, flagella and colony characters such as color, margin size, elevation, consistency. 4.4.3 Gram reaction The bacteria are grouped into Gram positive and Gram-negative categories, based on their ability to retain the counter stain safranine. Gram-positive bacteria appear blueviolet while Gram-negative bacteria appear pink-red. 4.4.4 KOH solubility test A simple KOH solubility test also helps in separating the Gram-positive and Gramnegative bacteria. The bacterial colonies are mixed with 3% KOH solution and the suspension is raised with a toothpick. A Gram-negative bacterium gives a mucoid thread. 4.4.5 Pigmentation Pigmentation of bacteria is an important criterion for identification. Pseudomonas species produce several kinds of pigments. Based on fluorescence of colonies under UV light Pseudomonads could be categorized as fluorescent and non-fluorescent. Kings medium B is the most preferred for this purpose. 4.4.6 Pathogenicity tests For many plant diseases, this test is the only method for confirming or negating an initial presumptive diagnosis. The suspected bacteria are inoculated onto the susceptible host at optimum concentrations and incubated at suitable conditions that favour expression of symptoms

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4.4.7 Hypersensitive tests Tobacco is the preferred plant. But for Xanthomonads, tomato is preferred. An aqueous suspension (108 - 109 cfu/ml) from a 24-28 hour culture is injected into the leaf and observed for necrotic symptoms. Pictorial representation of a few biochemical tests and isolation of seed-borne bacteria on selective media are presented in plate 7. 4.4.8 Basic tests for identification of seed-borne bacteria
Crop/Pathogen
Phaseolus vulgaris/ Pseudomonas syringae pv syringae

Description
On semi selective medium for fluorescent Pseudomonads (KBC), colonies fluorescent under UV light, off-white, semi-transparent colonies, Gram ve rods, oxidase ve, LOPAT (+----+), utilize sucrose, inositol, mannitol, erythitol, and sorbitol not homoserine; on pod testgreen, sunken lesions after 3 days. On KBC, colonies fluorescent under UV light, off-white, semitransparent colonies, Gram ve rods, oxidase ve, LOPAT (+----+), utilize only sucrose, not inositol, mannitol, erythitol, and sorbitol and homoserine; pod test-water soaked lesions after 3 days confirms the pathogen.

Pseudomonas savastanoi pv. phaseolicola

Xanthomonas axonopodis pv. phaseoli Non-fluorescent, yellow colonies on Milk Tween (MT) medium, Gram ve rods, no brown pigment, starch +ve, Tween 80 +ve, pathogenicity test positive confirms the pathogen Lycopersicon esculentum / Pseudomonas syringae pv. syringae On KBC, colonies fluorescent under UV light, off-white, semitransparent colonies, Gram ve rods, oxidase ve, LOPAT (+----+), utilize sucrose, inositol, mannitol, erythitol, and sorbitol not homoserine; on pod test-green, sunken lesions after 3 days. On KBC, colonies fluorescent under UV light, off-white, semitransparent colonies, Gram ve rods, oxidase ve, LOPAT (+----+), utilize sucrose, inositol, mannitol, and sorbitol not erythitol and homoserine; pathogenicity test-leaf spots. Yellow colonies on MT media, Gram ve, two zones around colonies, bigger, clear, smaller, opaque, pathogenicity test-leaf spots. subsp. Semi selective medium for Clavibacter michiganensis subsp. michiganensis (SCM)-fluidal, mucoid, yellow colonies with grey flecks; TGA-yellow, mucoid, circular colonies, Gram +ve, conduct pathogenicity test by inoculating tomato plants with 107 cfu/ml suspension of pathogen. Symptoms develop after 14 days at 25 oC. Pathogenicity test-positive. On media semi selective for Xanthomonas campestris pv. campestris (SX)-clear zones around colonies, MT-yellow colonies with two zones, bigger, clear, smaller, opaque, Gram ve rods, pathogenicity test-leaf spot, black rot.

Pseudomonas syringae pv. tomato

Xanthomonas vesicatoria Clavibacter michiganensis michiganensis

Crucifers/ Xanthomonas campestris pv. campestris

4.4.8.1 Genus Acidovorax These are non-fluorescent, motile by a single polar flagellum, oxidase and catalase positive bacteria that accumulate poly--hydroxyburate. The colonies are white or colorless but some strains may produce a yellow or brown diffusible pigment. Some of the seed-borne bacteria in this genus are: A. avenae subsp. citruli (bacterial fruit blotch of watermelon); A. avenae subsp. avenae (bacterial leaf blight and stalk rot of maize).

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4.4. 8.7 Genus Burkholderia Bacteria are non-fluorescent, oxidase variable, catalase positive, and accumulate PHB and are motile by a single polar flagellum or polar tuft. All strains can grow on glucose, glyceraol, inositol, galactose, sorbitol and mannitol. 4.4.8.5 Genus Clavibacter Non-motile pleomorphic rods that are often in angular arrangements. Growth factors are required, nitrate and nitrite are not reduced, casein is not hydrolysed, and they are catalase positive and oxidase, lipase, tyrosinase and urease negative. Important phytopathogens in the genus are: C. michiganense subsp. michiganense - bacterial canker of tomato is a cosmopolitan, economically important, seed-borne disease of tomato; C. michiganense subsp. nebraskensis-bacterial wilt and blight on maize. 4.4.8.6 Genus Curtobacterium Usually motile by lateral flagella. Rods also pleomorphic, showing bending-type cell division. Produces small (2-4mm) colonies after 4 days that are smooth, entire, convex and semi-fluid. Yellow-orange or pink pigments may be produced. 4.4.8.2 Genus Pseudomonas This is a very heterogeneous genus that can be conveniently divided into 2 groups: Fluorescent Non-fluorescent

The fluorescent pseudomonads produce a yellowgreen to blue fluorescent pigment on Iron-deficient media (KB medium) and have been further divided into 5 groups using 5 key tests: Levan production Oxidative reaction Potato rot Arginine dihydrolase production Tobacco hypersensitivity

These are generally known as the LOPAT tests. Group I (LOPAT + - - - +); Group II (LOPAT - - + - +); Group III (LOPAT - + - - +); Group IV (LOPAT + + + + -) and Group V (LOPAT - + - + -). 4.4.8.3 Genus Ralstonia The bacterium accumulates PHB, does not form levan from sucrose, does not hydrolyse starch and aesculin, reduces nitrate, oxidase positive, arginine dihydrolase negative and does not grow at 40 oC. The species can be divided in 5 biovars according to acid production from 3 disaccharides and 3 sugar alcohols (table 8). One of the most destructive bacterial pathogens of plants Ralstonia solanacearum, belongs to this genus. Previously this pathogen was considered a non-fluorescent Pseudomonas but was later assigned to the genus Burholderia and then Ralstonia.

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Table 8: Tests to differentiate between biovars of Ralstonia solanacearum Utilization Maltose Lactose Cellobiose Mannitol Sorbitol Dulcitol I II + + + Biovar III + + + + + + IV + + + V + + + + -

4.4.8.4 Genus Xanthomonas Most species in this genus produce smooth, circular and butyrous or viscid colonies with a typical yellow pigment. A few members lack pigment and produce white colonies. The bacteria are motile by a single polar flagellum and are catalase positive, oxidase negative or weakly positive; nitrate is not reduced; extra-cellular polysaccharides are commonly produced. 4.4. 8.8: Media and tests for preliminary identification of seed-borne bacteria
Medium/ Test Nutrient agar (NA) (Catalase) Observation Add a drop of 3% hydrogen peroxide. Bubbling indicates positive reaction.

Tetrazolium chloride Bright red smear indicates tolerance to TTC TTC) (Tolerance test; 0.1, 0.02%) Kings medium B (KMB) Blue-green fluorescence under ultraviolet (UV) light, or in bright fluorescence test sunlight is positive Sucrose nutrient (SNA) (Levan test) agar Thick, creamy, mucilaginous growth indicates levan-positive stain

Soluble starch agar (SSA) (Starch hydrolysis test) Nitrate reduction test Potato rot test Nutrient agar (NA) (Color, Gram reaction) NA for oxidase test Gelatin hydrolysis test Oxidation/fermentation test

Add iodine. Clear zones in the blue iodine reaction indicate starch hydrolysis. One drop of each of the nitrate test reagents should be added in the usual way. Positive stains will break down the plug of freezedried potato. Observe color on the NA slope. Use a loop to remove culture from the slope for the gram reaction. (The 3% potassium hydroxide solubility test is recommended) Use oxidase touch sticks on NA slope. A deep purple coloration within 30 seconds indicates a positive result. Gelatinpositive strains will liquefy the medium. Compare with non-inoculated control and/or a known positive. After stab inoculation, the anaerobic tube is closed tight. Change from blue to yellow indicates acid production. Some bacteria may not metabolize glucose (aerobic tube positive)

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4.4.8.9: Tests used to characterize some genera of bacteria

PSEUDOMONAS LOPAT Levan production Oxidase Potato soft rot Arginine dihydrolase Tobacco hypersensitive reaction Gelatine liquefication Aesculin hydrolyisis Tyrosinase activity Tartarate utilization Carbon sucrose utilization Pectolytic enzyme Toxin production Maximum growth temperature Ice nucleation activity Hugh- Leifson test Acetoin production Catalase Indole production Lecithinase production Growth at 36 0C Potato soft rot Acid from organic compounds H2 S from cystine Urease production Milk proteolysis Acid production from carbohydrates Starch hydrolysis Tween 80 lypolysis Aesculin hydrolysis Gelatine liquefaction Urease production Growth at 35 0C

GATTa

Other

SOFT ROT ERWINIAS

XANTHOMONAS

4.4.9 Serological tests Serological test has become an important tool in the diagnosis of plant pathogenic bacteria. Enzyme-linked immunosorbent assay (ELISA) and Dip-stick assays (DSA) are practiced in seed health testing for bacteria. Immunofluorescent microscopy is another useful technique in the identification of bacteria. 4.5 Seed health test of viruses Biological tests are time and labour-consuming compared to molecular assays, but still important in detection and diagnosis of seed-borne viruses. 4.5.1 Growing-on test A simple and efficient method to determine whether seeds contain seed-transmitted viruses is to subject the seed sample to a growing-n test. Planting of seeds and growing of the progeny seedlings 20-25 days under optimal conditions in many cases reveal seedtransmitted virus infection by appearance of symptoms (Plate 8a-c). Further, and most importantly, only seed-to-plant-transmissible virus infections will be revealed. The only requirement for conducting a growing-on test is the availability of vector-free growth facility and optimal germination and growth conditions. An advantage of growing-on tests is that the virus content is normally higher in seedlings than in seeds and germinated seeds, and therefore easier to detect in a subsequent infectivity, serological or molecular assay. 28

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4.5.2 Indicator plants and tests A wide range of herbaceous plant indicator species and cultivars has been identified for virus diagnostic purposes, especially within the genera Chenopodium (plate 8d) and Nicotiana and some Cucumis, Phaseolus, Vigna and Vicia spp. Indicator hosts may react either with local symptoms or with symptoms of systemic infection or sometimes both, depending on the virus and indicator host. Species reacting locally to a virus are useful since symptoms appear quickly, i.e., from 2 to 7 days post-inoculation, while symptoms on systemically reacting plants appear only after 7 to 14 days postinoculation or more. In mechanical inoculation of plant parts, an abrasive, such as carborundum or celite, is used to create micro-wounds in epidermis cells. 4.5.3 Electron microscopy Transmission electron microscopy is very essential to observe virus particles. The virus particles are placed on a copper grid supported with polyvinyl farmvar film and stained by negative heavy metal stains such as ammonium molybdate, phosphotungstic acid, and uranyl acetate. The particles are identified based on morphology (plate 9a-c). 4.5.4 Serological testing methods One of the most important methods for diagnosis and identification of virus diseases is the employment of specific reaction between antibody and its corresponding antigen. Enzyme-linked immunosorbent assay (ELISA), introduced in plant virology by Clark and Adams (1977) is the most widely used serological methods for routine detection of plant viruses. Despite detection sensitivity and specificity less than that of the nucleic acid based methods, ELISA and other immunoassays have great potential for used in routine seed-health testing for viruses (plate 8e). Direct and indirect are two main types of ELISA. In the direct form, the detecting virus-specific antibody is labelled with an enzyme. In the indirect format, the detecting virus-specific antibody is not labelled with an enzyme, but is detected by an anti-antibody labelled with enzyme. This second antibody is produced against the IgG of the animal in which the virus-specific antibody was produced. The virus-specific antiserum could, for instance, be produced in rabbits and the anti-rabbit serum in goats. The double-antibody sandwich (DAS)-ELISA, a direct ELISA was the first ELISA procedure developed for plant virus detection. A single enzyme conjugate, used for detecting any virus is the main advantage of indirect ELISA. Such conjugate are commercially available. Dot blot immunoassay (DIBA) is another enzyme-amplified serological detection system. The main differences between DIBA and ELISA are that, the antigen samples are applied as dots on a nitrocellulose membrane (NCM), the consequent reaction takes place by submerging the whole NCM in the reagent dilutions and the signal indicating the presence of virus appears as an insoluble colored product, which binds to the membrane at the sites of dot application and therefore can be used as a permanent record. The protein binding capacity of a nitrocellulose membrane is much higher than that of a polystyrene surface, as little as 1/50 or less of the antigen amount required in ELISA can be detected by Dot blot immunoassay. However, Dot blot immunoassay generally gives higher background than ELISA, unless certain precautions are taken. Tissue blot immunoassay and tissue print immunoblotting are detection methods similar to DIBA, except that it does not involve tissue extraction. Instead of dotted extracts, freshly cut tissue surfaces are printed directly onto nitrocellulose membranes. The antigen trapped in the tissue blots are then reacted with the antibodies, conjugate and

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substrate in the same way as in DIBA (Huth, 1997). Immunosorbent electron microscopy (ISEM) is a highly sensitive technique, which combines electron microscopy (EM) and serology. The electron microscopy specimen carrier, the grid, whose support film is first coated with specific antibodies, is exposed to a virus-containing extract for few min, followed by washing off unbound host-plant material and contrast staining. Thus, the virions are trapped specifically instead of passively, resulting in an increased concentration of virions per unit area of the grid examined in the EM (plate 9d). The technique enables detection of viruses that occur at low concentrations in their hosts or of viruses in extracts from pooled samples. 4.6 Molecular techniques for detection of seed-borne pathogens The molecular assay methods rely on the detection of their nucleic acids unlike serological detection techniques, which build on the specific interaction between antibodies and the protein surface of disease agents. The techniques described so far are the traditional, common procedures for identification and detection of seed-borne pathogens. Significant advances have been made in molecular techniques related to diagnosis of seed-borne pathogens. Nucleic acid based detection techniques such as restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR) has gained significance because of its ease in application and its high sensitivity compared to the other DNA-based techniques. To develop a PCR-based detection procedure for seed-borne pathogens requires knowledge of sequences of at least a part of the target DNA region to design specific primers. Ribosomal genes and the spacers between them provide attractive targets for detection and phylogenetic studies, because more than 50 copies may be present per genome. This suggests a high sensitivity of detection. The ribosomal genes and spacers between them possess conserved sequences as well as variable sequences, so they can be easily amplified and sequenced with universal primers based on their conserved sequences (White et al. 1990). Nuclear rDNA consists of the small and large subunits, 5.8S, and the internal transcribed spacer (ITS) region in fungi, and each sequence is variable at the family, genus, or species level. The ITS regions between the 18S and 28S r DNA subunits, which are nontranscribed regions, have considerably greater sequence variation between species. For these regions, ITS regions have been widely used for developing speciesspecific probes or for designing primers for detection of pathogens on interest (Beck and Ligon, 1995; Goodwin et al. 1995; Kageyama et al. 1997; Lovic et al. 1995; Lee et al., 2001). In general, bacterial assays can be very fast since extraction of DNA is not needed a simple 2-3 min hot start is adequate to break the cells. Also, direct PCR reduces the chances of cross contamination. However, even when used as a direct assay, real-time PCR has several disadvantages compared with isolation techniques when used to detect low levels of bacteria as often occur in seeds. False negatives can result from using small volumes and presence of PCR inhibitors is a problem (Weller et al., 2000). False positives can result from presence of dead cells or free DNA. To avoid these inherent disadvantages and to utilize the best of both techniques, PCR can be used in combination with pathogen isolation on agar media. By using a combined viable enrichment (growth on media) with an enzymatic amplification (PCR), termed BIO-PCR (Schaad et al., 1995), the target bacterium can be enriched in liquid or solid media and detected at extremely low levels in seeds. A typical BIO-PCR assay includes the following simple steps, extracting a sample, plating a sample onto agar media or adding sample to liquid medium, incubating for 15-72

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hrs, depending on the growth rate; washing plates to remove bacteria or centrifuge liquid medium and 1 or 10 l for direct PCR. BIO-PCR has several advantages over traditional PCR, including increased sensitivity, elimination of PCR inhibition, and detection of viable cells only. The majority of plant viruses have ribonucleic acid (RNA) genomes; when running PCR on RNA viruses, it is necessary to transcribe the virus RNA into complementary DNA (cDNA). This is done by use of reverse transcriptase enzyme, which uses the RNA string as a template for the complimentary DNA string. RNA consists of nucleotides built of ribose-phosphate and the bases Guanine (G), Cytosine (C), Adenine (A), and Uracil (U). Guanine is complementary to Cytosine and Uracil to is complimentary to Adenine. When constructing cDNA from RNA, the Uracil will be replaced by Thymine (T). As a result, we end up having deoxyribose-phosphate strings to which the bases A, T, G and C are connected. Usually RT-PCR involves extraction and purification of total RNA from the sample under test. RNA extraction is laborious procedure and includes a risk of loosing the RNA. This is because RNA is very easily broken down by RNases, which are everywhere in our environment. One way to avoid extraction of the RNA is to combine ELISA with the PCR procedure. The immunocapture (IC) procedure uses antibodies (IgG) to capture the antigen of interest. The IgG is bound to the surface of a micro tube or a PCR tube. After the antigen has been trapped the RT-reaction is possible by adding a RTreaction mix into the tube and place it at the right temperature. During this step the viral RNA will be transcribed into DNA and thus be ready for PCR. The PCR will take place in the same tube as the IC and the RT-reactions. Therefore, it is called an IC-RT-PCR one tube reaction (plate 8f) (Albrechtsen, 2006). Real-time PCR has many important advantages over classical PCR: (i) it eliminates the need to do a southern blot to confirm identification of PCR product; (ii) it is more contained system and less prone to cross contamination; (iii) less labour is required; (iv) it is more user friendly; (v) it provides data useful for selecting primers; (vi) opimization of PCR protocol is quicker; and (vii) it can be used for multiplex PCR. 5. Seed treatment strategies Seed treatment is an important management strategy against seed-borne diseases. Seed treatment not only protects the seed from the seed-borne pathogens but also protects against the soil borne pathogens that may infect the growing seedlings. In most of the cases, seeds are given blanket treatment with the recommended fungicide and dosage. The seed health assessment helps in taking a precise decision about the appropriate fungicide targeting the pathogen of interest. In some cases, the seed lot may be free from seed-borne pathogens, wherein no seed treatment is required. Hence seed health testing becomes an important pre-requisite in taking decision on appropriate seed treatment. Usually the seed lots are treated in bulk in the processing plant after clearance for quality. 5.1 Seed treatment Depending on the nature and purpose, the seed treatment can be categorized as seed disinfection, seed disinfestation and seed protection. Seed disinfection refers to the elimination of pathogen that has penetrated the cells and established in the seed. Seed disinfestation kills / removes the spores and other forms of pathogens present on the surface of the seed. Seed treatment protects the seed and young seedlings from pathogens in the soil. The fungicides may be treated as dust, slurry (wettable powder) or liquids. The

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fungicides may be organic or inorganic and metallic or non-metallic. The mercurial fungicides have been banned because of environmental pollution problems. Fungicides may also be classified as contact or systemic. Some fungicides used for seed treatment like ridomil used against downy mildew pathogens are systemic as they penetrate and persist in the seed/seedling for a longer time and protect from the pathogens for an extended period. Usually the seeds are treated with insecticides along with fungicide, which reduces the insect damage during storage and germination. When applying two or more pesticides, the compatibility of the chemicals must be determined as otherwise, the seed germination may be seriously reduced. Thiram, captan, maneb, carbendizim, benomyl, metalaxyl, and carboxin are the common fungicides used in seed treatment at the rate of 2 to 4 g kg-1 of seed. Benomyl is the most effective systemic fungicides against fusarial infections, whereas, maneb is effective against dematiaceous hypomycetes like Alternaria, Curvularia and Drechslera spp. Metalaxyl is an effective systemic fungicide effectively being used against downy mildew pathogens (Singh and Shetty, 1990). Thiram, captan and carboxin are the broadspectrum fungicides (Table 9). Table 9: List of important fungicides used as seed treatment and their target diseases Fungicides Benomyl Captan Carboxin Difenoconazole Fludioxonil Imazalil Mancozeb Hymexazole Maneb Mefanoxam Metalaxyl Oxadilyl PCNB Streptomycin Thiabendazole (TBZ) Triabendazole Triadimenol Thiram Disease Wheat bunt, wheat loose smut, blackleg of canola Seedling blight Wheat bunt, loose smut, covered smut, barley stripe, seedlings blight, sunflower rust, Rhizoctonia root rots of dry beans/soybeans Bunt, loose smut, seedling blight, common root rot, take all root rot Seedling blights, Rhizoctonia root rot sugar beets Seedling blights, common root rot, barley stripe Bunt, smut, seedling blights, safflower rust Aphanomyces, Pythium in sugar beets Bunt, smut, seedling blight Pythium seed rots, seedling blight, Phytophthora root rot soybean, lentils Seedling blights, Phytophthora root rot, Pythium seed rots, Downy mildew Phytopthorra root rots, Pythium seed rot. Bunt, covered smut, seedling blight, safflower rust, Rhizoctonia root rot Bacteria in beans Bunt, seedling blights, Ascochyta (chickpea) Bunt, loose smut, covered smut, seedling blight, Root rot, Barley stripe Bunt, loose smut, covered smut, seedling blights, root rot. Seedling blights, safflower rust

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5.2 Seed treaters Commercial seed treaters ensure application of accurate quantities of pesticides, as too much pesticide may injure seed or too little is often ineffective. The commercial seed treaters are of three types-liquid treaters, slurry treaters and dust treaters. Seed treatment may be of three types- physical, biological and chemical. Physical treatment usually refers to heat treatment of seeds. Heat treatment is the oldest method used to eradicate deep-seated infection of seeds. Hot water treatment was effective against Xanthomonas axonopodis pv. malvacearum (Syn. X. campestris pv. malvacearum infection in cotton seeds (Honervogt and Lehmann-Danzigen,1992). Dry heat is also reported to be useful in elimination of X. camtestris pv. translucens from barley seeds at temperature ranging from 71 to 84 C for 11 days (Fourest et al., 1990). Heating in macrowave reduced transmission of soybean mosaic virus. Low energy electronic beams eliminate Tilletia caries and Septoria nodorum from wheat seeds (Burch et al., 1991). 5.3 Biological control Several potential biological control agents have been identified in the recent past and have reached even the shelves of commercial traders. Many plant growth-promoting rhizobacteria (PGPR) consisting mainly Pseudomonas fluorescens and Bacillus species have been tried as biological control agents. Bacillus subtilis is marketed as Kodiak for seed treatment by Gustafsm Inc. Dallas, Texas (Rhodes and Powell, 1994). Even nonpathogenic fungi like Trichoderma and Chaetomium spp. are effective as biocontrol agents. However the effectiveness of biological control is affected by environment. There is still scope for identifying newer strains, especially endophytes as biological control agents and their active metabolites. 5.4 Application In most of the cases, seeds are treated in the processing plant to achieve precision coating and penetration of the seeds with an appropriate dosage of chemicals. Only in some instances, the chemicals are applied to seeds just before sowing at farmers level. But later approach will not ensure appropriate dosage and effective coverage. Seed pelleting was developed in the 1940s to give uniform shape to seeds and aimed at precision sowing of vegetable seeds. Pelleting is done by inert materials like chalk, peat or sand mixed with adhesive. Fungicides may also be mixed with pelleting material. Film quoting with polymer binders may be formulated with colorants and plasticizers (Halmer, 1994). This method is used to apply fungicides, especially in vegetable seeds (Ester, 1994), as well as field crops including maize, oil seeds, rye and sunflower (Barlett, 1994). This may also protect seeds from storage fungi. In fluid drilling method, the seeds are primed in aerated water in controlled environment to initiate germination. Such seeds are mixed in a liquid gel and planted in field. Chemical or biological control agents may be delivered through fluid drilling. For instance, metalaxyl, etridiazole and captan are applied by fluid drilling to control Pythium damping-off in tomatoes (Taylor and Harman, 1990). In solid matrix priming, the seeds are mixed with solid material and water in known proportions facilitating controlled uptake of water. This method is used to deliver chemical or biological control agents. Trichoderma spp. population applied to seeds increased due to solid matrix priming (Taylor and Harman, 1990).

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6. Seed certification The certification of seed health is assessed at two stages During the crop production stage as per field standards, based on field inspection by authorized seed inspectors of seed certification agency. During seed quality assessment by the seed testing station, based on seed inspection as per seed standards. The seed-borne inoculum acts as an important contributing factor for disease epidemics. Since the viral disease cannot be controlled by any chemicals, use of healthy seed lots is an alternative and feasible strategy for controlling seed-borne viral diseases. This needs an effective seed health certification scheme and fixing of tolerance levels, as well a simple and reliable seed testing procedure for routine seed health testing. Attempts have been made in the past to fix tolerance limits to some of the seed borne viruses; Lettuce mosaic virus (LMV) in lettuce (Grogan, 1983), Cucumber mosaic virus (CMV) in narrow leafed lupin (Bywe et al., 1995), Alfalfa mosaic virus (AMV) in burr medic (Medicago polymorpha) (Jones and Nicholas, 1998) and Alternaria leaf spot and charcoal rot in sunflower (Bhutta et al., 1999). Limited information is available for some other economically important seed-borne viruses with regard to contribution of seedborne inoculum to the disease spread and its impact on yield. The study covers Bean yellow mosaic virus (BYMV; Kaiser, 1973), Broad bean stain virus or Broad bean true mosaic virus in faba bean (Jones, 1980), BYMV in yellow lupin (Corbett and Edwardson, 1957), Peanut mottle virus in groundnut (Adams and Kuhn, 1977), cucumber mosaic virus in lentil (Fletcher et al., 1999) and Pea seed borne mosaic virus in pea (Masmoudi et al., 1994). Fixing of inoculum threshold to seed-borne diseases is difficult. It is influenced by many factors, such as macro and micro-environment, cultural practices, agricultural systems, locations, certification requirements and class of seed, breeder, foundation, or certified seed. For example, lettuce mosaic virus can be controlled by using seed certified to have 0 infected seed in 30,000 in California and 0 in 2,000 in the Netherlands (Grogan, 1980). Seed-borne viruses that are also transmitted by aphids are of particular concern when infected seed serves as the primary source of inoculum. Some of the most economically important plant virus diseases are in this category. This appears to be the case for many potyviruses (Smith and Hamilton, 1988). Information on germination of infected seeds and survival of resulting plants, virus disease progress during the growing season, magnitude of yield loss and amount of infection in harvested seed in replicated field experiments is required to establish acceptable threshold levels of seed-borne infection (Thackray et al., 1998). Infection of one-week-old cowpea plants resulted in a higher proportion of seed infection than in those infected later as reported by Sumana and Murthy (1992) and Nain et al. (1994). Puttaraju et al. (2004) reported that sowing cowpea seeds with 4 to10% infection of BCMV-BlCM resulted in 65 to 100% infection at maturity. A tolerance of one Xanthomonas campestris pv campestris infected seed per 1000 is fixed for direct sowing of Brassica seed, whereas zero tolerance is required for transplanting (Schaad, 1980). Testing of 30, 000 seed as three-sub sample of 10,000, is

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required for establishing freedom from black rot as per industry requirement (Franken et al., 1991). Only one infected seed among 10,000 seeds is sufficient to cause severe crop loss, particularly in transplant seedlings (Williams, 1980; Roberts et al., 1999) In tomato single Clavibacer michigiganensis sub sp. michiganensis infected seed over 10,000 seeds can initiate an epidemic (Chang et al., 1991; Gitaitis et al., 1991). 7. Seed health testing facilities Seed health testing laboratory should include equipment required for seed health test for fungi, bacteria and viruses. A brief list of essential equipments is given in table 10 to12. Table 10. List of equipment needed to set up a seed health-testing laboratory fungal pathogens
Sl. No. 1 2 3 4 Equipment Low speed centrifuge (Clinical centrifuge) Stereo binocular microscope Compound binocular microscope Laminar flow Units One Four Two One Use Sedimentation of debris in seed washing test Observation of fungal colonies on seeds Observation of fungal conidia Isolation of pure culture Approx. unit price US $* 100 4000 2000 2000

Other facilities Incubation room

Specifications Air-conditioned room that could be maintained at 20-25 oC. Shelves with two near ultraviolet light tubes hung at 40 cm height from the bench and 20 cm apart, light cycle controlling device.

Table 11. Bacteriology

Sl. No. 1 2 3 4 5 6 7 8 9 10 11 12 Equipment Fluorescent microscope Universal shaker Compound microscope Autoclaves 2 size Bacteriological incubators capacity 200 liters Hot air oven Electronic balanceanalytical Electronic balanceprecision Laminar flow (Class II) UV lamp Quartz distillation unit Deep freezer (- 20 oC) Units One One Two 20 liters 50 liters One One One One One One One One Use Observe immunofluorescence of bacteria Extraction of bacteria Observation of bacteria for identification/Grams staining Wet sterilization Incubate bacterial culture plates Dry air sterilization Weigh chemicals Weigh chemicals Pure culture isolation Viewing of fluorescent bacterial colonies Distilled water Store fine chemicals Approx. unit price US $* 10000 500 2000 1000 400 400 1000 1200 2000 200 2000 1500

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Table 12. Virology

Sl. No. 1 2 Equipment Micro plate reader (ELISA reader) Finn Pipettes Units One Set Use Conduct ELISA test Dispensing exact micro volumes if reagent Approx. unit price US $* 4000-8000 2000

Optional Sl. No. 1 2 Equipment Ultra centrifuge with two rotors Electron microscope One One Units Use Approx. unit price US $* 40,000

Purification of virus View virus 200,000 particles/Immunosorbent electron microscopy

* Price may vary depending on model. Other facilities Screen house Climate room US $ 20,000 US $ 20,000

Molecular detection of seed borne pathogens Thermo cycler Ultra freezer (-80 oC) Gel documentation unit 8. Conclusion Seed is a vital input in agriculture and use of quality seed contribute substantially to agricultural production and quality of produce, globally. The use of healthy seed is a key factor to boost agricultural production, also in developing countries. With the advent of new high yielding varieties and in particular, hybrid seeds, the farmers greatly depend upon various agencies for continuous seed supply to get maximum yields. Further, the high yielding varieties of cereals, pulses and oil seeds etc., demand greater requirements of basic inputs such as fertilizers, pesticides, etc., and improved cultural practices to maximize agricultural production and thereby improve the agricultural economy of the country. However, many of the high yielding varieties that are released in the past are highly susceptible to pests and diseases, thereby resulting in the built up of pests and diseases leading to epidemics, causing serious crop losses. Seed-borne pathogens are of critical concern in seed production and quality as they adversely affect the germination and vigour of the seedlings, and thus yield and quality of the product. 38 US $ 8000 Preservation of reference culture US $ 6000 Scan and store gel data US $ 8000

Therefore, seed health testing is of key concern in seed production. Sound seed certification programs greatly contribute to production of good and healthy seed which require proper management of seed crops for obtaining disease free seed. To this end private seed industries and co-operatives play a larger role in ensuring adequate production and supply of quality seed to farmers through their effective management skills, marketing ability and modern seed technology practices. Though seed treatments are pivotal in control seed-borne pathogens, blanket seed treatments should be avoided as they pose health hazards, therefore post evaluation of treated seed is of utmost concern in crop production. It is recommended to use an integrated approach in production of healthy seed, taking into consideration prophylactic as well as curative measures in crop cultivation, harvest, storage and seed treatment. With the advent of new policies on seed development, the demand for import of quality seed and plant material is increasing. The increasing scale of trans-national exchange of germplasm constitute potential risk of introducing new races or strains of pest or pathogen and therefore require efficient plant quarantine strategies to deal effectively with the accidental infiltration of pests and pathogens of concern for safe conduct of imports. LITERATURE CITED Adams, D.B. and Kuhn, C.W. 1977. Seed transmission of peanut mottle virus in peanuts. Phytopathology 67:1126-1129. Albrechtsen, S. E. 2006. Testing methods for seed-transmitted viruses: Principles and protocols. CABI publishing, UK. Barlett, D.H. 1994. Review of current and future seed treatment usage in oilseed rape. Pages 159- 168 in Seed Treatment: Progress and Prospects Mono. 57, BCPC, Thornton Health UK Beck, J.J., and. Ligon, J.M. 1995. Polymerase chain reaction assays for the detection of Stagonospora nodorum and Septoria tritici in wheat. Phytopathology 85:319-324. Bhutta, A. R, Rahber Bhatti, M. H, Ahmed, I and Shakoor, C. A, 1999. A studies of correlation between seed and field infection/intensity for establishing tolerance limit in sunflower. HELLIA 22(31):137-142. Burth, U., Gaber, K., Jahn, M., Lindner, K., Motte, G., Panzer, S., Pflaumbaum, J., and Scholze, F. 1991. Seed treatment with electron beams - a new method for the control of seedborne pathogens on winter wheat. Nach. Deutsch Pflanzenschutzdienstes 43:41-45 Bwye, A.M, Jones, R.A.C. and Proudlove, W. 1995. Cucumber mosaic virus in lupins. Western Australian Journal of Agriculture, Fourth Series 36, 124-130. Chang, R. J, Ries, S. M., Pataky, J. K. 1991. Dissemination of Clavibacter michiganensis subsp. michiganensis by practices used to produce tomato plants. Phytopathology 81, 127681. Clark, M.F. and Adams, A.N. 1977. Characteristics of the microplate method of enzymelinked immunosorbent assay for the detection of plant viruses. Journal of General Virology 34: 475-483.

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Corbett, M.K. and Edwardson, J.R. 1957. Virus diseases of yellow lupins: Preliminary investigation on control by use of a protecting border. Proc. Soil Sci. Soc. Florida 17, 294301. Ester, A. 1994. Film coating of leek with insecticides: effects on germination and on the control of onion fly (Delia antiqua (Meigen)). Pages 195-199 in Seed Treatment: Progress and Prospects Mono. 57, BCPC, Thornton Health, UK. Fletcher, J. D, Russell, A. C, Butler, R. C. 1999. Seed-borne Cucumber mosaic virus in New Zealand lentil crops: yield effects and disease incidence. New Zealand Journal of Crop and Horticultural Science 27, 197204. Fourest, E., Rehms, L.D, Sands, D.C., Bjarko, M., and Lund, R.E.1990. Eradication of Xanthomonas campestris pv. translucens from barley seed with dry heat treatments. Plant Disease 74:816-818. Franken, A. A. J. M. van Zeijl, C., van Bilsen, J. G. P. M., Neuvel, A., Devogel, R., van Wingerden, Y., Birnbaum, Y. E., van Hateren, J. and van der Zouwen, P. S. 1991. Evaluation of a planting assay for Xanthomonas campestris pv. campestris. Seed Sci. & Technol. 19, 215-226. Gitaitis, R., Beaver, R. and Voloudakis, A. 1991. Detection of Clavibacter michiganensis subsp. michiganensis in symptomless tomato transplants. Plant Disease 75, 834-838. Goodwin, P. H., Hsiang, T., Xue, B. G., and Liu, H. W. 1995. Differentiation of Gaeumannomyces graminis from other turf-grass fungi by amplification with primers from ribosomal internal transcribed spacer. Plant Pathol. 44:384-391. Grogan, R. G. 1980. Control of lettuce mosaic virus with virus-free seed. Plant Disease 64, 446-449. Grogan, R. G. 1983. Lettuce mosaic virus control by use of virus-indexed seeds. Seed Sci. & Technol. 11, 1043-1049. Hadas, R. 2000. International Seed Health Initiative-Veg Reference method: Detection of Tobamoviruses (TMV; ToMV; PMMV) on Pepper(Capsicum annum). International Seed Health Initiative-Veg. Halmer, P.1994. The development of quality seed treatments in commercial practice objectives and achivements. Pages 363-374 in seed treatment: Progress and Prospects Mono. 57, BCPC, Thornton Health, UK. Hampton, R. O., Ball, E. M. and De Boer, S. H. (eds) 1990. Serological methods for detection and identification of viral and bacterial plant pathogens, a laboratory manual. APS press, American Phytopathological Society, St. Paul, Minnesota. Honervogt, B, Lehmann-Danzinger, H., 1992. Comparison of thermal and chemical treatment of cotton seed to control bacterial blight (Xanthomonas campestris pv. Malvacearum). J. Phytopathol. 134:103-109. Hull, R. 2002. Matthews Plant Virology, 4tyh edn. Academic Press, London.

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Huth, W. 1997. Tissue print immunoassay-a rapid and reliable method for routine evidence of Gramineae viruses. Nachrichtenblatt des Deutschen Pflanzenschutzdienstes 49, 185-191. ISTA. 2003. International Rules for Seed Testing. Seed Science and Technology 24: 2842. Jones R A C, Nicholas D A. 1998. Impact of an insidious virus disease in the legume component on the species balance within self-regenerating annual pasture. Journal of Agricultural Science, Cambridge 131:155-170. Jones, R. A. C. 1980. Seed-borne viruses and the possibility of producing seed free from broad bean strain virus and Echtes Ackerbohnenmosaik-Virus. In: Bond, D. A. (Ed.), Vicia faba: Feeding value, processing and viruses. Martinus Ninjhoff, The Hague, pp 319-333. Kageyama, K., Ohyama, A., and Hyakumachi, M. 1997. Detection of Pythium ultimum using polymerase chain reaction with species-specific primers. Plant Dis. 81:1155-1160. Kaiser, W. J. 1973. Biology of bean yellow mosaic and pea leaf roll viruses affecting Vicia faba in Iran. Phytopath. Z. 78, 253-263. Kohler, G. and Milstein, C.1975. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495-497. Lee, H. K., Tewari, J. P., and Turkington, T. K. 2001. A PCR-based assay to detect Rhynchosporium secalis in barley seed. Plant Dis. 85:220-225. Lovic, B. R., Martyn, R. D., and Miller, M. E. 1995. Sequence analysis of the ITS region of rDNA in Monosporascus spp. to evaluate its potential for PCR-mediated detection. Phytopathology 85:655-661. Masmoudi, K., Duby, C., Suhas, M., Guo, J. Q., Guyot, L., Oliver, V., Taylor, J and Maury, Y. 1994. Quality control of Pea seed for pea seed borne mosaic virus. Seed Sci & Yechnol 22, 407-414. Mullis, K. B., and F. A. Faloona. 1987. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 155 :335-350. Nain, P. S. Rishi, N. and Bishnoi, S. S. 1994. Profile of viral diseases of cowpea in Northern India. India J. Virol. 10, 128-136. Oliver, V., Germain, R., Cucuzza, J., Linders, H. and Gregoire, S. 2001. Final report: Detection of lettuce mosaic virus in lettuce seeds (comparative tests). International Seed Health Initiative-Veg. Puttaraju, H. R., Prakash, H. S. and Shetty, H. S. 2004. Seed infection by Blackeye cowpea mosaic potyvirus and yield loss in different cowpea varieties. J. Mycol. Pl. Pathol. 34, 41-46. Rhodes D.J., Powell, K.A. 1994. Biological seed treatments - the developmental process. Pages 303-310 in Seed treatment: Progress and Prospects Mono. 57, BCPC, Thornton Health, UK.

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Roberts, S. J., Hiltunen, L. H., Hunter, P. J. and Brough, J. 1999. Transmission from seed to seedling and secondary spread of Xanthomonas campestris pv. campestris. Brassica transplants: effects of dose and watering regime. European Journal of Plant Pathology 105, 87989. Rybicki, E. 2001. PCR primer design and reaction optimization. In: Coyne, V. E., James, M. D., Reid, S. J. and Rybicki, E. P. (eds) Experts from a Molecular Biology Techniques Manual, 3rd edition, University of Cape Town, Department of Molecular and Cell biology. Schaad, N. W. 1980. Gram-negative bacteria: Xanthomonas. In: Schaad, N. W., Jones, J. B. and Chun, W. (Eds.) Detection of bacteria in seed and other planting material. St. Paul, MN, USA: APS Press, 68-75. Schaad, N.W., Cheong, S.S., Tamaki, S., Hatziloukas, E., and Panopoulos, N.J. 1995. A combined biological amplification (BIO-PCR) technique to detect Pseudomonas syringae pv. Phaseolicola in bean seed extracts. Phytopathology, 85: 243248. Singh, S. D. and Shetty, H. S. 1990. Efficacy of systemic fungicide metalaxyl for the control of downy mildew (Sclerospora graminicola) of pearl millet (Pennisetum glaucum). Indian Journal of Agricultural Science. 60, 575-581. Stace-Smith, R. and Hamilton, R. I. 1988. Inoculum thresholds of seed-borne pathogens: viruses. Phytopathology 78, 875-880. Sumana and Murthy, K. V. 1992. Reaction of cowpea cultivars to blackeye mosaic virus . Legume Res. 15, 141-142. Taylor, A.G, Harman, G.E. 1990. Concepts and technologies of selected seed treatments. Annu Rev. Phytopathol, 28:321-39. Thackray, D. J., Diggle, A. J., Berlandier, F. A., and Jones, R. A. C. 1998. Deveoplemt of a simulation model forecasting aphid outbreaks and spread of cucumber mosaic virus in lupins. In: Zalucki, M. P., Drew, R. A. I., White, G. C. (Eds.), Pest Management-Future challenges. Proceedings of The Sixth Australina Applied Entomological Research Conference, vol. 2. University of Queensland Press, Brisbane, pp. 99-106. Weller, S.A., Elphinstone, J.G., Smith, N.C., Boonham, N., and Stead, D.E. 2000. Detection of Ralstonia solanacearum strains with a quantitative, multiplex, real-time, fluorogenic PCR (TaqMan) assay. Appl. Environ. Microbiol. 66: 28532858. White, TJ., Bruns, T., Lee, S. and Taylor, JW. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR Protocols: A guide to methods and applications (Innis, M.A., Gelfand, D. H., Sninsky, J. J. and White, T. J., Eds.), pp. 315-322. Academic Press, San Diego, CA. Williams, P. H., 1980. Black rot: a continuing threat to world crucifers. Plant Disease 64, 73642.

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