Fungal and Bacterial Seed Health Testing Protocols for

Vegetables

Edited By

Muhammad Boota Sarwar

https://www.facebook.com/BootaSarwar?ref=tn_tnmn msarwar80@hotmail.com

92-3003003405

Federal Seed Certification & Registration DepartmentFungal and Bacterial Seed Health Testing Protocols for Vegetables

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A high quality seed is an essential prerequisite for sustainable and profitable vegetable production. Farmers have to deal with significant loss due to infection with serious seed-borne pathogens on their plants, which may start from germinating seed, seedling in the nursery, grown plants in the field and proceed till the products are kept in the storage. Health conditions of seeds are crucial information for all users, especially to make up decision of necessary treatment. High quality seed is not only need to be true to type, but also clean from contaminants, should have a high viability, and should be healthy (free from any pathogen) .

In order to achieve a high quality seed, many countries have applied a routine testing of seed-borne diseases as an integral part of seed testing, seed certification and are considered as a tool for entire seed-borne disease control program. The requirement of Phytosanitary Certificate for Seed trade among regions and countries has also increased importance of seed health testing. Vegetable seeds produced in Pakistan or imported have not yet applied international standards for the presence of plant pathogens, neither to eradicate these pathogens despite of the fact that vegetable seeds are contaminated with various seed-borne pathogens. Government agencies are in position to have a better understanding of producing and controlling healthy seed. Therefore, there is dire need to make an effort to develop and apply detection methods for economically seed-borne diseases on vegetables, diseases control in the field and seed lot, seed treatment to minimise and avoid pathogen contaminant, and to allow establishment the standard of health vegetable seeds based on the international regulation adopted from International Seed testing Association (ISTA).

Vegetables' are not only vital for healthy diets and productive lives but also vital for strong economies. According to FAO World production of vegetables is over 193919100 million pounds while China, Egypt, India, U.S.A are the largest vegetables producers all over the world (6).

Potato, Tomato, onion, egg plant, carrot, lettuce, pumpkin, squash, guards are the major vegetables grown through out the world. (28).Production of vegetables is becoming profitable Agri. business in Pakistan.Agro ecological conditions of Pakistan are best suited for most of the vegetables. In the year 2006 these were grown over an area of 2386000 hectares with production of 30485000 tones. The major Kharif vegetables include Lady Finger,Squash (Tinda),Brinjal,Bitter Gourd,Bottle Gourd,Pumpkin,Lufa ,Long Melon,Cucumber,Pursilano ,Arum ,Beans,Field. Vetch, Lotus and Tomatoes. While Rabbi vegetables include Radish Turnip,Carrot,Spinach,Cauliflower,Cabbage Sweet Patato,Peas Garden Peas,Kanol Kohl, Fennu Greek, Lettuce, Sugar Beet and Tomatoes (1).

1. Diseases of vegetables, identification and management:

1.1.1. Wherever the vegetables are grown they are subjected to different diseases that seriously reduce both the yield and market value. Before establishing and applying any management practice it is important to understand the biology of seed borne disease. Seed borne diseases are caused by fungi, bacteria, viruses and nematodes. In most of the seed borne diseases infected seed is the primary source of inoculum and if it is controlled the disease is controlled (33). In some diseases the pathogen is devastating in field but the disease is not seed borne. Some Pathogens are also infecting seeds in fields or in storage, and reduce seed quality. Some important vegetables seed borne diseases are given in Table 1.

Table 1 Some important seed borne fungal and bacterial diseases of vegetables[Type text]

S.No. Family/crop(3) Botanical name(3) Diseases(26,31,19) Causal organism

  Umbelliferae   Bacterial BlightXanthomonas compestris pv. Carotae

1 Carrot Daucus carota Alternaria diseasesAlternaria dauci, Alternaria radicina

2 Coriander Coriandrum sativum Cercospora leaf spot Cersospora carotae  Alliaceae   Grey mold Aspergillus flavus, A.niger

3 Onoin Allium cepa Scape blight/neck rotBotrytis alli, B. squamosa, B. cinerea, B. byssoidea

4 Garlic Allium sativum Bacterial soft rot

Dickeya zeae (Pectobacterium chrysanthemi)

      Fusarium basal rotFusarium oxysporum f. sp. cepae

  Cruciferae   Alternaria diseasesAlternaria brassicae, Alternaria brassicicola

5 Raddish Raphanus sativus Ring spot Mycroshaerella brassicicola

6 Turnip Brassica rapa Black leg Phoma lingam

7 Cabage Brassica rapa Black rotXanthomonas compestris pv compestris

8 cauliflower Brassica oleracea Leaf blightXanthomonas compestris pv raphani

      wilt

Verticillium dahliae, V. alboatrum, Fusarium moniliforme, F. oxysporum

  Chenopodiacea  Cladosporium leaf spot Cladosporium variable

9 Spinach Spinacia oleraceaStemphylium leaf spot Stemphyllium botryosum

10 Beet Beta vulgaris AnthracnoseColletotrichum dematium, C. spinaciae

  Solanaceae   Black leg/soft rot Erwinia carotovora11 Potato Solanum tuberosum Black scurf Rhizoctonia solani

      Dry rotFusarium sambucinum, F. solani

      Brown rot Ralstonia solanacearum      Scab Streptomyces scabies      wilt Ralstonia solanacearum

12 TomatoLycopersicum esculentum wilt Ralstonia solanacearum

      Early blight Alternaria solani      Fruit rot Alternaria tenuis

13 Chillies Capsicum annuum Fruit rot Alternaria tenuissima      wilt Ralstonia solanacearum

14 Brinjal

Solanum melongena wilt Ralstonia solanacearum

  Leguminosae   Ascochyta complex

Ascochyta pisi, Phoma medicagnis, Mycosphaerella pinodes

15 Peas Pisum sativum Grey mold Botrytis cinerea

      Bacterial blight

Psudomonas syringae pv pisi

2. Seed health testing

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2.1.1. This has two important components i.e. suitable seed health assay and disease tolerance standards. Developing a seed health testing method is complex and requires consideration of the disease dynamics. The epidemiology of a disease, a change in the disease in time and space, depends on the infection level in the seed, the climatic zone where the seed is grown and the effect of such zones on infection rates.

2.1.2. Disease tolerance standards are established by appropriate statistical analysis of inoculum thresholds for seed borne diseases, incidence of infection on seeds and its correlation with plant infection in field conditions(10).

3. Methods for seed health testing

Different techniques/methods are used for the seed health testing are given below (4):-

3.1. Field inspections3.2. Direct visual examination3.3. Incubation3.4. Grow-out3.5. Indicator tests3.6. Serological3.7. DNA hybridization

4. Detection techniques for seed borne fungi:

4.1. Direct and indirect Inspection of seed with out incubation

This method is used to have first hand information about the abnormalities on seed surface. But it also helps for selection of particular method for detection of seed- borne pathogens.

Procedure: Usually seeds are examined under a hand lens, preferably a stereo microscope. Visual symptoms are examined on seed showing discoloration and malformation, including bodies of fungi and resting hyphae on the seed. The method is applicable to many pathogens such as Aschochyta, Botrytis, Botryodiplodia, Cercospora, Colletotrichum, Diplodia, Fusarium, Pleospora, Macrophomina and Rhozoctonia spp.

Stem gall Disease of Coriander (Protomyces macrosporus) (8): Dry Seeds of whole samples can be examined both by naked eye and by stereo microscope at a magnification of 6-12 X. Four hundred cremocarps of coriander are taken from a seed lot and examined for infected seeds which are partially or completely atrophied. The diseased tissue then may be crushed to a powder and examined in water drop on a glass slide under compound microscope at 40-200X. Clamydospores of fungus are golden yellow with a three layered wall more or less spherical in shape (45-55 µ diameters).

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4.2. Incubation Tests:

Incubation tests are primarily used to determine the kind, amount and distribution of inoculum. Major types of tests based on incubation of seed sown on a suitable medium and substrates are:

a) Agar plate method:- This test is based on growth of pathogen only. b) Blotter method: - It determines the growth of seedlings and pathogens. c) Seedling symptom tests: - In this test, seeds are grown to a seedlings seeds stage for

development of disease symptoms. For .this purpose, seeds are sown in soil and sand. d) Growing on check: - This test is based on the growth of host and on development of

symptoms. e) Indicator test: - An indicator plant is inoculated by test pathogens. It is mostly used

for detection of viruses. Incubation tests produce good results under standard conditions of incubation only.

4.2.1. The Blotter Paper Test (11): This is a broad-spectrum method having combination of the in vitro and the in vivo principles of investigation. The basic advantage of blotter test is quick identification of habit characters such as length and arrangement of conidiophores, size, septation, color, chain formation, appearance of spore masses, characters of mycelium etc.

Procedure: The seeds are sown in Petri dishes or in other suitable containers on moistened absorbent paper (blotter), usually in three layers to provide enough moisture during test. In case of light colored fungus like Verticillium or Cephalosporium spp., black or gray blotter paper may be used to facilitate observations. The seeds are placed at a fixed distance according to their size. Small seeds are plated @ 20-25 and large seeds 10-16 per plate (Plate diameter 9 cm.). The seeds are incubated for a fixed period of time and temperature. Usually one week at 20°C ±2 (standard temperature for most seed germination tests). The day and night cycle of 12/12 hrs. is used under fluorescent light. Recording of the fungal growth is made by a low power stereoscope with up to 50 or 60 X magnification and the data is taken on infection percentage.

The infection percentage is based on presence of the pathogenic signs of disease in/on the seedlings which may have germinated during incubation. For noting of fungi (disease incidence), a color pencil that writes on wet paper is used to facilitate recording of observations. Stereoscope with two opposite light sources should be used to visualize the growth of pathogens.

Note: Some times blotters are soaked in 0.1 - 0.2 per cent 2, 4-D solution (2, 4- dichoro phenoxyacetate) to retard seed germination. For certain kinds of seed, which are heavily contaminated with saprophytes. Pre-treatment with some suitable disinfectant e.g. 1-3 per cent chlorine solution is used.

Application: The blotter method is widely used where regular seed health testing is carried out. It is applicable to wide range of crop seeds including cereals, vegetables, and oil seeds. Ornamentals and forest seeds for detection of different spp. of Acremoniurn, Alternaria, Ascochyta, Botryodipiodia, Botrytis, Cephalosporium, Colletotrichum, Curvularia. Diplodia, Drechslera, Fusarium, Nigrospora, Macrophomina, Myrotheciuin, Phoma, Phomopsis, Pyricularia, Sepioria, Stemphylium and Trichoconis spp.

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4.2.1.1. The Freezing Method (22,11): This method is a modification of the blotter paper method. Seeds are kept at low temperature the seedlings are then frozen overnight (at -20°C to retard germination of seed) and then incubated at 20°C for 5-7 days.

Application: The deep freezing retards the germination process of seed and the subsequent fungal development is often more abundant but it depends on particular host- pathogen combination. This method is preferred for detecting fungi such as Phoma lingam in cruciferous seeds, Septoria nodorum in wheat and Alternaria porn in onion seeds.

4.2.2. Ragdoll (Rolled Towel) Method (22): The seeds (100 in number) are paced at a distance. This method works in two ways, firstly it gives germination percentage of a test seed lot and secondly disease symptoms are observed on the seedlings.

Application: This method is used for detection of those pathogens which produce symptoms on roots, e.g. detection of F. solani in gram, many others pulses and leguminous crops

Procedure: Hundred seeds are placed on two well moistened blotter papers (48 x 48 cm.) and then covered with another well moistened blotter of the same size. The blotters are rolled in many folds or wrapped in plastic bags and incubated at 20°C ±2°C for seven days. Cotton towel or anchor brand brown paper can be used instead of blotters. Evaluation: Germinated and ungerminated seeds are examined by naked eye or under the stereoscope. Discoloration on cotyledon leaves, stems and roots may be observed due to Fusarium, Rhizopus and Mucor spp. etc.

4.2.3. The Agar Plate Test (Incubation in Agar Media)(17): It is a common method, which is used to study the fungal growth and colony character on agar medium. Low pH media such as potato dextrose agar (PDA) or malt extract agar (MA) are best suited for field fungi and common saprophytes. The method is preferable where the blotter method does not provide adequate conditions for development of mycelial growth, sporulation or symptoms of pathogens on seedlings and seeds. Muskett and co-workers in Northern Ireland used this method for the first time in a large scale test scheme for predicting health condition of 25000 flax seed lots in 15 years.

Procedure: Four hundred seeds are taken as working sample. Number of seeds and space depend on seed size and pathogen to be tested. For most kinds of seeds, tested with the agar plate method, a hypochlorite solution @ 0.5 to 1.0 per cent is used for 10 minutes as disinfectant to check the growth of saprophytes. Seeds are plated on sterilized agar medium. Selective media may he used for specific test. Usually, 10 seeds are plated in a 9 cm. diameter Petri dish. Seeds are incubated at 20°C ±2°C for eight days under fluorescent day light tubes.

Evaluation: Microscopic examination of fungus is done for various characters of fungal growth. Colony color is a very important character for many fungi such as development of spore shape and size depends on light and temperature.

Application: The agar plate method is applicable to those kinds of seeds in which saprophytic species do not materially impair quick identification of pathogens. This can be often overcome by pre treatment with a suitable disinfectant. This procedure is relatively sensitive for revealing minor amount of inoculum and in detailed studies of the particular fungal species. Agar plate method can be used for detection of all pathogens that are detected by blotter method keeping in view the economics of seed health testing.

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4.2.3.1. Agar Plate Method for detection of Fusarium Oxysporum in Tomato Seed (5,14)Four hundred seeds are plated on Littman Agar medium @ 10 seeds per plate . The incubated seed are incubated for 5 days at 25 oC. Colonies showing variable pigmentation from pale to rose, purple and vinaceous are produced by the pathogen. Stereomicroscope can be used for observation of clear colony characteristics. Microscopic examination reveals macroconidia in false beads, mostly 1 celled, oval to reniform measuring 5-12 × 2.2-3.5. Macro-conidia in globose sporodochia or in pinnotal slime, falcate, gradually tapering toward both ends with a pointed or highly hooked apical cell and foot shaped cells from 0-7, mostly 3 septate from 4-13×2.0-3.5 to 34-78 ×3.0-6.0 predominantly 27-46×3.5. Phialides monophialide, chlamydospores globose, generally abundant in hyphae and conidia, terminal or intercalary both smooth and rough walled, single in pairs or in short chains.

4.2.3.2. Special Agar Plate procedure for Detection of Phoma betae in Beat ( 16) In some cases modified agar plate method is used e. g in case of Phoma betae in Beet seeds. ater agar (1.2% agar) containing 50 ppm of sodium salt of 2, 4-D (2, 4- dichiorophenoxyacetate) is poured at the rate of 1 5m1/9 cm. Hundred seeds without any pre treatment are plated @ five seeds per plate. The plates are incubated for 7 days at 20°C in darkness. Hold fast like structure or hyphae clumps of Phoma betae. The hold fast is formed only at the bottom of the dish and correct focusing is important. They are typically formed at the tips of hyphae as a number of haline swellings. In the presence of bacteria the holdfast may be restricted in development and later on become brown in color.

4.2.4. Water Agar Seedling Test (Test Tube Agar Method) (23): Application: The procedure was originally developed for detection of Xanthomonas campestris in seeds of cabbage and crucifers and is now being used for seed health testing in barley and wheat for observing symptoms of Drechslera graminea, D. teres and D. sorokiniana on seedlings. It is also an economical procedure for observing symptoms of Septoria nodorum and Didymella bryoniae in wheat and cucurbit respectively.

Procedure: The seeds are sown in test tube of 16 mm diameter. One seed is placed in a test tube containing 10 ml of water agar medium. Tubes are covered in-group by aluminum foil to retain moisture and are incubated at 20°C under artificial day light tubes at 12/12 hrs. The aluminum foil is removed when the seedlings have reached the cover. The length of incubation is determined by the time needed for development of symptoms. Note: Water may be replaced by any nutrient or selective medium.

Evaluation of seedlings: Conidia develop on the agar and at the tip of rotted seedlings as discrete brown spots and pustules on shortened and malformed coleoptiles which is a clear indication of infection in both cases

4.2.5. Seedling Symptom Test (13) Seedling symptom test provide information pertaining to field performance and planting value of seed lots in relation to seed-borne seedling diseases. As approach to establish natural conditions during test, seeds are sown in autoclaved soil, gravel or sand etc.

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Application: This method is useful for testing of valuable materials for quarantine purpose and for those pathogens, which produce symptoms on roots and seedlings. The test also gives good performance for testing of treated seed samples. Pathogens which produce good symptoms under seedling symptom test are

- Drechslera spp., Fusarium spp, Septoria spp.

5. Seed Health Testing For Bacterial Diseases

After fungi, bacteria cause large number of diseases, which are transmitted through seed in the world. In Pakistan, only two dozen bacterial diseases have been reported in various crops by different workers. Out of these, 17 diseases that are known to be seed-borne in nature(4). Practically testing for seed-borne bacteria for vegetables is not in general practice is so far restricted to a few major crops.

Detection Techniques for Seed-Borne Bacteria .

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a) Direct inspection of seeds.b) Seedling symptom tests on various substrates. c) Water Agar seedling symptom tests, using different agar media. d) Direct plating of seed on selective media. e) Seed washing liquid plating tests and confirmation by biochemical and

Pathogenicity tests. f) Detection and identification of bacteria using serological tests (ELISA etc.) g) Bioassay techniques i.e. phage plague techniques.

5.1. Direct Inspection of Seed

Some bacteria produces discoloration on seed surface and rotting may be produced in the presence of suitable humidity and temperature. It is a useful method for inspection of vegetatively produced seeds e.g. seed potato.

Direct Inspection Of Potato Seed Tubers (2,9)

Potato tubers (200 in No.) are taken at random from a seed lot and examined using a hand lens or a stereoscopic microscope. To examine the first disease symptoms inside, the tubers are cut at the stolen end either transversely or longitudinaly with a sharp knife or scalpel.

Application: The following potato tuber diseases can be observed with visual symptoms:- Bacterial wilt / Brown rot (Pseudoomonas solanacerum); A grayish bacterial slime may ooze through eyes or stolen end of tubers and from the vascular rings of cut tubers. Soft rot/ Black leg (Erwinia carotovora sp. carotovora and Ec. sp. atroseptica). New tubers some times rot at the stolen end. Soft rot bacteria may infect lenticels when tuber surfaces are wet, causing circular depressed area.

Ring rot (Corynebacterium sepedonicum). In most of the tubers, vascular ring becomes rotted and turns gray, yellowish tan or radish brown.

Common scab Streptomyces scabies,(Distinguished raised, sunken corky, brownish to blackish lesions on the surface of tubers.

Dry rot ( distinguished by hard tissues of the tuber with whitish mycelial growth inside caused by Fusarium sambucinum and F. solani )

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5.2. Growing on Test (Seedling Symptom Test) (11)

Seeds are placed on wet towels or sown in sand and soil. The resulting cotyledons are observed under stereomicroscope for symptom study.

5.2.1. Detection of Xanthomonas campestris pv. Campestris from Crucifer seeds using Blotter Papers:

In crucifer seeds, no visible symptoms for bacterial disease are produced on seed. The purpose of this test is to detect the black rot bacterium by recording symptoms from seedlings grown on blotter paper placed in plastic boxes.

Materials Plastic boxes, 30 x 22 x 8 cm., I ml syringes; Hypodermic needles 22, gauge 2.5 cm., polythene bags; Petri dishes; Blotter papers; Test tubes; Sterile saline 0.85%; Reference isolate of X campestris; Scalpel; Forceps; Needles; Bunsen burner; Greenhouse or Growth chamber.

1. A total of 500 seeds are plated on 2-3 layers of moist filter papers in plastic boxes of internal dimensions of 30 x 22 x 8 cm. Seeds are sown at a density of 2.5 seeds/cm2 at 22 - 25 °C temperature in daylight for 8 - 18 days.

2. Dead, black and collapsed seedlings are recorded. If symptoms are not observed, seedlings can be incubated up to 21 days.

3. Seedlings showing symptoms are selected and crushed in sterile saline; the suspension is left for 10 - 15 minutes.

4. About 4 - 6 weeks old Brassica seedlings are inoculated with the suspensions from the diseased seedlings by injecting below the cotyledonary leaves. Plants are injected with a reference isolate of X campestris. Plants are incubated at 22-25°C and covered with a polythene bag for 24 hours.

5. Seedlings showing yellow discoloration, water soaked areas and browning of the stems and veins are recorded after 5 - 7 days.

5.2.2. Seedling Symptom Test for Pseudomonas lachrymans from Cucumber seed using Paper Towel Method (30):

Procedure: Cucumber seeds (400) are placed on a paper towel where after another towel is placed on the top. The towels are rolled and placed in incubation room.

Incubation: Rolled towels with seeds are incubated at 20-30°C with 12/12-hrs. Light cycle for eight days. For development of disease lesions on seedling, 92-98% relative humidity is required.

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Examination: Examination of seedlings is preferably made after the eight days. Water soaked dark stripes elongated creases, or round irregular spots develop on the under surface of cotyledons. In some instances, the epidermis is eaten out and gray sediment, typical of P. lacrymans, appears in the form of small heads in the lesion. When the rate of infection is low, a lesion may appear as a small, round water soaked spot, or as a stripe along the margin of the under surface of the cotyledon. In doubtful cases, bacterium identification is confirmed by inoculation on cucumber plants.

5.2.3. Detection of Xanthomonas vesicatoria From Chilli and Tomato Seeds (25)

Procedure: Four hundred seeds are sown in sterile soil in 15 cm. pots at 25 seeds per pot. After 10-15 days, when seedlings have reached the cotyledon stage, the pots are transferred to humidity chamber (95-100 per cent relative humidity) for 3-5 days and then again placed in green house at 20-30°C.

Evaluation: The plants are removed from humidity chamber after 10-12 days. Symptoms appear as round greenish small spots which later become ash colored and cause yellowing and dropping of the cotyledons.

5.2.4. Detection of Pseudomonas viridiflava In Seedlings of Radish, Cauliflower Tomato, Sweet Pepper and Bean (24)

Four hundred seeds are plated @ 20 seeds per Petri dish on blotter paper water agar. In case of seed sowing in peat soil, one seed per plastic pot is sown.

Incubation: Seeds are incubated at 21-25°C in growth chamber with 6 hrs. Light (philips 36w/84 day light tubes) and 8 hrs darkness for 7 days.

Evaluation I Examination: Necrotic lesions are produced on cotyledons. Black or brown discoloration is also produced on roots. Tissues from and around the lesion are taken and put in sterile water. A loop full of this suspension is streaked on to King’s B medium (King et al, 1954). Bacterial isolate may be confirmed by biochemical test i.e., soft rot of potato and Levan production. Bacterial isolate (approximate 108 cells/ ml) can also be inoculated in wound with needle prick in leaves of radish, cauliflower, tomato, sweat pepper and beans. Water soaked spots are produced around the needle prick.

5.3. Water Agar Seedling Symptoms Test

In this test, seedlings are grown on different agar media. Symptoms are observed both on seedlings, stem, leaf and root.

5.3.1. Technique for detection of Xanthomonas campestris from crucifer seed ( 23)

Procedure: In this test, seedlings are grown in agar medium. Working sample of 1000 seeds is soaked for 3-4 hrs. The solution is drained off and the seeds are planted on 1.5 per cent water agar. Twenty seeds are plated in each Petri dish of 90- mm diameter.

Incubation I Examination: The dishes are incubated at 20°C in darkness. The germinated seeds and seedlings are examined for black rot infection on the eighth and on the twelfth day by scanning under 6X on 12X magnification of a stereo-binocular microscope.

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Note: Prolonged incubation for more than 12 days is not advisable due to attack by saprophytic soft rot organisms and height of seedlings up to the lid of Petri dishes.

Observation: Two types of symptoms are generally observed:

1. Rotting of cotyledon and hypocotyls.

2. Appearance of V. shaped black rot lesions on young seedlings.

1. Severely attacked seedlings showed delayed germination and stunted growth, cotyledon and hypocotyls appeared yellowish and pulpy. Such seedlings collapse on the agar surface. When examined in water drop under the compound microscope. The diseased parts show bacterial ooze as a yellowish mass all over the surface. Such systemically attacked seedlings are readily distinguished in the agar plate from healthy seedlings on the eighth day of incubation.

2. Brown to black localized V. shaped and diffused lesions are seen on margins of infected cotyledons and on the first true leaf. The infected paris in a drop of water when examined under compound microscope show slow to fast oozing of bacteria from the vascular bundles. Xanathomonas is easily isolated from this kind of lesion.

Pathogenicity: Confirmation is made by spraying of purified bacterial isolate on three weeks old plants. First sign of infection appear after eight days in the form of water soaked lesion extending from the margin of the leaf in to the laminar tissue through the vein ending. Typical black rot symptoms develop on the infected leaves during the subsequent days of incubation.

5.4. Isolation Techniques Using Selective Media: Saprophytes usually grow faster than phytopathogens and create an unfavourable environment for the others in isolation media through competition for nutrients, production of an adverse p1-I or exertion of an antibiotic effect. These problems can be overcome through selective media.

5.4.1. Direct Plating of Crucifer Seeds On To SX Agar Procedure (21): Surface disinfected 100 crucifer seeds are plated directly on Petri dish containing SX Agar media. Plates are incubated at 30°C for 5 days. After three days small light yellow colonies surrounded by clear zone are observed.

5.5. Seed Washing Liquid Plating Test

The purpose of extraction is to get the bacteria, which are present in or on the seeds in order to isolate them on suitable media. Washing, grinding, maceration and soaking of seed depend on the location of bacteria in or on the seed surface. Anyhow soaking for long period and grinding of whole seed give better results.

Procedure: Seed washing are spread over the agar medium generally nutrient agar by dilution plate technique and pure culture is prepared on a suitable selective media. Pure cultures of the bacterium (108 cells/ml) can be inoculated in the leaf of the plant at seedling stage for observing symptoms of disease.

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5.5.1. Detection of Xanthomonas campestris in Crucifer Seeds by Liquid Plating Test (20)A working sample of 10,000 seeds is added to the flask containing 75ml saline water (0.85% NaCl), One drop Tween 20, 5 mg Benomy (benlate, 75 %a active) and O.5 ml of 1:1000 dilution of chloroethonil ( Bravo, 75 % active). The mixture is shacked on a rotary shaker for 1- 2 minutes and filtered through cheese cloth. Washing is re suspended in 2 ml saline and centrifuged at 400 rpm for 5 minutes. O.1 ml of centrifuged and suspended solution is spread on NSCA agar with L shaped glass rod. (Nutrient Starch Cycloheximide Agar media,15 gm potato starch and 250 mg Cycloheximide added to Nutrient Agar media constituents).The plates are incubated at 30 oC for 2- 3 days and examined for small light yellow colonies surrounded by clear zone of starch hydrolysis. Yellow mucoid colonies can be further purified on Yeast dextrose Agar media. Further confirmation of the pathogen can be done through pathogenicity tests.

5.5.2. Detection of Pseudomonas in Pea and Bean Seeds ( 7,32)One thousand seeds are soaked in distilled water for 24-48 hrs at 4 oC. Dilution series is prepared from seed suspension. From this suspension, 0.1 ml is spread on Kings B medium with the help of bent glass rod. Petri dishes are then incubated at 25-28 oC for 48 hrs. Green or blue fluorescent colonies are observed under UV light. These colonies can be purified on YDC (Yeast extract dextrose carbonate medium). Confirmation of the bacterium can be made by hypersensitivity and pathogenicity tests. Water soaked spots on the surface of hosts with in two weeks of inoculation.

5.6. Serological Techniques for Identification of Seed-Borne Bacteria

Serological techniques are protein based assays which use antigen antibody reactions. Such tests can be carried out on glass slides, in test tubes, Petri plates, Polystyrene plates etc. Isolates of suspected pathogen serve as antigen. When the antigen (foreign protein) is injected in the body of worm blooded animal (mostly rabbit, mice) antibodies are produced against it in the blood of animal. Antiserum containing antibodies is used for further purification of specific antibody.

Agglutination Test

When many bacterial cells and homologous antibodies are present in a suspension agglutinates will be formed. This principle is used in the slide agglutination test. The test is not suitable for direct application on seed extracts, because rather high concentrations of bacterial cells are necessary.

Preparation of materials i) Slide or small tubes with 2m1 PBS ii) PBS having pH7.2

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For preparing PBS (Phosphate buffer Saline) following solutions are made:

Solution i: Na2 I-1P04, H02 - 26.7gm. NaC1 - 8.5gm. Water - I000ml

Solution ii: KH2PO4 - 20.41gm. NaC1 - 8.5gm. Water - l000ml

The above two solutions are mixed in proportions 67 ml (solution 1)& 33 ml (solution 2)

Procedure: This test can be performed either on a slide or in a test tube. A bacterial suspension is prepared in Phosphate buffer saline (PBS) and drop of antiserum of origin is added. The mixture is allowed to react for a couple of hours at 35- 40°C. If agglutination appears in the drop, this means that result is positive.

5.7. Double Diffusion Test

In this test, the diluted antiserum and the bacterial suspension are placed in separate wells in a layer of water agar. The antibodies and the antigens will then diffuse out in to the agar, and when they meet, a precipitate band is formed which means positive result. This test is most frequently used to compare the reaction of several possible different antigens with the antiserum.

Materials

1) Water agar Plates2) PBS – l00 ml3) Distilled Water - 300 ml 4) Sodium azide - (NaN3) - 0.2% as preservative 5) Agar puncher device

Procedure

In a water agar plate, 3 mm diameter wells are made in circular fashion with one in centre. The outer wells are filled with antigen and the central well with antiserum. The plate is covered with the lid and incubated in moist chamber at 25-28°C. After 2-3 days Immuno precipitation lines will become visible due to diffusion of antigen and antibodies. The presence of Pseudomonas solanacearum and Agrobacterium tumefaciens in potato can easily be detected by this test.

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5.8. Test with Labeled Antibodies

The methods included in this group are characterized by the use of antibodies labeled with a specific compound. An enzyme usually alkaline phosphate is used for Enzyme linked Immunosorbent Assay (ELISA) and radioactive isotops are used for Radio Immuno Assay (RIA). The best-known Methods for plant bacteriological seed health testing are:

i) Enzyme linked immunosorbent Assay (ELISA). ii) Radio immuno Assay (RIA) iii) Immuno isolation Technique (1ST) iv) Immuno Fluorescence Microscopy (IFM)

5.8.1 Enzyme linked immunosorbent Assay (ELISA)(15)

ELISA is one of the most common serological assays used for the detection of viruses but currently this technique is also widely being used for detection of for bacteria and fungi also. This technique is advantageous in many ways, it is quick, specific and economical. With availability of mono and poly antibodies we can even detect specific races, biovars and strains of bacterial pathogens. Polystyrene plates having 96 wells are used for coating, we can even store these plates after coating with antibodies, moreover pre coated plates with specific antibodies are also available. We can use ELISA strips (coated with labeled antibodies) which give results in minutes, such results are qualitative. For quantitative assays diagnostic kits are also available which give results in 2-3 days.

There are various forms of ELISA i.e. DAS (Double Antibody Sandwich)DAC (Direct Antigen Coating)TAS (Tipple Antibody Coating).

Now a days ready to use Kits (antibodies, positive and negative controls, washing buffers, extraction buffers, substrate buffers, coating buffers) are available commercially ( Adgen Phytodiagnostics, Bioreba etc.) provided with protocol instructions. However detailed composition of Reagents and Buffers used in the technique is given.

DAS is the most common technique. In this technique we coat the plate with specific antibody followed by coating with antigen (bacterial isolate), Enzyme linked conjugate, Substrate (P.nitophenyl phosphate PVP is being used for color development). For appropriate reaction washing and incubation of 2-3 hrs (37 oC) and 12-14 hrs (4 oC) is required after each step. At the end of reactions a yellow color develops in specific wells of ELISA plate which is an indication of positive reaction. With the help of ELISA plate reader population of a specific bacterium can be quantified.

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6. The International Seed Health Initiative for Vegetables ISHI-Veg

In order to achieve uniformity in seed health testing ISHI have established “Guidelines for the Use of Seed Health Methods by the Vegetable Seed Industry”. Different protocols described for Veg seed health testing in the manual are being reviewed time to time. The described methods have a well-established track record in the seed industry and are recognized as reference methods by seed pathologists from testing laboratories of inspection services, private testing labs and the vegetable seed industry. In addition, some ISHI-Veg methods have been accepted as International seed health testing association rules (ISTA) and as standards by the US Department of Agriculture- Animal and Plant Health Investigation Services (USDA-APHIS) and National Seed Health System (NSHS). 

Table 2 illustrates some methods described by ISHI- Veg manual (10).

7. Disease management in seed crops

The basic aim for diagnosis and identification of different disease causal organisms is their management and control in order to save plants from the potential loss due to various diseases. Disease management in seed crops can be done through cultural methods, chemical applications, seed treatments, and disease resistant varieties (12,27).

7.1. Cultural practices

7.1.1. Crop rotations with non host or antagonistic crops. The duration of rotation depends upon host range and disease cycle of the pathogen. It is also important to eliminate alternate hosts, such as weeds or adjacent crops that may come in host range of controlled pathogen.

7.1.2. Removing Infected debris or remaining seed after harvest is also a source of inoculum for further disease spread. It must be removed from the field immediately after harvest and should be burnt or incorporated in to the soil. The fields can be fumigated or solarized thereafter.

7.1.3. Irrigation practices during crop production can also serve as a source of disease spread. Water splash, increased leaf wetness, increased relative humidity favors disease development in case of many seed borne pathogens. Timing and duration of irrigation is also important in this context.

7.1.4. Planting practices such as date of planting, plant spacing, row orientation and ventilation practices such as thinning and canopy management for maximum air circulation can also help to escape disease inoculums.

7.1.5. Filed transplanting hygiene practices are also important. One should avoid dipping transplants in water, mechanical injuries during transplanting.

7.1.6. Growing seed crops in environments unfavorable for disease can also be a strategy for disease management.

7.2. Seed treatmentsSeeds can be subjected to different treatments in order to seed treatments to minimize transmission of pathogens and manage seed borne diseases. Such methods include physical, chemical, biological methods.

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7.2.1.1. Physical seed treatments: these include hot water treatments (e. g cabbage seed at 50C for 30 minutes for controlling Phoma lingam and Xanthomonas campestris pv. Campestris), the seeds may be exposed to hot dry air, aerated steam, microwaves, cathode rays, etc.(27)

7.2.1.2. Chemical seed treatments:Different type of chemical treatments include surface sterilization (e.g with chlorine), different types of Fungicides e.g., vitavax, benlate,metalaxy, captan,fludioxonil,PCNB, ethazole,thiram (18).

7.2.1.3. Bio control agentsSome of the microbial taxa that have been successfully commercialized as biorational” pesticides. these are include bacteria belonging to the genera Agrobacterium, Bacillus, Pseudomonas, and Streptomyces and fungi belonging to the genera Ampelomyces, Candida, Coniothyrium, and Trichoderma, currently marketed as EPA-registered biopesticides in the United States e.g. Kodiak, HiStick N/T ,Serenade, Companion (Bacillus spp.), AQ10(Ampelomyces quisqualis),Contans(Coniothyrium minitans) for control of various seed borne diseases (29). Biocontrol ahgents have problems with consistency and only few are available commercially.Some natural plant products are also being used such as oils, plant extracts, composts etc.

7.3. Post harvest disease management: 7.3.1. Disease management is not only important when the crop is in the field but also

during harvesting, storage and conditioning. Any injury, damage during these processes can lead to devastating losses. It is also imperitive to remove pathogen structures from seed lots during processing (sclerotia, teliospores, etc.), controlled storage conditions i.e humidity, temperature are important to avoid development of storage molds i.e Aspergillus, Penicillium spp.etc.

REFFERENCES

1. Agriculture Statistics of Pakistan. 2006. Federal Bureau of Statistics, Ministry of economic affairs and statistics, Government of Pakistan. http://www.statpak.gov.pk.

2. Ahmad, S. I. and Bhutta, A. R. 1990. Potato Seed Health Testing and Application. SHT. Tech Bull. No. II. FSC &RD, Islamabad, Pakistan.

3. Botanical names, Common names and edible plant parts of some important east Texas. http://horticulture.sfasu.edu/hrt212/notes/BOTANICALNAMES.pdf. (Last viewed 25-12-2006).

4. Bhutta, A. R and I. Ahmad. 2000. Seed Pathological Techniques and their Application. National Book Foundation, Islamabad.

5. Elliote, J. A. and Craford, R. F. 1922. The spread of tomato wilt by infected seed. Phytopathology. 12:428-434.

6. Food and Agriculture Organization, United Nations. 2006. FAO Stat. http://faostat.fao.org/site/336. (Last viewed 05/23/06)

7. Grogan, R. G. and K. N. Kinble. 1967. The role of seed contamination in transmission of Pseudomonas phaseolus vulgaris. Phytopath. 57:28-31.

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8. Hashmi, M. M. 1986. Studies on seed-borne fungi of Capsicum annum and Coriandrum sativum. A monograph submitted to the Danish Govt. Institute of Seed Pathology for Developing Countries, Copenhagen, Denmark.

9. Hooker, W.J. 1981. Compendium of Potato Diseases. American Phyto pathological Society, Minnesota 55121, USA.

10. International Seed Federation.2007. The International Seed Health Initiative for Vegetables. The Manual of Seed Health Testing Methods. http://www.worldseed.org/ISHI-Veg_Manual.htm. ( Last update. 18-01-2007).

11. ISTA, 1976. International rules for seed testing. Proc. Int. Seed Test Assoc. Zurich, Switzerland.

12. Jiskani, M. M. 2006.Diseases of Vegetables. Department of Plant Pathology, Faculty of Crop Protection, Sindh Agriculture University Tandojam.Pakistan. http://www.nationalpak.com/diseasevege.asp.

13. Karlberg, S. 1974.Gronings. OchSundhet sun ndersokningar ienlets Jord enlight multipotmeto den, Medd Statens certrala frokontrollans talt No. 49: 45-51.

14. Kendrick, J. B. 1944. Fruit invasion and seed carriage of tomato Fusarium wilt. Phytopathology. 34:1005-1006.

15. Maclaughlin, R.J., T.A. Chen and J.M. Well. 1989. Monoclonal antibodies against Erwinia amylovora, characterization and evaluation of a mixture for detection by ELISA. Phytopathology. 79:610-613.

16. Mangan, A. 1978. The use of plain agar for detection of Phoma betae on beet seed. Seed Sci. & technol. 11:607-614.

17. Muskett, A. E. and Malone, J. P. 1941. The ulster method for the examination of flax seed for the presence of seed-borne parasites. Ann-Appl.Biol. 28:8-13.

18. Pedersen, P. 2006. Chemicals for seed treatment. Iowa State University Extension.USA.

19. Richardson, M. J. 1990.An Annotated List of Seed-borne Diseases, 4th Ed. The international Seed Testing Association. Switzerland.

20. Schaad, N. W. 1982. Detection of seed borne bacterial pathogens. Plant disease. 66(10):885-890.

21. Schaad, N. W. and Kendrick, R. 1975. A qualitative method for detecting Xanthomonas campestris in crucifer seed. Phytopathology. 65:1034-1034.

22. Singh, D.V, Mathur, S. B. and Neergaard, P. 1974. Seed health testing of maiz. Evaluation of testing technique with special reference to Drechslera maydis, Seed Sci & Technol. 2: 340-365.

23. Srinivasan, M.C., Neergaard, P. and Mathur, S. B. 1973. A technique for detection of Xanthomonas compestris in routine seed health testing of crucifers. Seed Sci. and Technol.1:853-859.

24. Shakya, D.D.and Vinther, F. 1989. Occurrence of Pseuodomonas viridiflava in seedlings of raddish . Phytopathology. 124:123-127.

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25. Shekhwat, P.S.and Chakarwati, B.P. 1979. Comparison of agar plate method and cotyledon methods for detection of Xanthomonas vesicatoria in Chilli seed. Phytopathology. 94:80-84.

26. Taxas A & M University. 2006.Taxas plant disease hand book, Vegetable diseases. Plant Pathology and Microbiology. plantpathology.tamu.edu/Texlab/Vegetables/veg1.html - 28k

27. Toit, L. D.2006.Diseases in vegetable seed crops, identification, Biology and Management. Mount Vernon Research & Extension Unit.Washington State University.USA.

28. United Stated Department of Agriculture. 2006. Vegetables and Melons Outlook /VGS-318. 2006. Economic Research Service. www.ers.usda.gov/publications/vgs/2006/12dec/vgs318.pdf.

29. United States Environmental Protection Agency. 2006.Terms: Biopesticides. Online. (Office of Pesticide Programs). (http://www.epa.gov/pesticides/biopesticides/).

30. Volcani, Z. 1966. A Quantitative method for assessing cucumber seed infection caused by Psendomonas lacrymans. Israel J. Botany. 15:192-97.

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32. Watson, D.R.W. and Dye, D. W. 1971. Detection of bacterial diseases in garden pea stocks. Plant Disease Reports. 55(6):519-521.

33. Whiteaker, G. 2006.Vegetable Seed Quality: A Local and International Perspective. Sakata Seed America, Inc.Morgan Hill, CA. WWW.Sakata.com.

Table 2 Methods of vegetable seed health testing

Sr. No

Crop Pathogen Method

1 Bean (Phaseolus spp.) Pseudomonas syringae pv. phaseolicola

Plate TestKBBC, MSB, BBD Media

Pseudomonas syringae pv. syringae

Agar Plate AssayModified Agar Plate AssayKBC, MSP Media

Xanthomonas axonopodis pv. phaseoli

Selective Media Assay (BBD)PCR

Colletotrichum lindemuthianum

Be5.1-ISTA Method

2 Pea (Pisum sativum) Ascochyta pisi, A. pinodella, A. pinodes

Be6.1-ISTA Method

3 Brassicas (Brassica) Xanthomonas campestris pv.campestris

Br1.1 ISHI-Vegetables Method

Phoma lingam Freezing Blotter Method (STA Laboratories)

4 Carrot (Daucus carotae) Xanthomonas campestris pv. carotae

Seed wash in Saline/WaterXCS, MD5 Media

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Alternaria dauci Direct plating/Blotter

Alternaria radicina Freezing Blotter

5 Celery (Apium graveolens) Septoria apiicola Seed Wash in water, Centrifugation

Phoma apiicola Kleb. Grow-out, Seedling evaluation

6 Watermelon (Citrullus lanatus var. lanatus).Cantaloupe (Cucumis melo var. cantalupensis)Melon (Cucumis melo)

Acidovorax avenae ssp. citrulli Seedling Grow-outSeminis Inc. PCR-Wash method

7 Cucurbits (Cucurbitacea) Didymella bryoniae ISHI-Vegetables Method

8 Spinach (Spinacia oleracea) Peronospora farinosa f.sp. spinaciae

Wash Test

Verticillium dahliae Agar Plate Test (CDFA)Modified Agar Plate Test (ISU)

9 Coriander/Cilantro (Coriandrum sativum)

Pseudomonas syringae pv. coriandricola

Selective Media Test

10 Pepper (Capsicum annuum), Tomato(Lycopersicon esculentum)

Xanthomonas campestris pv.vesicatoria

ISHI Seed wash

11 Tomato (Lycopersicum esculentum)

Clavibacter michiginensis pv. michiginensis

ISHI-Veg (2001) Method

Pseudomonas syringae pv. tomato

CDFA Method(Slective media)

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