Field crops

FIELD CROPS PATHOGEN: Clavibacter michiganensis subsp. nebraskensis HOSTS: Maize (Zea mays) METHOD Cb1.1 sCNS Culture Plate Method (Shepherd, 1999) METHOD Cb1.1 sCNS Culture Plate Method (Shepherd, 1999) METHOD CLASS: STANDARD PATHOGEN: Clavibacter michiganensis subsp. nebraskensis SAMPLE: 400 seeds PROCEDURE: 1. Divide a representative sample of 400 corn seeds into 4 sub-lots of 100. 2. Surface sterilize seeds with 0.5% sodium hypochlorite (NaOCl) for 3 minutes. Rinse thoroughly with sterile water. 3. Grind each sub-sample until finely pulverized and add to 100ml phosphate buffer saline solution (seeds may be ground dry or soaked overnight at 6ºC before grinding). 4. Shake sub-samples at room temperature for 1 hour. 5. Prepare a dilution series comprising the original suspension and two 10-fold dilutions. 6. Plate each of the three dilutions onto sCNS in triplicate. 7. Incubate at 28°C for 5 to 7 days. 8. Any orange colonies that appear in the first 1 to 3 days should be disregarded. 9. Round and peach colored colonies (Figure 1) that appear after 3 days and by 9 days are putative colonies of C. michiganensis subsp. nebraskensis (Cmn). 10. Cmn can be confirmation by plant injections of culture suspensions into seedlings of Goss’ wilt susceptible corn varieties (such as A632). Seedlings are then examined for typical Goss’ wilt symptoms on leaves. 11. Alternatively, Cmn can be confirmed by CORYNE strip tests, performed according to manufacturer’s (bioMeriux-Vitek Inc., Hazelwood, MO) directions on colonies plated on TSA agar. The positive code for Cmn is 6550004 or 6150004. ELISA kits (Agdia, CMM kit) can also be used to eliminate negatives, but are known to give false positives, so any positives should undergo further testing. MEDIA PREPARATION: sCNS: Distilled water 1 liter Nutrient broth 8.0g Yeast extract 2.0g K2HPO4 2.0g KH2PO4 0.5g Dextrose 5.0g MgSO4* 7H2O 0.25 Agar 15g Bravo 720 (Chlorothalonil) 0.2ml diluted 1:50 in water Mertect 340F (Thiabendazole) 50µl (1 drop) Potassium dichromate 0.02g After autoclaving filter sterilize the following adding to media just prior to pouring: Naladixic acid 40mg Cycloheximide 100mg REFERENCES: Shepherd, LM. 1999. Detection and transmission of Clavibacter michiganensis subsp. nebraskensis of corn. Ms Thesis, Iowa State University, Ames, IA. PATHOGEN: Cochliobolus carbonum HOSTS: Maize (Zea mays) METHOD: Cf2.1 ISU Freezing Blotter Method (McGee 1994) METHOD: Cf2.1 ISU Freezing Blotter Method (McGee 1994) METHOD CLASS: STANDARD PATHOGEN: Cochliobolus carbonum SAMPLE: 400 seeds PROCEDURE: 1. Randomly select 400 seeds. 2. Wash thoroughly in running water to remove chemical seed treatment. 3. Immerse seeds in 0.5 % (v/v) sodium hypochlorite (NaOCl) for 3 minutes; then immerse in distilled water containing 200 mg/liter of benomyl (Benlate 50w) and 100mg/liter of streptomycin sulfate (735 units/gram) for 30 minutes. 4. Place two autoclaved (sterile) blotters in each polystyrene box (25cm x 15cm x 4cm deep). 5. Moisten the blotters with 70 ml of sterile distilled water containing 0.035 grams of Botran fungicide 75W (dicloran) 6. Aseptically place seeds on blotters. 7. Incubate samples at 25 C for 2 days. 8. Transfer to -20 C for at least 15 hours. 9. Transfer to 25 C for 7 additional days with 8 hours of light per day. 10. Examine seeds for the presence of Cochliobolus species. 11. Suspect black to brown mycelium with fruiting bodies should be examined with a light microscope to confirm species, as described by Ellis & Holliday (1972). REFERENCES: Ellis, M. B. & Holliday, P. (1972) Cochliobolus carbonum. CMI Descriptions of Pathogenci Fungi and B acteria. No. 349. McGee, D. C. 1994. Seed assays for Stewart's wilt and other seed-borne diseases of corn. Pages 161 - 168 in: Proc. Annu. Corn Sorghum Res. Conf. 48. American Seed Trade Association, Washington D. C. PATHOGEN: Cochliobolus heterostrophus HOSTS: Maize (Zea mays) METHOD: Cf3.1 ISU Freezing Blotter Method (McGee 1994) METHOD: Cf3.1 ISU Freezing Blotter Method (McGee 1994) METHOD CLASS: STANDARD PATHOGEN: Cochliobolus heterostrophus SAMPLE: 400 seeds PROCEDURE: 12. Randomly select 400 seeds. 13. Wash thoroughly in running water to remove chemical seed treatment. 14. Immerse seeds in 0.5 % (v/v) sodium hypochlorite (NaOCl) for 3 minutes; then immerse in distilled water containing 200 mg/liter of benomyl (Benlate 50w) and 100mg/liter of streptomycin sulfate (735 units/gram) for 30 minutes. (optional) 15. Place two autoclaved (sterile) blotters in each polystyrene box (25cm x 15cm x 4cm deep). 16. Moisten the blotters with 70 ml of sterile distilled water containing 0.035 grams of Botran fungicide 75W (dicloran) 17. Aseptically place seeds on blotters. 18. Incubate samples at 25 C for 2 days. 19. Transfer to -20 C for at least 15 hours. 20. Transfer to 25 C for 7 additional days with 8 hours of light per day. 21. Examine seeds for the presence of Cochliobolus species. 22. Suspect black to brown mycelium with fruiting bodies should be examined with a light microscope to confirm species, as described by Ellis & Holliday (1972). REFERENCES: Ellis, M. B. & Holliday, P. (1972) Cochliobolus carbonum. CMI Descriptions of Pathogenic Fungi and Bacteria. No. 349. McGee, D. C. 1994. Seed assays for Stewart's wilt and other seed-borne diseases of corn. Pages 161 - 168 in: Proc. Annu. Corn Sorghum Res. Conf. 48. American Seed Trade Association, Washington D. C. PATHOGEN: Pantoea stewartii, Erwinia stewartii. HOSTS: Maize (Zea mays) METHOD Cb2.1 ELISA (Enzyme-linked immunosorbent assay) (Lamka et al, 1991) METHOD Cb2.1 ELISA (Enzyme-linked immunosorbent assay) (Lamka et al, 1991) METHOD CLASS: STANDARD PATHOGEN: Erwinia stewartii, Pantoea stewartii SAMPLE: 400 seeds PROCEDURE: Diagnostic kit: This method uses an ELISA kit developed by Agdia, Elkhart, Indiana. The monoclonal antiserum used in the kit was developed at Iowa State University as described (Lamka et al, 1991). Extraction of the bacterium: 1. Grind 4 replicates of 100 corn kernels, per sample and add 100ml of General 2. Or place 4 replicates of 100 corn kernels, per sample, into 100ml of General Extraction Buffer and soak overnight at room temperature, then grind. 3. Disinfect the grinder between samples by rinsing with distilled water, followed by a rinse with a laboratory or hospital standard detergent, then a final rinse with distilled water. Running the ELISA kit: Run kit according to manufactures instructions. Please cont) if you need a copy of instructions or assistance with the protocol. REFERENCES: Lamka GL, Hill JH, McGee DC and Braun EJ. 1991. Development of an immunosorbent assay for seed-borne Erwinia stewartii. Phytopathology 81:839-846. PATHOGEN: Fusarium moniliforme HOSTS: Maize (Zea mays) METHOD Cf8.1 Culture Plate (McGee 1994) Cf8.2 Blotter (Singh et al., 1974) METHOD: Cf8.1 Culture Plate (McGee 1994) METHOD CLASS: TEMPORARY STANDARD PATHOGEN: Fusarium moniliforme SAMPLE: 400 seeds PROCEDURE: 1. Randomly select 400 seeds; 4 replicates of 100. 2. Wash thoroughly in running water to remove chemical seed treatment. 3. Immerse seeds in 1.0% (v/v) solution of sodium hypochlorite (NaOCl) for 1 minute; then triple rinse with sterile water. 4. Aseptically place seeds on acid PDA (potato dextrose agar), usually 5 to 10 seeds per petri plate (9 cm diameter plates). 5. Incubate samples at 25 C with 8 hours of light per day for 7 to 21 days. Check periodically to determine stage of colony growth. 6. Suspect mycelium should be examined for the presence of micro- or macrocondia of Fusarium moniliforme. REFERENCES: McGee, D. C. 1994. Seed assays for Stewart's wilt and other seed-borne diseases of corn. Pages 161 - 168 in: Proc. Annu. Corn Sorghum Res. Conf. 48. American Seed Trade Association, Washington D. C METHOD: Cf8.2 Blotter (Singh et al., 1974) METHOD CLASS: TEMPORARY STANDARD PATHOGEN: Fusarium moniliforme SAMPLE: 400 seeds PROCEDURE: 1. Four replicates of 100 seeds are surface disinfected in 2.0% sodium hypochlorite. 2. Incubation on a damp blotter at 20°C for 24 h, followed. 3. Freezing at -20°C for 24 h. 4. Incubation for 7 days at 26°C under cycles of 12 h NUV light/12 h darkness. REFERENCES: Singh, D. V., Mathur, S. B., and Neergaard, P. 1974. Seed health testing of maize. Evaluation of testing techniques with particular reference to Drechslera maydis. Seed Sci. Technol. 2:349-365. PATHOGEN: Gibberella zeae HOSTS: Maize (Zea mays) METHOD Cf7.1 Culture Plate (McGee 1994) Cf7.2 Blotter (Singh et al., 1974) METHOD: Cf7.1 Culture Plate (McGee 1994) METHOD CLASS: TEMPORARY STANDARD PATHOGEN: Gibberella zeae. SAMPLE: 400 seeds PROCEDURE: 1. Randomly select 400 seeds; 4 replicates of 100. 2. Wash thoroughly in running water to remove chemical seed treatment. 3. Immerse seeds in 1.0% (v/v) solution of sodium hypochlorite for 1 minute; then triple rinse with sterile water. 4. Aseptically place seeds on acid PDA (potato dextrose agar), usually 5 to 10 seeds per petri plate (9 cm diameter plates). 5. Incubate samples at 25 C with 8 hours of light per day for 7 to 21 days. Check periodically to determine stage of colony growth. 6. Suspect mycelium should be examined for the presence of perithecia or macroconidia of Gibberella zeae. REFERENCES: McGee, D. C. 1994. Seed assays for Stewart's wilt and other seed-borne diseases of corn. Pages 161 - 168 in: Proc. Annu. Corn Sorghum Res. Conf. 48. American Seed Trade Association, Washington D. C METHOD: Cf7.2 Blotter (Singh et al., 1974) METHOD CLASS: TEMPORARY STANDARD PATHOGEN: Gibberella zeae SAMPLE: 400 seeds PROCEDURE: 5. Four replicates of 100 seeds are surface disinfected in 2.0% sodium hypochlorite. 6. Incubation on a damp blotter at 20°C for 24 h, followed. 7. Freezing at -20°C for 24 h. 8. Incubation for 7 days at 26°C under cycles of 12 h NUV light/12 h darkness. REFERENCES: Singh, D. V., Mathur, S. B., and Neergaard, P. 1974. Seed health testing of maize. Evaluation of testing techniques with particular reference to Drechslera maydis. Seed Sci. Technol. 2:349-365. PATHOGEN: Penicillium oxalicum HOSTS: Maize (Zea mays) METHOD Cf9.1 Blotter test (Handoo and Aulakh, 1979) METHOD: Cf9.1 Blotter test (Handoo and Aulakh, 1979) METHOD CLASS: STANDARD PATHOGEN: Penicillium oxalicum SAMPLE: 400 seeds, 4 replicates of 100 seeds PROCEDURE: 1. Surface sterilized in 2% NaOCl for 10 min. 2. Incubate on moist blotter at 20°C for 2 hr. 3. Transfer to a freezer at -20°C for 24 hr 4. Incubation at 20°C for 7 days under cycles of 12 h UV light and 12 h darkness. REFERENCES: Handoo ML, Aulakh KS, 1979. Seed-borne fungi of maize in India. Seed Research, 7(1):41-47. PATHOGEN: Peronosclerospora sorghi HOSTS: Maize (Zea mays) METHOD Cf6.1 Grow –out (Adenle & Cardwell, 2000) METHOD: Cf6.1 Grow –out (Adenle & Cardwell, 2000) METHOD CLASS: STANDARD PATHOGEN: Peronosclerospora sorghi SAMPLE: 400 seeds, in 4 replicates of 100 seeds. PROCEDURE: 1. Four replicates of 4 pots of 25 maize seeds in each replicate are planted in plastic pots in a greenhouse. 2. Downy mildew –infected seedlings should be detectable 7 days after seedling emergence REFERENCES: Adenle V.O. & Cardwell, K. E. 2000 Seed transmission of maize downy mildew (Peronosclerospora sorghi) in Nigeria. Plant pathology 49:628-634. PATHOGEN: Sclerophthora macrospora HOSTS: Maize (Zea mays) METHOD Cf5.1 Direct Visual Method (CIMMYT) METHOD: Cf5.1 Direct Visual Method (CIMMYT) METHOD CLASS: STANDARD PATHOGEN: Sclerophthora macrospora SAMPLE: 400 seeds, 8 replicates of 50 seeds PROCEDURE: 1. Place 50 seeds / replicate in a 250 ml beaker containing a combined solution of 5% NaOH and 0.02% Aniline Blue. 2. Soak seeds for approximately 36 hours to obtain adequate softening for internal tissue examination. 3. Pour resulting material into a 15 x 23 cm plastic box. 4. Squash embryos with a spatula and spread into a thin layer. 5. Examine under a stereomicroscope (at 50X magnification) for characteristic oospores that are hyaline to yellowish and 45-75 µm diameter. REFERENCES: E.J. Warham,, L.D. Butler, and B.C. Sutton 1995. Seed Testing of Maize and Wheat: a laboratory guide. CIMMYT. 1995. CIMMYT and International Mycological Institute. ISBN: 968-6923-70-5 PATHOGEN: Stenocarpella maydis HOSTS: Maize (Zea mays) METHOD Cf1.1 Culture plate (Iowa State University) METHOD: Cf1.1 Culture plate (Iowa State University) METHOD CLASS: STANDARD PATHOGEN: Stenocarpella maydis (Diplodia zea-maydis) SAMPLE: 400 seeds PROCEDURE: 1. Test a total of 4 replications of 100 maize seeds. 2. For each replication, select at random 100 seeds (follow an internationally accepted procedure for mixing and preparing samples). 3. If seeds are chemically treated, wash thoroughly in running water for 10-20 minutes to remove seed treatment. 4. Surface sterilize the seed in 1.0 % sodium hypochlorite (bleach) for 1 min, rinse three times with 400 ml sterile water per rinse. 5. Prepare Potato Dextrose Agar (acidified to pH 4.5 with 85% Lactic Acid. Pour agar in 90x15mm or 100x15 ml aseptic disposable plastic petri plates. Cool and let stand for at least 1 day before use. 6. Place 5 or 10 seeds on each agar filled 90x15mm or 100x15 ml aseptic disposable plastic petri plate . 7. Incubate plates at 25 C with a photoperiod of 8hrs under white fluorescent light. Incubate for a total of 5 days. 8. Examine seeds at 3 days looking for characteristic hyphae and/or pycnidia. Stereomicroscopic examination should be used to confirm the identification. Record results (for most quarantine tests the presence of characteristic hyphae and/or pycnidia would result in a yes or + being recorded for that sample). 9. Re-examine seeds at 5 days again looking for characteristic hyphae and/or pycnidia. REFERENCES: Iowa State University, Seed Testing Laboratory, Ames, IA PATHOGEN: Ustilago maydis and Sphacelotheca reiliana HOSTS: Maize (Zea mays) METHOD: Cf4.1 Washing test (McGee, 1988) METHOD: Cf4.1 Washing test (McGee, 1988) METHOD CLASS: STANDARD PATHOGEN: Ustilago maydis and Sphacelotheca reiliana SAMPLE: 400 PROCEDURE: 1. Wash 400 seeds in sterile water for 15 minutes in a shaker. 2. Centrifuge at 3000 rpm for 15 minutes. 3. Remove the pellet and examine for with a light microscope to confirm the identity teliospores, as described by Ainsworth (1965a) for U. maydis (see Document 4) and Ainsworth (1965b) description for S. reiliana (see Document 5) REFERENCES: Ainsworth, G. C. (1965a) Ustilago maydis. CMI Descriptions of Pathogenic Fungi and Bacteria, No. 79. Wallingford, UK: CAB International. (GET IMAGES) Ainsworth, G. C. (1965) Sphacelotheca reiliana. CMI Descriptions of Pathogenic Fungi and Bacteria, No. 73. International Mycological Institute: Kew, UK. (1965b) (GET IMAGES) McGee, D.C., 1988. Maize diseases. A reference source for seed technologists. St. Paul, Minnesota, USA; APS Press, 149 pp. PATHOGEN: Cercospora kikuchii HOSTS: Soybean (Glycine max) METHOD Sf1.1 Culture Plate (Jordan et al., 1986) METHOD Sf1.2 Culture Plate (McGee and Nyvall, 1984) METHOD Sf1.1 Culture Plate (Jordan et al., 1986) METHOD CLASS: STANDARD PATHOGEN: Cercospora kikuchii SAMPLE: 400 seeds PROCEDURE: 1. Surface sterilize seeds in 0.5% sodium hypochlorite (NaOCl) for 4 minutes; wash in distilled water. 2. Plate on potato dextrose agar. 3. Incubate at 25°C in the dark for 10 days. REFERENCES: Jordan EG, Manandhar JB, Thapliyal PN, Sinclair JB, 1986. Factors affecting soybean seed quality in Illinois. Plant Disease. 70(3):246-248. METHOD Sf1.2 Culture Plate (McGee and Nyvall, 1984) METHOD CLASS: STANDARD PATHOGEN: Cercospora kikuchii SAMPLE: 400 seeds PROCEDURE: 1. Seeds are surface sterilized in 1% sodium hypochlorite (NaOCl) for 30 seconds and then rinsed in sterile water. 2. Incubate on a moistened blotter at 25°C for 10 days under continuous light. 3. Seeds are evaluated for the presence of Cercospora kikuchii as indicated by purple staining on the seed coat under fungal growth. REFERENCES: McGee DC, Brandt CL, Burris JS, 1980. Seed mycoflora of soybeans relative to fungal interactions, seedling emergence, and carry over of pathogens to subsequent crops. Phytopathology. 70(7):615-617. McGee DC, Nyvall RF, 1984. Soybean seed health. Coop. Ext. Serv. Iowa State University, Pm-990. PATHOGEN: Curtobacterium flaccumfaciens pv. flaccumfaciens HOSTS: Soybean (Glycine max) METHOD Sb 1.1 Grow-out method (Dunleavy, 1986) METHOD Sb 1.1 Grow-out method (Dunleavy, 1986) METHOD CLASS: TEMPORARY STANDARD PATHOGEN: Curtobacterium flaccumfaciens pv. flaccumfaciens SAMPLE: 1000 seeds PROCEDURE: 1. Surface-sterilized soybean seeds are planted in sterile soil. 2. They are grown at 30°C under growth-chamber conditions until characteristic large tan-colored lesions leaf symptoms are visible. REFERENCES: Dunleavy JM, 1986. Effect of temperature on systemic spread of tan spot of soybean from seed to unifoliate leaves. Phytopathology 76:1079. PATHOGEN: Phomopsis/Diaporthe complex HOSTS: Soybean (Glycine max) METHOD Sf2.1 Culture plate (McGee, 1986) METHOD Sf2.2 Blotter (McGee and Nyvall, 1984) METHOD Sf2.1 Culture plate (McGee, 1986) METHOD CLASS: STANDARD PATHOGEN: Phomopsis/Diaporthe complex SAMPLE: 400 seeds PROCEDURE: 1. Seed are surface sterilized in 0.5% NaOCl for 1 minute and then rinsed in sterile water. 2. Plate seeds on potato dextrose agar (PDA) adjusted to pH 4.5. 3. Seeds incubation at 25°C for 7 days under continuous light. 4. Seeds are evaluated for presence of the three fungi associated with the Phomopsis/Diaporthe complex in soybeans according to the following descriptions. cultures on PDA are floccose and rope-like, turning tan to brown with age. In reverse, the colonies are tan to dark brown with black, pulvinate stromata. Pycnidia are black, stromatic, solitary or aggregated, and usually unilocular, Note: There is considerable controversy regarding the taxonomy of this group. These descriptions should, therefore only be used to identify the Diaporthe/Phomopsis complex and not the individual pathogens. Phomopsis longicolla, previously known as Phomopsis sp. are floccose, dense and white, with occasional greenish yellow areas on PDA. In reverse, colonies are colorless, with black, spreading stromata. Pycnidia are black, stromatic, solitary or aggregated, unilocular or multilocular, with prominent necks more than 200µm long. Alpha conidia are hyaline, ellipsoid to fusiform, guttulate, and measure 5-9 x 1.5-3.5µm. Beta conidia, which are rarely formed, are hyaline, filiform, and hamate. No perithecia are formed on PDA. Diaporthe phaseolorum var. caulivora cultures on PDA are white, closely appressed, with tufted aerial mycelium. In reverse, colonies are colorless, with black circular stromata, less than 2mm in diameter. Perithecia are black and globose, measuring 165-340 x 282-412µm, and have a long protruding beak. Asci measure 30-40 x 4-7µm. Ascospores are hyaline, elongate-ellipsoidal, two-celled, slightly constricted at the septum, biguttulate in each cell, and measure 8-12 x 3-4µm. Pycnidia rarely occur on PDA. Diaporthe phaseolorum var. meridionales cultures on PDA are white even colonies with brown chlamydospores. Stromata are irregular in shape (2-10mm long). Perithecia are similar in to those of D. phaseolorum var. caulivora, but they have wider neck width, i.e. 100 compared to 55µm. Diaporthe phaseolorum var. sojae with no beak, or a beak less than 200µm long. Alpha conidia are hyaline, usually fusiform, guttulate, and measure 5.5-10.5 x 1.3-3.5µm. Beta conidia, which are more commonly produced, are hyaline, filiform, and hamate. Perithecia are nearly spherical and measure 148-282 x 185-346µm. They have long, tapered beaks, measuring 60-100 x 60-150µm, and usually are solitary, not clustered. Asci are elongate and clavate, measuring 35-51 x 3.3-10µm. Ascospores are similar in shape to alpha conidia, but are larger, 9-13 x 2-6µm, and bicellular. They are biguttulate in both cells. REFERENCES: McGee DC, 1986. Prediction of Phomopsis seed decay by measuring soybean pod infection. Plant Disease 70:329-333. METHOD Sf2.2 Blotter (McGee and Nyvall, 1984) METHOD CLASS: STANDARD PATHOGEN: Phomopsis/Diaporthe complex SAMPLE: 400 seeds PROCEDURE: 1. Two sterilized blotters are placed in plastic boxes. 2. Moisten blotters with 80ml of sterile water, containing 40mg of 2,6 dichloro-6-nitroaniline (Botran 75W). 3. Four sets of 100 seeds are surface sterilized in 1% NaOCl for 30 seconds and then rinsed in sterile water. 4. Seeds are incubated at 25°C for 10 days under continuous light. 5. Seeds are evaluated for the presence of fungi in the Phomopsis/Diaporthe complex characteristic dense, white fungal growth. REFERENCES: McGee DC, Brandt CL, Burris JS, 1980. Seed mycoflora of soybeans relative to fungal interactions, seedling emergence, and carry over of pathogens to subsequent crops. Phytopathology. 70(7):615-617. McGee DC, Nyvall RF, 1984. Soybean seed health. Coop. Ext. Serv. Iowa State Univ, Pm-990. PATHOGEN: Pseudomonas syringae pv. glycinea HOSTS: Soybean (Glycine max) METHOD Sb 2.1 Soaked bulk seed – Biochemical confirmation (Chauveau, 1988) METHOD Sb 2.2 Ground bulk seed – Serological and pathogenicity confirmation (Alvarez et al, 1995) METHOD Sb 2.1 Soaked bulk seed – Biochemical confirmation (Chauveau, 1988) METHOD CLASS: STANDARD PATHOGEN: Pseudomonas syringae pv. glycinea SAMPLE: 5000 seeds PROCEDURE: 1. Five subsamples of 1000 soybean seeds are soaked for 24 hr at 4-5 C in 600 ml of sterile tap water adjusted to pH 6.5 with a phosphate buffer solution. 2. Threefold serial dilutions are made from the soaking solution and 0.1-ml aliquots plated on King's B medium amended cephalexin (KBC). 3. After incubation at 25 C for 2-3 days, presumptive colonies of P. s. glycinea, exhibiting a blue fluorescence under UV light (370 nm), are re-isolated onto KBC. 4. Five presumptive colonies of each subsample are subculture onto King’s B medium. 5. These subcultures are then confirmed as P. s. glycinea by a positive reaction for levan production and negative reactions in oxidase and esculin hydrolysis tests. Media King’s B Medium DI water 1 liter Proteose peptone #3 20g K2HPO4 2.5g Glycerol 15ml MgSO4 * 7H2O 6g Agar 20g *4ml cephalexin from the stock solution per liter applied after autoclaving. (Stock = 1g per 100ml water) Esculin Hydrolysis Agar DI Water 1 liter NH4H2PO4 0.5g K2HPO4 0.5g MgSO4 * 7H2O 0.2g NaCl 5g Yeast extract 5g Ferric ammonium citrate 0.5g Esculin 1g Agar 12g *pH should be about 6.8. Levan Agar DI Water 1 liter Sucrose 50g Nutrient agar 23g REFERENCES: Chauveau, J. F. 1988, personal communication METHOD Sb 2.2 Ground bulk seed – Serological and pathogenicity confirmation (Alvarez et al, 1995) METHOD CLASS: STANDARD PATHOGEN: Pseudomonas syringae pv. glycinea SAMPLE: 5000 seeds PROCEDURE: 1. Five subsamples of 1000 dry soybean seeds were ground in a Stein Mill for 1 min , then added to 600 ml of sterile saline (0.85% NaCl) and the suspension placed on a rotary shaker for 2 hr at 25 C at 220 rpm. 2. Threefold serial dilutions are made from the suspension and 0.1-ml aliquots plated on King's B medium amended with cephalexin. 3. After incubation at 25 C for 2-3 days, presumptive colonies of P. s. glycinea, exhibiting a blue fluorescence under UV light (370 nm), are re-isolated onto KBC. 4. Presumptive colonies of each subsample were confirmed as P. s. glycinea by the following pathogenicity and slide agglutination tests. Pathogenicity 1. Pathogenicity was determined by inoculating 15-day-old, greenhouse-grown soybean seedlings (cvs. Oakland, Beeson, Acme, and Flambeau) by rubbing leaves with a sterile cotton swab dipped in an aqueous suspension of the presumptive colony (approximately 105 cfu/ml). 2. The seedlings were incubated in light for 48 hr at 90% relative humidity in a mist chamber at 25 C, then transferred to the greenhouse and observed for necrotic lesions on leaves 4-7 days after inoculation. Agglutination 1. Ten microloters of bacterial suspension of each colony (105 cfu/ml) was mixed in polystyrene Micro ELISA plates (Dynatech Corp.) with 10 µl of a 1:1,000 aqueous dilution of the antiserum obtained from A. Calzolari (Osservatorio Regionale per le Malattie delle Plante, Bologna, Italy). 2. The plates were agitated for 1 hr at 25 C on a rotary shaker at 220 rpm, and agglutination was determined under a stereoscopic microscope. Media King’s B Medium DI water 1 liter Proteose peptone #3 20g K2HPO4 2.5g Glycerol 15ml MgSO4 * 7H2O 6g Agar 20g *4ml cephalexin from the stock solution per liter applied after autoclaving. (Stock = 1g per 100ml water) REFERENCES: Alvarez, E., Braun. E. J., and McGee, D.C. 1995. New assays for detection of Pseudomonas syringae pv. glycinea in soybean seed. Plant Dis. 79:12-14. PATHOGEN: Sclerotinia sclerotiorum HOSTS: Soybeans (Glycine max) METHOD Sf3.1 Culture plate (Totir, 2000) METHOD Sf3.1 Culture plate (Totir, 2000) METHOD CLASS: STANDARD PATHOGEN: Sclerotinia sclerotiorum SAMPLE: 400 seeds PROCEDURE: 1. Four sub-samples of 100 seeds are surface sterilized in 1.75% NaOCl for 30 seconds. 2. Seeds are rinsed three times in sterile water. 3. Incubate seeds on potato dextrose agar for 10 days at 25ºC (5 seeds/plate) 4. Seeds with characteristic white mycelium of Sclerotinia sclerotiorum are marked after 3, 5, and 7 days to account for overgrowth of colonies compromising the final count. 5. A final count is made of seeds with characteristic white mycelium and/or large black sclerotia, at 10 days. REFERENCES: Totir, C. 2000. Seed transmission and control of Sclerotinia sclerotiorum in soybean seeds. Ms. Thesis, Iowa State University, Ames, IA. PATHOGEN: Xanthomonas axonopodis pv. glycines HOSTS: Soybean (Glycine max) METHOD Sb 3.1 MXG selective media (Prathuangwong, et al., 1997) METHOD Sb 3.1 MXG selective media (Prathuangwong, et al., 1997) METHOD CLASS: TEMPORARY STANDARD PATHOGEN: Xanthomonas axonopodis pv. glycines SAMPLE: 5000 seeds PROCEDURE: 1. Seeds are surfaced sterilized with 95% ethyl alcohol for 30 sec 2. Seed are plated on MXG selective medium amended with 1 ml of a 50 mg/ml stock solution of polymixin B sulfate/ liter. 3. Plates are incubated at room temperature for 2 weeks. 4. Presumptive colonies of X. campestris pv glycines are distinguished by morphological characteristics including color and size. Biochemical tests 1. Subcultures on plates of nutrient glucose agar were identified by a range of biochemical tests including oxidase reaction, acid and gas production from carbon sources, gram staining, and production of indolactetic acid Pathogencity test 1. Cultures are grown on nutrient glucose broth overnight and adjusted to a concentration of 107 cfu/ml. 2. Leaves of a 3-5 day old soybeans (cultivar SJ14 or other susceptible variety) were inoculated as described (see Document 6 for inoculation method). 3. Plants were evaluated for symptom development after 7-10 days at 30-34 C and 79-95% relative humidity. Medium recipes: MXG Prepare MXP agar Distilled water 1000 ml K2HPO4 0.8 g KH2HPO4 0.6 g Yeast extract 0.7 g Soluble starch 0.8 g Glucose 1.0 g Agar 15.0 g Additional ingredients 1 ml methyl green (1% in 20% ETOH) 1 ml per liter of MXP, pH = 7.2-7.4 and autoclaved. After cooling add filter sterilized: Cycloheximide (50 mg/ml in 12.5% methanol) 1 ml Cephalexin (50 mg/ml aqueous) 1 ml Bacitran (10 mg/ml aqueous) 1 ml Kasugamycin (50 mg/ml aqueous) 1 ml REFERENCES: Prathuangwong, S., Khandej, K., & Goto, M. 1997. Development of new methods for ecological study of soybean bacterial pustule: A semiselective medium for detecting Xanthomonas campestris pv. glycines in contaminated soybean seed. pp 197-202 in: Banpot Napompeth (ed). Proceedings of World Soybean Research Conf. V, Chiangma, Thailand 1994. PATHOGEN: Bean Pod Mottle Virus HOSTS: Soybean (Glycine max) METHOD: Sv4.1 ELISA (Iowa State University adaptation of Agdia kit) METHOD: Sv4.1 ELISA (Iowa State University adaptation of Agdia kit) METHOD CLASS: TEMPORARY STANDARD PATHOGEN: Bean Pod Mottle Virus SAMPLE: 400 seeds PROCEDURE: Diagnostic kit: This method uses an ELISA kit developed by Agdia, Elkhart, Indiana. Extraction of the virus. 1. Grind 4 replicates of 100 soybeans, per sample and add 100ml of General Extraction 2. Or place 4 replicates of 100 soybeans sample, into 100ml of General Extraction Buffer and soak overnight at room temperature, then grind. 3. Disinfect the grinder between samples by rinsing with distilled water, followed by a rinse with a laboratory or hospital standard detergent, then a final rinse with distilled water. Running the ELISA kit: Run kit according to manufactures instructions. Please cont) if you need a copy of instructions or assistance with the protocol. Evaluating ELISA plates: Evaluate visually, or measure on an ELISA plate reader at 405nm. The threshold for a positive reaction on the plate reader should be >2X that of the negative control. PATHOGEN: Soybean Mosaic Virus HOSTS: Soybean (Glycine max) METHOD: Sv3.1 ELISA (Iowa State University adaptation of Agdia kit) METHOD: Sv3.1 ELISA (Iowa State University adaptation of Agdia kit) METHOD CLASS: TEMPORARY STANDARD PATHOGEN: Soybean Mosaic Virus SAMPLE: 400 seeds PROCEDURE: Diagnostic kit: This method uses an ELISA kit developed by Agdia, Elkhart, Indiana. Extraction of the virus. 1. Grind 4 replicates of 100 soybeans, per sample and add 100ml of General Extraction 2. Or place 4 replicates of 100 soybeans sample, into 100ml of General Extraction Buffer and soak overnight at room temperature, then grind. 3. Disinfect the grinder between samples by rinsing with distilled water, followed by a rinse with a laboratory or hospital standard detergent, then a final rinse with distilled water. Running the ELISA kit: Run kit according to manufactures instructions. Please cont) if you need a copy of instructions or assistance with the protocol. Evaluating ELISA plates: Evaluate visually, or measure on an ELISA plate reader at 405nm. The threshold for a positive reaction on the plate reader should be >2X that of the negative control. PATHOGEN: Tomato Ringspot Virus HOSTS: Soybean (Glycine max) METHOD: Sv2.1 ELISA (Iowa State University adaptation of Agdia kit) METHOD: Sv2.1 ELISA (Iowa State University adaptation of Agdia Inc Kit) METHOD CLASS: TEMPORARY STANDARD PATHOGEN: Tomato Ringspot Virus SAMPLE: 400 seeds PROCEDURE: Diagnostic kit: This method uses an ELISA kit developed by Agdia, Elkhart, Indiana. Capture by: polyclonal antibody; detection by: polyclonal antibody. Extraction of the virus. 1. Grind 4 replicates of 100 soybeans, per sample and add 100ml of General Extraction 2. Or place 4 replicates of 100 soybeans sample, into 100ml of General Extraction Buffer and soak overnight at room temperature, then grind. 3. Disinfect the grinder between samples by rinsing with distilled water, followed by a rinse with a laboratory or hospital standard detergent, then a final rinse with distilled water. Running the ELISA kit: Run kit according to manufactures instructions. Please cont) if you need a copy of instructions or assistance with the protocol. Evaluating ELISA plates: Evaluate visually, or measure on an ELISA plate reader at 405nm. The threshold for a positive reaction on the plate reader should be >2X that of the negative control. PATHOGEN: Tobacco Ringspot Virus HOSTS: Soybean (Glycine max) METHOD: Sv1.1 ELISA (Iowa State University adaptation of Agdia kit) METHOD: Sv1.1 ELISA (Iowa State University adaptation of Agdia kit) METHOD CLASS: TEMPORARY STANDARD PATHOGEN: Tobacco Ringspot Virus SAMPLE: 400 seeds PROCEDURE: Diagnostic kit: This method uses an ELISA kit developed by Agdia, Elkhart, Indiana. Capture by: polyclonal antibody; detection by: polyclonal antibody. Extraction of the virus. 1. Grind 4 replicates of 100 soybeans, per sample and add 100ml of General Extraction 2. Or place 4 replicates of 100 soybeans sample, into 100ml of General Extraction Buffer and soak overnight at room temperature, then grind. 3. Disinfect the grinder between samples by rinsing with distilled water, followed by a rinse with a laboratory or hospital standard detergent, then a final rinse with distilled water. Running the ELISA kit: Run kit according to manufactures instructions. Please cont) if you need a copy of instructions or assistance with the protocol. Evaluating ELISA plates: Evaluate visually, or measure on an ELISA plate reader at 405nm. The threshold for a positive reaction on the plate reader should be >2X that of the negative control. PATHOGEN: Maize Dwarf Mosaic Virus HOSTS: Maize (Zea mays) METHOD: Cb3.1 ELISA (Iowa State University adaptation of Agdia kit) METHOD: Cb3.1 ELISA (Iowa State University adaptation of Agdia Inc Kit) METHOD CLASS: TEMPORARY STANDARD PATHOGEN: Maize Dwarf Mosaic Virus SAMPLE: 400 seeds PROCEDURE: Diagnostic kit: This method uses an ELISA kit developed by Agdia, Elkhart, Indiana. Capture by: polyclonal antibody; detection by: polyclonal antibody. Extraction of the virus. 1. Grind 4 replicates of 100 corn kernels, per sample and add 100ml of General 2. Or place 4 replicates of 100 corn kernels sample, into 100ml of General Extraction Buffer and soak overnight at room temperature, then grind. 3. Disinfect the grinder between samples by rinsing with distilled water, followed by a rinse with a laboratory or hospital standard detergent, then a final rinse with distilled water. Running the ELISA kit: Run kit according to manufactures instructions. Please cont) if you need a copy of instructions or assistance with the protocol. Evaluating ELISA plates: Evaluate visually, or measure on an ELISA plate reader at 405nm. The threshold for a positive reaction on the plate reader should be >2X that of the negative control. Method #2 Grow-out (Williams et al., 1968; Hill et al., 1974; Mikel et al., 1984). 1. Maize seeds were planted in sterile soil and grown under various environmental conditions for different periods of time. 2. Seedlings were examined for MDMV symptoms. 3. MDMV symptoms were conformed either by inoculation of indicator plants or by ELISA. Note: Due the very low rates of transmission of MDMV indicated in these studies and the large numbers of seeds that would be required in a grow-out test to have any reasonable chance of detecting infected seedlings, this procedure has not been adopted as a routine seed health test for MDMV. PATHOGEN: Maize Chlorotic Mottle Virus HOSTS: Maize (Zea mays) METHOD: Cb4.1 ELISA (Iowa State University adaptation of Agdia kit) METHOD: Cb4.1 ELISA (Iowa State University adaptation of Agdia kit) METHOD CLASS: TEMPORARY STANDARD PATHOGEN: Maize Chlorotic Mottle Virus SAMPLE: 400 seeds PROCEDURE: Diagnostic kit: This method uses an ELISA kit developed by Agdia, Elkhart, Indiana. Capture by: polyclonal antibody; detection by: polyclonal antibody. Extraction of the virus. 1. Grind 4 replicates of 100 corn kernels, per sample and add 100ml of General 2. Or place 4 replicates of 100 corn kernels sample, into 100ml of General Extraction Buffer and soak overnight at room temperature, then grind. 3. Disinfect the grinder between samples by rinsing with distilled water, followed by a rinse with a laboratory or hospital standard detergent, then a final rinse with distilled water. Running the ELISA kit: Run kit according to manufactures instructions. Please cont) if you need a copy of instructions or assistance with the protocol. Evaluating ELISA plates: Evaluate visually, or measure on an ELISA plate reader at 405nm. The threshold for a positive reaction on the plate reader should be >2X that of the negative control.

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