Indian Phytopath, 49 (4) : 319-328 (1996)

Methods to test seeds for associated fungi*

M.N. KHARE

Department of Plant Pathology. Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur (MP.) 482 004

Pathogen free sound seeds are preferred forsowing to have desired germination, emergence,healthy seedlings and plant population. Many dis-ease causing pathogens are seed borne and seedtransmitted. It is essential to test seeds for associ-ation of such pathogens in order to either rejectthe seed or if the association is within tolerancelevel, to treat the seed before planting. Severalmethods have been evolved for testing seeds forassociated microorganisms which have been re-viewed from time to time (de Tempe, 1961, 1963,1964; Agarwal, 1976; Neergaard, 1977; Agarwaland Sinclair, 1987; Gaur and Dev, 1988). Whileselecting a method, it is necessary to see that it isreliable, less time consuming in obtaining results,economical, reproducible and sensitive. The selec-tivity also depends upon the purpose of the test.Certain times more than one method is used fortesting a pathogen. Fungi form the largest groupamong such microorganisms causing seed dam-age, seed rot, seedling rot, diseases at later stagesof crop growth till maturity.

Different types of fungi are associated withseeds as intraembryal, extraembryal, contaminantor with inert matter mixed with the seed sample.Various methods are used for the detection offungi based on their location in or on the seed. Itis essential to mix the seed of a sample thoroughlybefore testing.

*Prof.M.S. Pavgi Lecture delivered during 46th AnnualMeeting of I.P.S. held at Coimbatore (January 27-29,1994)

VISUAL INSPECTION OF SEED SAMPLE

The seed sample is examined as such in atransparent petriplate with naked eye. Plant partslike pieces of stem, leaf, fruit are separated andexamined for associated fungi. Seeds withmechanical damage are recorded. Fungal sclerotia,galls, smut balls and other fungal bodies areseparated and percentage recorded. Sorghum andpearl millet seed samples are examined for theextent of prevalence of ergot sclerotia. Soil clods,sand, stone pieces are also separated. As per 1STArules (ISTA, 1993), these impurities are consideredinert matter.

INSPECTION UNDER STEREOSCOPICMICROSCOPE

The seed samples are examined under 'stereo-scopic microscope for seed discolouration, mor-phological abnormalities and fungal structures notobserved with naked eye.

Seed discolouration

Seed coat in many cases exhibit different typesof discolouration depending upon the associatedfungus. The legume seeds develop browndiscolouration when infected with anthracnose fun-gus; ashy brown discolouration in seeds of lentiland chickpea is due to Assochyta fabae f.sp. lentisand A. rabiei respectively; purple colour seed coatin soybean due to Cercospora kikuchi; black pointof wheat due to Alternaria alternata and severalother fungi; blackish seed coat in chickpea due toA. alternata. When infected with Bipolaris oryzae

320 Indian Phytopathology

paddy seed develops light brown to blackish patch-es. Maize seeds' exhibit white streaks when infect-ed with Fusarium moniliforme.

Morphological abnormalities

Seeds of some crops loose weight, remainsmaller, become shrivelled and malformed due tofungal association. Chickpea seeds from wiltedplants (Fusarium oxysporum f. sp. ciceri) remainsmall and wrinkled. Coriander seeds are hypertro-phied when infected with Protomyces macrosporus.Paddy seeds get deformed due to false smut.

Fungal structures

The plant pieces and seeds of some cropsexhibit fungal structures. Hyphae and sclerotia ofMacrophomina phaseolina are visible on seed coatof sesame, mycelium and oospores of Peronosporamanshurica on soybean seed, pycnidia of Phomasorghina on sorghum seed, Phoma betae onsugarbeet seed, Macrophomina phaseolina on le-gume seed, acervuli of anthracnose fungi on sor-ghum and legume seeds are few examples.

Examination under UV or NUV light

Seed infections due to some fungi can berecognised under ultraviolet and near ultravioletlight source. Pea seeds infected with Ascochytapisi exhibit yellow green fluorescence, wheat seedsinfected with Stagonospora nodorum producegreenish fluorescence and loose smut infectedwheat seeds give bluish fluorescence. The indica-tion is further confirmed by other tests.

Seed soaking in water

Seeds are soaked in water and examined un-der stereoscopic microscope for oozing of sporesfrom pycnidia and other spore containing struc-tures like Gloeotinia temulenta in rye grass,Aureobasidium lini in linseed and Septoria in cel-ery.

Seed washings

Seeds are washed in water and the washingsare examined for the presence of fungal sporeswhich are identified based on their morphology.

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The test is made quantitative by shaking a definitequantity of seeds in a specific volume of waterwith detergent in a flask. It is shaken in an auto-matic shaker for 10 minutes. The suspension iscentrifuged at 2500-3000 rpm. The pellet is dilut-ed with known quantity of water or lactophenol.The contained spores of fungi are counted in ahaemocytorneter and load of spores per gram orper seed is calculated. Conidia of several fungican easily be identified. Load of oospores, smutspores, chlamydospores can also be determined.The viability of conidia and other spores can bechecked by germinating them in cavity slides.Sharma (1990) used washing test for estimatingseed borne ergot inoculum with pearl millet seed.Shetty et al. (1980) detected and estimatedoospores of Sc/erospora graminicola on pearlmillet seeds by this method.

The viability of P. manshurica oospores wastested by Pathak et al. (1978) by tetrazolium dye.They used 1% solution of 2,3,5-triphenyltetrazolium chloride at pH 6.5-7 by add-ing in 2 parts of a solution containing 9.078 gKHl04 per litre distilled water and 3 parts ofanother solution having 1l.876g Na2HP04. 2Hpper litre distilled water. Oospore scraping of P.manshurica from soybean seed was added in a testtube containing one ml distilled water and. incu-bated at 30°C for 48h. One ml of the dye wasadded and the tube was again incubated at 30°Cfor 48 h in darkness. The viable oospores devel-oped orange red colour.

Shetty et al. (1978) checked the viability ofoospores of Sclerospora graminicola causingdowny mildew of pearl millet by collecting oos-pores by seed washings and keeping them in oneml distilled water in a test tube and incubating at30°C for 48 h. One ml tetrazolium chloride solu-tion was added in the tube and again incubated at30°C for 48 h in darkness. The oospores wereexamined, the viable oospores developed redcolour.

Roongruangsree et al.(l988) used phloxine Btest for the viability of oospores of Peronospora

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manshurica. Aqueous solutions (0.05 and 0.1%)of phloxine B are prepared in de-ionized water.An oospores suspension is presoaked in de-ion-izedwater for one hour, then centrifuged and theoosporepellet is resuspended in phloxine B solu-tion.The oospores are examined after 20 minutesat room temperature. The viable oospores do nottake the stain, whereas non-viable oospores getbright red stain.

Incubation test

Standard blotter test and agar plate test areusually followed where seeds are incubated for adefinite period under specific conditions. The as-sociated fungi are identified based on their mor-phological and habit characters on seed surfaceand colony characters on the medium.

Standard blotter test

Usually glass or plastic transparent petriplatesof 9 em diameter are used in the test. Three blot-ters of the size of the petriplate are dipped insterilized water and placed in the petri plate afterdripping off extra water. Untreated 25 small or 10large seeds are plated at equal distance in eachpetriplate, Four hundred seeds of each sample areexamined. The plates are incubated under NUVor day light tubes with arrangement for alternateperiods of 12 hours light and 12 hours darknessat 20±2°C. The distance between the tubes andplates is kept 40 ern. The plates are removed onthe eighth day and the seeds are examined understereoscopic binocular microscope fitted with coollamps. The inert matter is also examined in thesame way for the associated fungi. The standardblotter method has been modified in several waysfor specific purpose. Michail et al. (1977) recom-mended 5 ern plastic petriplates with one seed perplate for the detection of Rhizoctonia solani.

2, ./-D method

Due to germination of seeds in blotter methodthe seed coats are lifted at different levels whichmake tile seed examination for associated fungidifficult. To overcome this problem, the blottersare dipped in 0.1 to 0.2 per cent solution of sodi-

Indian Phytopathology 321

urn salt of 2,4-dichlorophenoll:yacetic acid in wa-ter. Neergaard and Saad (1962) observed higherpercentage of Pyricularia oryzae in paddy seeds.Shukla et al. (1990) also reported higher countsof Phoma medicaginis f. sp. sojae from soybeanseeds. In another modification, the blotter papersare dipped in water and the seeds are pretreatedwith 2,4-D solution. Strandberg (1988) had max-imum detection ofAlternaria dauci associated withcarrot seeds pretreated with 0.1% Ca (OC12) andplated on blotters dipped in 2,4-D solution.

Deep freeze blotter method

The seeds are plated as in standard blot-ter method and incubated for 24 hours at 20±2°Cas usual, for next 24 hours tile plates are incubat-ed at -20°C in dark and then kept back underoriginal conditions for five days (Limonard. 1966,1968). The test is better for the detection ofCephalosporium acremonium and Fusariummoniliforme associated with maize seed andColletotrichum deniatium with capsicum seed.Singh et al. (1982) found this method useful forthe detection of Alternaria padwickii from paddygrains.

Water pH: Change of pH of water used in blottertest becomes specific for the detection of fungi.pH 4 gives higher counts of A. padwickii associ-ated with paddy grains (Singh et aI., 1982).

Seed soaking in calcium chlorideRenukeswarappa and Sethna (1985) soaked chilliseeds in 5% calcium chloride solution for 24 hoursand plated as in standard blotter method. It yield-ed 50% more Col/etotrichum dematium.

Agar plate method

Two media pota 0 dextrose agar and maltextract agar are usually used in petri plates forplating seeds after pretreatment with 1% sodiumhypochlorite solution without further washings.Pretreatment can also be done with 0.1% mercu-ric chloride followed by 2-3 washings with sterilewater. Ten small seeds and five large seeds areplaced in each 9 em diameter petriplate. The in-cubation is done as in standard blotter method

322 Indian Phytopathology

and the seeds are examined for associated fungiafter five to eight days. The developing fungalcolonies are examined. The col.rur of the colonyon the reverse side is also examined as it is char-acteristic for some fungi which are identified onthis basis.

Plant parts and other inert matter found mixedwith seed are also incubated by this method. Sev-eral fungi grow on and around the seed on agarplate and make the identication difficult, henceattempts were made to make the medium selectiveallowing growth of a particular fungus.

Ulster method

The seeds are plated directly on the maltextract agar without pretreatment for the detec-tion of Guighardia fulvida and Colletotrichum liniassociated with flax seed. The colony charactersare examined on both the sides of the plates(Muskett and Malone, 1941). Malone (1962) re-ported easy detection of Pyrenophora avenae whenoat seeds were exposed to dry heating at 100°Cfor one hour before plating.

Phoma betae : Sugarbeet seeds pretreated or un-treated \\,j111 1% sodium hypochlorite are platedon l.2% water agar containing SO ppm 2, 4-D inpetriplates. The incubation is done at 20°C forsev~n days in dark. On examination under stereo-scopic binocular microscope holdfasts are observedwith clumps of swollen cells at the hyphal tips.These holdfasts are formed at the bottom of themedium (Mangan, 1971, 1974). Bugbee (1974)developed a medium ~HP04 4g, KHl04 1.5g,soil extract 2S ml, boric acid 0.2g, sucrose 109,agar 17g, streptomycin sulphate O.lg,chlorotetracyclin O.lg, benomyl O.lg in one litrewater) in which holdfasts are brown to black andcan easily be identified.

Alternaria triticina : The medium used is nutrientagar containing K2HP04 1.36g, Na2C03 1.06g,MgS04 Sg, dextrose Sg, asparagine 19, agar 20gin one litre distilled water. Wheat seeds were pre-treated with 0.1% mercuric chloride for S minutesfollowed by washings with distilled water and plat-

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ed and incubated at 2SoC for 10 days in completedarkness. Colonies of tile fungus appear olive buffcovered with chains of conidia (prabhu andPrasada, 1967).

Drechslera oryzae (Bipolaris oryzae) : GA me-dium can be used for the detection of B. oryzaeassociated with paddy seeds as devised by Kulik(197S).

Pyricularia oryzae : Kulik (197S) used guaiacolagar (GA) medium (GA O.12Sg, agar Sg, strepto-mycin sulphate O.Sg or lactic acid 0.8 ml in I litrewater) for the detection of P. oryzae from paddyseeds. Seeds pretreated with 1% NaOCI for 10minutes are pressed in the medium in petriplateand incubated for four days in complete darknessat 22-2SoC. Infected seeds exhibit light pink toreddish halo.

Alternaria padwickii (Trichoconiella padwickii) :GA medium can be used for the detection of A.padwickii associated with paddy seeds (Kulik,1975).

Fusarium moniliforrne : Agarwal and Singh (1974)used modified Czapek-Dox agar (NaN03 2g,K2HP04 19, MgS04 O.Sg, KCI O.Sg,FeS04 O.Olg,sucrose 30g, quintozene O.Sg, yeast extract 2g,agar 20g, malachite green O.OSg as fresh solutionin water, dicrysticin 0.7Sg, water 1 litre) as selec-tive medium in petriplates for F. moniliforme as-sociated with several crops. Seeds are plated after3 days and incubated under NUV light for 8 daysfor alternate periods of 16 hour light and 8 hourdarkness. The colonies of the fungus appear with-in S to 8 days which are whitish pink, fluffy sur-rounded by a pinkish powdery growth with smalldot like points. The reverse side of the colony ispurplish. According to Wu and Mathur (1987).Nash and Synder medium (Nash and Synder.1962) and modified Nash and Synder mediumwere better for the detecion of F. moniliformefrom sorghum seeds in which seeds were soakedin 2Sppm fusaric acid before plating.

Ascochyta fabae f. sp. lentis : Lentil seeds pre-treated with 2% available chlorine in an aqueous

solution of sodium hypochlorite for 2-3 minuteswere plated on potato dextrose agar medium andincubated for seven days under 12 hours alternat-ing cycles of NUV light and darkness at 22±2°C.Rusty brown colonies of the fungus developedaround the seed with abundant pycnidia (Singh et01.,1993).

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Macrophomina phaseolina : Sesame seeds arepretreated with 0.1% mercuric chloride, washedthoroughly in sterile distilled water and plated onPDA-PCNB medium. The fungus grew aroundthe seed (Kushi and Khare, 1978). The mediumwas found useful in the detection of M phaseolinaassociated with cowpea seeds also (Sinha andKhare, 1977).

Curvularia lunata: In sorghum, C. lunata isimportant. CS medium with 200ppm carbendazim+ 200ppm streptomycin and CR medium with 200ppm carbendazin + 200 ppm rifampicin were se-lective for the detection of C. lunata associatedwith sorghum seeds (Deshpande, 1993).

Stagnospora nodorum (Septoria nodorum) : Anagarmedium SNAW (S. nodorum agar for wheat)is selective for the detection of S. nodorum asso-crated with wheat seed (Manandhar and Cunfer,1991).It contains Difco PDA 109, agar lSg, oxgalll.5g, peptone l g in 1 litre of deionised distilledwater.After autoclaving, chloroneb (Smg) copperhydroxide(Smg), dicloran (Smg), chloramphenicol(3.12mg), erythromycin (3.13 g), tetracycline hy-drochloride (12.S mg) and neomycin sulphate (10II1g)are added.

Mathur and Lee (1978) plated ten wheat seedsonoxgall agar in each 9 em plastic petriplate andincubatedat 20°C under 12 hours alternating cy-clesof NUV light and darkness for six days. Onelitre medium contained 109 dextrose, 109 pep-tone, lSg oxgall, 20g agar and one litre water.First three days, the plates were kept lids facingupand rest three days upside down. Pale green tobrightyellowish green fluorescence is observed inthe colonies of S. nodorum.

Cunfer and Manandhar (1992) used a selec-

Indian Phytopathology 323

tive medium for the detection of S. nodorum as-sociated with barley seeds. It contained Difco PDA(lOg), bactopeptone (2g), oxgall (1.5g), agar (l2g),chloroneb (Smg), copper hydroxide (lOmg),dichloran (7.Smg) dissolved in 20% ethanol andCGA-449 SO WS (lmg). The pretreated seeds wereplated and incubated at 20°C under alternatingperiods of light and darkness for 12 days underNUV light.

Seedling symptom test

The test is based on the distinguishing symp-toms produced by seed-borne fungi on growingseedlings under controlled conditions. The test isused for obligate as well as other pathogens whichcan produce symptoms on seedlings. Seeds areplanted in sterilized crushed bricks, soil, sand,agar, paper towel and blotters.

Hiltners method

Sterile crushed brickstone pieces of 3~4 mmsize are used in a container. One hundred cerealseeda are planted well spaced and covered withthe same material upto 3cm. Sterile water is usedfor providing required moisture. The containersare placed in darkness at room temperature. Seed-lings are examined after two weeks for diseasesymptoms. Sterile gravel, sand and soil can alsobe used. Wooden boxes, cardboard boxes, iron orwooden trays, earthern pots of various sizes canbe used for the test (Hiltner, 1917).

Multipots

Plastic multipot trays are filled with sterilizedsoil and one seed is planted in each pot. The trayis kept in a plastic bag for retention of moisturebefore placing in the incubation chamber at re-quired temperature. This method is used at SeedTesting Station, Solna, Sweden for routine testingof cereal seeds for germination and health. AtJabalpur, inverted egg trays have been successful-ly used as multipots for seed testing. The methodhas been modified for the detection ofColletotrichum gossypii in cotton seed by HalfonMeiri and Volcani (1971), and for Drechslera

324 Indian Phytopathology

ntaydis race T in maize by Kommedahl et al.(1971).

Test tube agar method

Culture tubes (160 x 16 mm) are filled withlOml 1% water agar and solidified to have slightslant. One seed is placed in each tube and incu-bated at required temperature needed for the patho-gen and the host under NlN or day light tubeswith 12 hours alternate cycles of light and darkperiods. The tubes are covered with an aluminiumfoil to retain moisture. The foil is removed whenthe seedlings reach the top. The required time forsymptom expression depends upon the pathogenand the host which is 10 to 14 days. The seedlingsdevelop distinguishing symptoms' (Khare et al.,1977). In case of anthracnose acervuli andMacrophomina phaseolin a, pycnidia are formedon hypocotyl in legumes and sesame. In wheat,characteristic symptoms develop at the base of thecoleoptile. Besides cultures tubes, the test can beconducted in plastic micro-culture plates andpetriplates also.

The method is most useful at quarantine sta-• lions for testing germplasm material which is usu-ally supplied in very small quantity. The healthyseedlings can be transplanted in pots and raisedunder controlled conditions for seed formation.The produce is checked and can be released.

Water agar can be replaced by a selectivemedium also.

Ragdoll method (rolled paper towel method)

The method is used for germination test ofseeds at seed testing stations. Two paper towelsare taken together, moistened with 'water, spreadon a glass plate and seeds are placed at equaldistance. Another moist paper towel is spread over'the seeds. The lower 5 em is turned over, thewhole unit is-rolled up and elastic bands arc ap-plied. The rolls are placed in a basket in standingposition and incubated at required temperature.The rolls are opened, and seed and seedlings areexamined for disease symptoms and fungi.

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Embryo count method

Rennie and Seaton (1975) developed NaOHsoak method for the detection of Ustilago nuda inwheat and barley seeds. The method was modifiedby Agarwal et af. (1981). They soaked wheat seedsin 5% NaOH and 0.02% trypan blue solution for18 hours at 25°C in an Erlemneyer flask. Embry-os are collected through flotation by adding 5%NaCi solution. The embryos are washed in water,boiled in lactophenol and examined in groovedplate under stereoscopic microscope. A workingsample of at least 2000 seeds is usually taken forthe test. Shetty et al. (1978) used embryo countmethod for the detection of hyphae of Sclerosporagraminicola in pearl millet seeds.

Agarwal and Srivastava (1981, 1985), andAgarwal and Verma (1982) modified this methodfor paddy bunt and wheat Kamal bunt respec-tively. Paddy seeds are soaked in 0.2% NaOH for24 hours at 18-25°C. The seeds are spread onblotters to remove excess moisture and examinedunder stereoscopic binocular microscope. The seedswith bunt disease (Tilletia barclayana) are jetblack. Bunt spores are released when seed is punc-tured by a needle in a drop of water. Same tech-nique is used for the detection of Kamal bunt ofwheat. Ahmad and Majumdar (1987) detected oos-pores and mycelium of Peronospora sorghi in sor-ghum seeds by NaOH soaking of seeds.

Histopathological techniques

To find the location of fungi in the seed,component plating was advised by Maden et al.(1975). The exact location of the pathogen in theseed and its further development is examined byhistological techniques. Usually, the seeds aresoaked in water, the soft seeds are processed formaking blocks in wax and microtomy is done.The sections are stained and examined. Seeds arealso cut through cryostat microtome to avoidlengthy process. Jacobsen and Williams (1971)studied the histopathology of Brasstca oleraceaseed infected with Phoma lingam, Singh et "al.·(1977) Alternaria tenuis in sunflower seeds, Sinhaand Khare (1977) Macrophomina phaseolina in

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cowpea seeds, Singh et al.(1980) Alternaria sesamiin sesame seeds, Singh and Sinclair (1985)Cercospora sojina in soybean seeds, Kunwar etal. (1985) Colletotrichum truncatum andPhomopsis sp. in soybean, Velcheti and Sinclair(1991) Fusarium oxysporum in soybean, and Singhet al. (1993) Ascochyta fabae f. sp. lentis in len-til.

Biochemical method

Gordon and Webster (1982) developed a tech-nique for detectin Drechslera graminea in barleyseed Jots by estimating the amount of ergostrol.They used standard methods for the extraction ofergostrol in barley seed lots and high performanceliquid chromatography analysis based on ultravi-olet absorbance.

Cleared whole mount

Velcheti and Sinclair (1991) cleared symp-tomatic 25 seeds of soybean by boiling for 5 min-utes in 10% KOH followed by staining with anilineblue. The seeds are dissected into different parts.Hyphae of F oxysporum were found in hourglasscell layer along with mircro and macro conidia.Chlamydospores were observed on the undersideof the seed coat.

Singh et. al. (1993) boiled lentil seeds in waterfor 10-15 minutes. The seed components wereseparated and boiled in lactophenol till softening,stained in cotton blue and mounted on glass slidein polyvinyl alcohol. The slides were dried in anoven at 60°C overnight. The seed coat, cotylendonsand embryonal axis exhibited the mycelium ofAscochyta fabae f. sp. lentis.

Maceration technique

Jang and Safeeualla (1990) used alkali mac-eration technique for the detection of seed bornenature of Peronospora parasitica in radish.

Sticky tape method

Fedovora (1987) used sticky tape to wrap thegrains or seeds individually. The tape is examinedfor the' spores and other fungal structures.

Indian Phytopathology 325

Immunodiagnostic assay

Lagerberg (1993) successfully used ELISAtechnique for the detection of Septoria nodorumand S. tritici associated with wheat seeds. Mille(1990) used immunofluorescence technique to de-tect Rhynchosporium secalis associated with bar-ley seeds. Dewey et. aJ. (1989, 1992) developedmonoclonal - antibody - ELISA - DOT - BLOTand DIP - STICK immunoassays for Humico!aJanuginosa in rice. Yao et al. (1991) detectedPeronosc/erospora sacchari by DNA hybridiza-tion in endosperm, pericarp and pedicel tissuesbut not in embryos of infected maize seeds.

Advancement of any branch of science de-pends upon the methods used in the study. Hence.it is essential to continue the investigations onnewer methods which may be more useful, reli-able, involving less expenses, simple so that thetechniques may easily conduct them, reproducibleand less time consuming. In modern agriculture.seed-borne pathogens playa vital role, hence needproper attention.

REFERENCESAgarwal, V.K and Singh O.V. (1974). Routine test-

ing of crop seeds for Fusarium moniliforme witha selective medium. Seed Research, 2 : 19-22.

Agarwal, V.K (1976) Techniques for the detection ofseed borne fungi. Seed Research, 4 : 24-31.

Agarwal, V.K., AganY~II, M., Gupta, R.K andVerma, H.S. (1981). Studies on loose smut ofwheat. 1.A simplified procedure for the detectionof seed borne infection. Seed Research, 9 : 49.

Agarwal, V.K and Srivastava, A.K (1981). A sim-ple technique for routine examination of rice seedlots for rice bunt. Seed Technology News., 11 : I.

Agarwal, V.K and Verma, H.S. (1982). A simpletechnique for the detection of Kamal bunt infec-tion in wheat seed samples. Seed Research, 11 :10.

Agarwal, V.K and Srivastava, A.K (1985). 'NaOHseed soak' method for routine examination of riceseed for rice bunt. Seed Research, 13 : 159-161.

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Agarwal, V.K. and Sinclair, J.B. (1987). Principlesof Seed Pathology, CRC Press, Inc. Boca Raton,Florida, U.S.A. pp. 29-76.

Ahmad, R. and Majumdar, S.K. (1987). Seed bornenature, detection and seed transmission of sorghumdowny mildew. In : Proc. Plant Quarantine andPhyto-sanitory Barries to Trade in the ASEAN.(Singh, K.G. and Manalo, P.L., Eds.), pp. 173-191.

Bugbee, W.M. (1974). A selective medium for theenumeration and isolation of Phoma betae fromsoil and seed. Phytopathology, 64 : 706.

Cunfer, B.M. and Manandhar, J.B. (1992) Use of aselective medium for isolation of Stagnosporanodorum from barley seed. Phytopathology, 82 :788-791.

Deshpande, G.D. (1993). Development of medium forselective expression of Curvularia lunata in sor-ghum seed health testing. J. Maharashtra Agri-cultural University, 18 : 142-143.

de Tempe, J. (1961). Routine methods for determiningthe health condition of seeds in seed testing sta-tion. Proc. Int. Seed Test. Assoc., 26 : 27-60.

de Tempe, J. (1963). Inspection of seeds for adheringpathogenic elements. Proc. Int. Seed Test. Assoc.,28 : 153-165.

de Tempe, J. (1964). Recent developments in seedhealth Testing. Proc. Int. Seed Test. Assoc., 29 :479-486.

Dewey, F.M., Macdonald, .M.M. and Phillips, S.l.(1989). Development of monoclonal-anti body-ELISA-DOT-BLOT-and-DIP-STICK immunoassaysfor Humicola lanuginosa in rice. Journal of Gen-eral Microbiology, 135 : 361-374.

Dewey, F.M., Twiddy, D.R.,Phillips, S.I., Grose, M.J.and Wareing, P.W. (1992). Development of amonoclonal antibody based immuno assay forHumicola lanuginosa on rice grains. Food andAgricultural Immunology, 4 : 153-167.

Fedorova, R.N. (1987). A rapid method of detectingspores on the surface of seeds and plants.Mikologiya i Fitopatologiya, 21 : 563-565.

Gaur, A. and Dev. U. (1988). Detection techniques forseed horne fungi. bacteria and viruses. In : Per-speclive in Mycology and Plant Pathology. (Eds.

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Agnihotri,' V., Sarbhoy, A.K. and Kumar, D.)Malhotra Publishing House, New Delhi. pp. 591-609.

Gordon, T.R. and Webster, R.K. (1982). Phytopa-thology, 72 : 932.

Halfon-Meiri, A. and Volcani, Z. (1977). A combinedmethod for detecting Colletotrichum gossypii andXanthomonas malvacearum in cotton seed. SeedSci. and Technol., 5 : 122-139.

Hiltener, L. (1917). Technische vorschriften fur diePrufung von saatgut, gultig vom I. Juli 1916 an BBesonderer Teil I. Getreide, Landwirtsch, VersoSIn., 89 : 1917.

ISTA (1993). International rules for seed testing. SeedSci. and Technol., 21(Supplement) pp 296.

Jacobsen, B.J. and Phillips, P.H. (1971).Histopathology and control of Brassica oleraceaseed infection by Phoma lingam. Plant Dis. Replr.,55 : 934.

Jang, P. and Safeeulla, K.M. (1990). Seed borne na-lure of Peronospora parasitica in Raphanussativus. Proc. Indian Academy of Sciences, 100 :255-258.

Khare, M.N., Mathur, S.B. and Neergaard, P. (1977).A seedling symptom test for detection of Septorianodorum in wheat seed. Seed Sci. and Technol., 5: 613-617.

Kommedahl. T., Lang, D.S. and Blanchard, K.L.(1971). Detection of Kernels infected withHelminthosporium maydis in commercial samplesof corn produced in t 970. PI. Dis. Repr., 55 :726.

Kulik, M.M. (1975). Comparison of blotters andguaiacol agar for .detection of Helminthosporiumoryzae and Trichoconis padwickii in rice seeds.Phytopathology, 65 : 1325.

Kunwar, I.K., Singh, T. and Sinclair, J.B. (1985).Histopathology of mixed infections byColletotrichum truncatum and Phomopsis spp. orCercospora sojina in soybean seeds. Phytopathol-ogy, 75 : 489.

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328 Indian Phytopathology

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