Notesandbriefarticles

REFERENCES

ALDRICH, D. T. A. & DOLING, D. A. (1967). Varietalresistance to clover rot on white clover. Nature,London 214, 946-917.

BJOR!.ING, K. (1942). Undersokningar rorande Klever-rotan. II. Studier av Utvecklingshistoriaoch Varia-tion hos Sclerotinia trifoliorum. Meddelanden fr8nStatens uaxtskyddsanstatt. Stockholm 37, 1-154.

ClRR, A. J. H. (1956). Studies on clover rot. Ph.D.Thesis, University of Wales.

DILLON WESTON, W. A. R.,LoVELESS,A. R.&TAYLOR,R. E. (1946). Clover rot. Journal of AgriculturalScience 36, 18-29.

FRANDSEN, K. J. (1946). Studier over Sclerotinia tri-foliorum Eriksson. Copenhagen: Det Danske Forlag.

KUHN, LINDA M. (1979). Delimitation of the economic-ally important plant pathogenic Sclerotinia species.Phytopathology 69, 881-886.

LOWRY, O. H., ROSENBROUGH, N. J., FARR, A. L. &RANDALL, R. J. (1951). Protein measurement withFolin phenol reagent.Journal of Biological Chemistry183, 265-275.

PuRDY, L. H. (1979). Sclerotinia sclerotiorum: History,diseases and symptomatology,host range, geographicdistribution and impact. Phytopathology 69, 875-880.

WONG, A. L. &WILLETTS, H. J. (1973). Electrophoreticstudies of solubleproteins and enzymesof Sclerotiniaspecies. Tran sactions ofthe British Mycological Society61,167-178.

WONG, A. L. & WILLETTS, H. J. (1975a). A taxonomicstudy of Sclerot inia sclerotioram and related species:mycelialinteractions.Journal of General Microbiology88, 339-344.

WONG, A. L. & WILLETTS, H. J. (1975b). Electro-phoretic studies ofAustralasian,North AmericanandEuropean isolates of Sclerotinia sclerotiorum andrelated species. Journal of General Microbiology 90,355-359·

POSSIBLE INVOLVEMENT OF PHYTOALEXINS IN

DURABLE RESISTANCE OF WINTER WHEAT TO YELLOW RUST

By D. W. CARTWRIGHT* AND G. E. RUSSELL

Department of Agricultural Biology, The University, Newcastle Upon Tyne, NE1 7RU

With the notable exception of the Gramineae, ithas been well established that the production ofantimicrobial compounds, or phyroalexins, is aprominent feature of the resistant responses ofseveral groups of plants to pathogenic attack (Kuc,1972). Although it has been shown that the resist-ance of rice to the blast fungus, Pyricularia oryzaeClV., can involve the accumulation of highly toxiclevels of two diterpene phytoalexins (Cartwrightet al., 1977; Langcake et al., 1978; Cartwright,Langcake & Ride, 1980), there is little evidence tosuggest that inhibitory compounds are producedby other cereals. However, studies on the nature ofdurable resistance of the winter wheat cultivarLittle Joss to the yellow rust fungus (Pucciniastriiformis Westd.) have indicated that hyper-sensitive browning and phytoalexin productionmight form the basis of one of the mechanismsresponsible for the durable resistance to thispathogen (Cartwright & Russell, 1980). This paperbriefly reports the detection and possible role ofpost-infectional antifungal compounds formed inLittle Joss leaves showing resistance to P. strii-[armis.

Adult plants of the winter wheat varieties LittleJOss, which has shown 'durable l resistance to

." Present address: Department of Applied Biology,Chelsea College, University of London.

yellow rust, and Nord Desprez, which is verysusceptible to most isolates of P. striiformis, weregrown in a glasshouse and inoculated with race104E137 of P. striiformis as described by Cartwright& Russell (1980).

Heavily infected flag leaf tissue (approximately1 g, taken 12 days after the initial inoculation withspores of P. striiformis) was weighed accurately,and ground in a mortar and pestle with sand andethanol (10 ml) . The supernatant was clarified bycentrifugation (3500 g) and the residue extracted afurther three times with aliquots of fresh ethanol.All supernatants were combined, rotary evaporatedto dryness (35 °C, in vacuo), and the resultingresidue taken up in distilled water (25 ml). Thisaqueous suspension was then extracted four timeswith ethyl acetate (25 ml aliquots). All organicfractions were combined, passed through phase-separating paper, rotary evaporated to dryness(25°, in vacuo) , and the residue redissolved in freshethyl acetate to give a final concentration equi-valent to 10 mg fresh weight/jzl. Subsequently,35 III of this extract was applied as a band to thebase ofa TLC plate (silica gel, 0'25 mm thickness),and the chromatogram developed in either 4 or10 % ethanol in chloroform. The plate was thendried and subjected to direct bioassay againstCladospor ium cucumerinum Ell. &Arth., as described

Trans. Br. my col. Soc. 76 (2), (1981). Printed in Great Britain

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Notes and briefarticles

Fig. 1.

Fig. 2.

Figs. 1-2. Cladosporium cucumerinum TLC bioassays of extracts of uninfected and yellow rust-infectedflag leaves of Nord Desprez (A and B respectively), and uninfected and infected flag leaves of Little Joss(C and D respectively). Fig. 1. shows two antifungal zones (Z1 and Z2) in a chromatogram developed in4 % ethanol in chloroform. The chromatogram in Fig. 2 was developed in 10 % ethanol in chloroformand shows a third zone (Z3).

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Notes and briefarticles

by Bailey & Burden (1973). Antifungal activitywas detected as white areas again st the greenbackground of fungal growth.

Examples of the antifungal zones detected onthin-layer chromatograms are shown in Figs 1 and2. The use of the more polar developing solvent(Fig. 2) invariably resulted in the masking of themajor inhibitory zones shown in Fig. 1. Theintensities of all zones varied greatly from experi-ment to experiment, but appeared to be directlycorrelated with the degree of browning of theinfected Little Joss leaves. Uninfected leaves, andinfected leaves of the susceptible variety NordDesprez, showed no areas of antifungal activity.The major antifungal area centred around Rf 0'65comprises two overlapping zones of activity.Although a range of solvent systems has failed toseparate the zones from each other, treatment ofcomparable chromatograms by spraying withvanillin in concentrated H 2S04 (1 % w/v) hasyielded two reactive spots at Rfs 0'63 and 0'69.The fact that these spots correspond to the majorzone of antifungal activity, and are absent fromsusceptible and uninfected tissue, tends to confirmthe accumulation of at least two, possibly closelyrelated, phytoalexin-Iike compounds. Apart fromthese colour reactions, preliminary studies of theirTLC mobilities have suggested that these com-pounds may be similar to the diterpene ricephytoalexins, momilactones A and B (Cartwrightet al., 1977). The more polar, minor band of anti-fungal activity at Rf 0'05 (Fig. 2) also reacted withvanillin-H 2SO4'

Although it seems highly unlikely that phyto-alexin production alone is responsible for durableresistance to P . striiformis, it appears that in themajority of race 104E137 infections of adult LittleJoss leaves, an initial hypersensitive-like responseoccurs during, or shortly after, the formation bythe fungus of a sub-stomatal vesicle (Cartwright &Russell, 1980). Detailed histological examination

has shown that this host response, which involvesthe progressive collapse and browning ofmesophylltissue, occurs without any apparent physical con-tact between the vesicle and the mesophyll cellssurrounding the sub-stomatal cavity. Not onlydoes this suggest that the host response might betriggered by the diffusion of substances from thesub-stomatal vesicle, but the subsequent browningand degeneration of the vesicle itself (before anyinfection hyphae are formed) points to the pro-duction and accumulation of antifungal compoundsof host origin. Further work will be needed to con-firm the hypothesis that phytoalexin production isone of the host defence mechanisms responsiblefor the durable resistance of Little Joss to P . stri-iformis.

REFERENCES

BAILEY, J. A. & BURDEN, R. S. (1973). Biochemicalchanges and phytoalexin accumulation in Phaseolusvulgaris following cellular browning caused bytobacco necrosis virus. PhysiologicalPlant Pathology3,171-177.

CARTWRIGHT, D., LANGCAKl!, P., PRYCE, R. J., LE-WORTHY, D. P. & RIDE, J. P. (1977). Chemicalactivation of host defence mechanisms as a basis forcrop protection. Nature, London 267, 511-513.

CARTWRIGHT, D., LANGCAKE, P. & RIDE, J. P. (1980).Phytoalexin production in rice and its enhancementby dichlorocyclopropane fungicides. PhysiologicalPlant Pathology 17,259-267.

CARTWRIGHT, D. & RUSSELL, G. E. (1980). Histologicaland biochemical nature of durable resistance toyellow rust in wheat. European and MediterraneanCereal Rusts Conference, Bari, Italy 1980. Pp. 25-28.

Kuc, J. (1972). Ph ytoalexins, Annual Review of Phyto-pathology 10,207-232.

LANGCAKE, P., CARTWRIGHT, D., LEWORTHY, D. P.,PRYCE, R. J. & RIDE, J. P. (1978). The dichlorocyclo-propanes - fungicides with an indirect mode ofaction. Netherlands Journal of Plant Pathology(Suppl. 1) 83, 153-155.

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