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The use of leaf transplants to study the cause of hypersensitivity to leaf rust, Puccinia recondita, in wheat carrying the Lr20 gene
PlgvioLqical Plant Path&g (1978) 12,311-319
The use of leaf transplants to study the cause of hypersensitivity to leaf rust, Puccinia recondifa, in wheat carrying the Lr20 gene D. R. JoNEst and B. J. DEVERALL Depormzmtof Plant Patho@ and Agricultural EntmoLogy, Uniocrsity of S’dncy, N.S. W. 2006, Australia (Accepedfor jwblication December1977)
Mycelia of virulent and normally avirulent races of leaf rust were established in cultivars carrying the temperature-sensitive Lr20 gene for resistance by incubating inoculated seedlings for 1 day at 20.5 “C and 3 days at 30.5 “C. Inoculated medhngs lacking this gene were incubated for 4 days at 20.5 “C. Epidermis-free transplants fiom uninfected fimgicide-fed leaves were placed under the lower epidermis of infected leaves and the two mesophylls held together for 21 h at 20.5 “C. Transplants were then removed, placed under the epidermis of uninfected leaves and exam&d for necrosis after a further 2 days at 20.5 “C. Widespread necrosis, seen to be free of hyphae, only occurred in transplants carryiq the Lr20 gene which had been held in leaves infected with avirulent mycelia. The genotype of the infected leaves did not affect the necrosis. The results are dkcmsed in relation to an hypothesis about the action of an Lr20 gene-specific toxin produced by rust with the complementary avirulence gene.
INTRODUCTION
In interactions between the wheat cultivar Thew, carrying the Lr20 gene for resistance, and an avirulent race of leaf rust Puccinia reconditaf. sp. t&i, the expression of resistance was closely associated with physiological changes leading to the collapse of host protoplasts [6]. The pattern of this necrosis including its tendency to spread along veins suggested that a phytotoxin was released from infection sites to kill surrounding tissue. Circumstantial evidence for the existence of a toxin was derived from experiments utilizing the temperature sensitivity of interactions involving the Lr20 gene to establish colonies of the avirulent rust in primary leaves of cv. Thew and then administering heat shocks [7l. Widespread necrosis occurred around inhibited avirulent, but not virulent, colonies and it also had the same temperature dependency as that associated with the normal expression of the Lr20 gene. The hypothetical toxin might be either a specific product of avirulent hyphae active only on wheat cells bearing the Lr20 gene or a non-specific product released after a recognition reaction between avirulent haustoria and invaded resistant cells. The experiments reported herein were designed firstly to detect the diffusion of a toxin from the exposed mesophyll of infected leaves of cv. Thew into the exposed t Present address: Plant Pathology Branch, Department Indooroopilly, Brisbane, Queensland 4068, Australia. 0048-4059/78/0501-0311 22
$01.00/O
of Primary
Industries,
Meiers Road,
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D. R. Jones and B. J. Deverall
312
mesophyll of transplants from uninfected leaves, using a procedure modified from that of Sharp & Emge [15]. Secondly, the source and specificity of the toxin were determined by comparison of the effects of several genotypes of host and transplant cultivars, including near-isogenic lines differing in presence and absence of the Lr20 gene. In the course of these experiments, there was a possibility for hyphae to grow into the transplant from the infected leaf, but this was minimized by incorporating a systemic fungicide, active against leaf rust, in the transplant cultivar. The transplant was held in the inoculated leaf for a pre-determined period and then transferred to an uninfected fungicide-treated leaf for a longer period. The transplant was then examined for necrosis and hyphae. MATERIALS
AND
METHODS
Wheat cultivars and leaf rust isolates Triticum aestivum L. cultivars Brevit (Sydney University Act. no. W972) carrying the Lr2c gene, Chinese Spring (W1806) carrying an undesignated gene for mature plant resistance [2], Kenya 744 (W744) carrying the Lr20 gene, Norka (W578) carrying the Lrl and Lr20 genes, Spica (W2341) carrying the Lrlla gene, Thew (W203) carrying the Lr20 gene, and two near-isogenic lines derived by Dr R. A. McIntosh [12] from Kenya 744 and differing in the presence or absence of the Lr20 gene were selected for this work [II]. Puccinia recondita Rob. & Desm. f. sp. tritici Eriks. & Henn. isolates 76-Anz-0 (Sydney University Act. no. SSSSS), 76-Anz-2,3 (70202)) 104-Anz-2,3,GT (74408)) 104~Anz-2,3,GTS (76694) and 122-Anz-2,3,4 (76348) avirulent on wheat cultivars containing the Lr20 gene and isolates 64-Anz-1,2,3 (70638), 104~Anz-1,2,3,GT (74606), 135-Anz-1,2,3,4,5 (64-L-3) and 162~Anz-1,2 (62408) virulent with respect to the Lr20 gene were used in this study [lo, 201. Avirulent isolates cause a ; or ;l infection type on cv. Thew and virulent isolates a 3+ infection type [9]. Isolate 104-Anz-2,3,GT causes a ; or ;l infection type on cvs Norka and Kenya 744 and a 3+ infection type on cvs Brevit, Chinese Spring and Spica [S, 91. Uredospores were stored in liquid nitrogen before use. Seedling treatmnzts Seedlings were grown and inoculated as previously described [6J and then incubated at 20.5 “C under incandescent and cool-white fluorescent light (12 h photoperiod, 20 000 Ix at soil surface) for 1 day. Colonies of races, avirulent with respect to the Lr20 gene, were established in leaves of cultivars carrying this gene by incubating seedlings for a further 3 days at 30.5 “C [7], a temperature which permits rust development [6]. Seedlings of cv. Thew infected with virulent races were similarly treated although this was not necessary to establish colonies. Seedlings of cultivars lacking the Lr20 gene were infected with race 104~Anz-2,3,GT and incubated at 20.5 “C for a further 3 days. Pots containing uninfected seedlings that were used as a source of transplants and as the final recipients of transplants were placed in a 0.035 g/l solution of a systemic fungicide (4butyl-4H-1,2,4triazole, RH 124, from Rohm & Haas Australia Pty.
Leaf rust in wheat
313
Ltd) 4 days after sowing. Transplants were excised from primary leaves 7 days after sowing. baf-transplant technique The positions of rust colonies in leaves were indicated by faint chlorotic spots 4 days after inoculation. Areas of primary leaves showing moderate to heavy infection densities (70 to 140 colonies/cm2) were selected as recipients for transplants. The transplant procedure was derived from the method of Sharp & Emge [15]. An infected leaf was placed between two cover slips held in a perspex block and cut transversely with a razor blade. Only the upper epidermis and mesophyll were severed as the coverslips prevented the blade from cutting completely through the leaf. Bending the leaf backwards, the lower epidermis was peeled back to expose infected mesophyll. The lower epidermis was removed from an uninfected primary leaf of a cultivar selected as the transplant donor in the same way as above. A 7 to 8 m m section of transplant was then excised and placed on the infected leaf so that the exposed mesophylls of both were in contact with each other. The lower epidermis of the infected leaf was finally placed in its original position covering the transplant. Seedlings with transplants were incubated at 20.5 “C in continuous light (20 000 lx) for 21 h ( f 0.5 h). Each transplant was then removed from the infected leaf and placed under the epidermis of an uninfected leaf of a seedling of the same cultivar as the transplant. After a further 2 days at 20.5 “C under continuous light, segments of leaves containing transplants were cut from seedlings, fixed, cleared and stained with trypan blue in alcoholic lactophenol and de-stained in chloral hydrate solution as previously described [6-j. Transplants were teased from the leaf segments and mounted in 50% glycerine. Assessnzentof necrosis Microscopic observations were limited to 20 to 32 randomly selected transplants for every rust-host-transplant combination. Wheat cells normally stained a uniform pale blue colour [6J. Cells with intensely stained and often collapsed protoplasts were considered to be dying or dead [q. The extent and distribution of this staining in each transplant was assessed visually and rated on a scale from 1 to 4 (Fig. 1). Necrotic cells associated with wounds made by forceps during handling were readily distinguished by their position and appearance and were disregarded in the rating. RESULTS
In early experiments, seedlings used as a source of transplants were not treated with fungicide and the transplants were held in the infected leaves for 2 days before examination. Necrosis occurred in these transplants in the same way as described below, but a high proportion of the necrotic areas were seen to contain small mycelia. However, the large areas of necrosis greatly exceeded the areas occupied by the hyphae. The modifications of feeding fungicide and of holding transplants in infected leaves for only 21 h were introduced to decrease the changes of hyphal growth into the transplants.
314
D. R. Jones and B. J. Deverall
Efect of rust rate on necrosisin tran.$lants Transplants from cv. Thew were put into leaves of cv. Thew infected with avirulent (76-Anz-0, 76-Anz-2,3, 104~Anx-2,3,GTS, 122-Am+2,3,4) and virulent (64~Anx1,2,3, 104~Anz-1,2,3,GT, 135-Anz-1,2,3,4,5, 162~Anx-1,2) races of leaf rust and also
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Fro. 1. Diagrammatic representation of level9 of necrosis in transplants after contact for 2 1 h and uninkcted tissue for 2 days. Rating : 1, with leaf tissue infected with P. recondika scattered and small groups of dead cells in the surface cell layer of the exposed meaophyll; 2, groups of dead cells extending from the surface of the exposed mcsophyll to the upper epidermis-up to 20% of the surface area necrotic; 3, larger groups of dead cells-between 20 and 40% of the surFace area necrotic; 4, very large groups of dead cells-over 40% of the surface arca necrotic.
in uninfected leaves. At the time of the removal of transplants after 21 h contact, necrotic areas were visible to the unaided eye around colonies of the avirulent rusts in the infected leaves. Microscopic examination of a few representative transplants in contact with infected tissue showed scattered dead cells on the surface of the exposed mesophyll and a few groups of cells with thickened walls caused by extracellular deposits. Areas of tissue that stained deeper than usual were noticeable in transplants that had been in contact with mesophyll infected with avirulent races. After incubation of transplants in infected leaves of cv. Thew for a further 2 days, scattered and small groups of necrotic cells were observed on the surface of exposed mesophyll of all transplants. Groups of living cells, some with translucent, wart-like deposits on their walls and others with heavily stained, extracellular deposits, were seen on the exposed mesophyll of transplants that had been in contact with infected leaf tissue (Plate 1). Large groups of necrotic cells were found in most transplants that had been in contact with mesophyll infected with avirulent races of rust (Plate 2). The necrosis in the transplants usually extended from the surface of the exposed mesophyll to the upper epidermis. Often the layer of surface cells appeared normal or had densely stained walls and the necrosis began in the second layer. A closer examination revealed that small hyphal segments, usually devoid of cytoplasm and
PLATES 1 to 4. Surface of exposed mesophyll of transplants in contact with leaf tissue infected with P. recondita for 21 h and uninfected tissue for 2 days. PLATE 1. Living cells of cv. Thew showing heavily stained, wall deposits after contact with cv. Thew infected with race 64-Am-1,2,3, virulent with respect to the Lr20 gene. x 176. PLATE 2. Necrosis (rating 2) in cv. Thew after contact with cv. Thew infected with race 76-Am-O, avirulent with respect to the Lr20 gene. Small fragments of hyphae lacking cytoplasm and haustoria were found on the surface of 500,h of the necrotic areas. x 27.
[facing page 3141
PLATE 3. Absence of extensive necrosis (rating: 1) in cv. Thew after contact with cv. Thew infected with race 64-Anz-1,2,3, virulent with respect to the Lr20 gene. A few small fragments of hyphae lacking cytoplasm and haustoria w-cre found on the surface of the transplant. Arrows indicate areas of living cells with extracellular deposits. x 26. PLATE 4. Necrotic area in cv. Kenya with race 104~Anz-2,3,GT, avirulent with 220 pm in length and lacking cytoplasm necrotic area. Arrows indicate the margin length and 700 pm wide. x 45.
744 after contact with cv. Chinese Spring infected respect to the Lr20 gene. A branched hypha about and haustoria was found on the surface of this of the necrotic area, which was about 2300 pm in
Leaf rust in wheat
315
lacking haustoria, were present on the surface of about 50% of the necrotic areas, distributed through many transplants. No hyphal fragments could be found on the necrotic areas in about 15% of transplants. No large areas of necrotic cells were seen in transplants that had been in contact with mesophyll infected with virulent rusts (Plate 3). Hyphae and sometimes haustoria were seen in a few places on the surface cells of about 75% of these transplants. The rating of necrosis in each transplant examined is presented in Table 1. TABLE 1 Effect of rDccof rust a nmosis in
Rust race infecting cv. Thew 64-Anz-1,2,3 (v) b 76-Anz-0 (a)” 76-Anz-2,3 (a) 104-k~z-1,2,3,GT (v) 104-Anz-2,3,GTS (a) 122~Anz-2,3,4 (a) 135-Anz-1,2,3,4,5 (v) 162~Anz-1,2 (v) Uninfected
I 20 2 2: 2 2: 20 20
tran$lant
Necrosis in 20 transplants from cv. Thew Ratinga 2 3 0 13 11 0 11 12 0 0 0
0 4 4 0 5 2 0 0 0
4 0 1 2 0 2 1 0 0 0
“SeeFig. 1. b Virulent with respect to the Lr20 gene, but carrying other avirulence genes. e Avirulent with respect to the ~920 gene.
Efect of host and tranrplant gem&be on necrosisin tranrp1ant.s Transplants from cvs Brevit, Chinese Spring, Kenya 744, No&a, Spica and Thew were put into leaves of each cultivar infected with the rust 104~Anz-2,3,GT (avirulent with respect to the Lr20 gene). At the end of each experiment, scattered, necrotic cells and groups of living cells with densely stained wall deposits were seen on the mesophyll surface of most transplants. Large areas of necrosis were observed in most transplants bearing the Lr20 gene (Plate 4). The presence or absence of the Lr20 gene in the infected leaves did not influence the necrosis in the transplant (Table 2). Hyphal fragments, lacking haustoria and usually cytoplasm, were associated with the surface cells of about 50% of the necrotic areas. Effect of near-isogenictransplant lines on necrosis Transplants from two near-isogenic lines of wheat derived from cv. Kenya 744 and differing in the presence or absence of the Lr20 gene were put into cv. Thew infected with avirulent race 104Anz-2,3,GT. Scattered necrotic cells and groups of living cells with stained wall deposits were observed on the surface of the exposed mesophyll of transplants of both lines. Large areas of necrotic tissue, however, were only seen in transplants bearing the Lr20 gene (Table 3). As before, empty hnphal segments were seen on the surface cells of about 50% of the necrotic areas.
D. R. Jones and B. J. Deverall
316 TABLE 2
Effid of host and transplant genotvp on m~osis in tr+&nt Host CultivaP Brevit (Lrzc)e
c. spring (MPR)”
Norka (Lrl, Lr20)
Transplant CUltiVar
1
Necrosis in 20 transplants Ratingb 2 3
Brcvit c. spring Kenya 744 Norka Spica Thew
20 20 4
0 0 12
2: 0
11 0 9
Brevit c. spring Kenya 744 No&a Spica
20 20 1
0 0
ThCW
2: 2
1; 0 7
Brevit c. spring Kenya 744 Norka Spica Thew
20 20 5 0 20 4
0 0 7 10 0 9
Brevit C. Spring Kenya 744 No&a Spica The-w
20 20 1 0 20 5
0
Brevit C. Spring Kenya 744 Norka Spica Thew
20 20 5 1 20 1
Brevit c. spring Kenya 744 No&a Spica Thew
20 20 5 2; 3
4
1: 1 1:
0 0 5 6 0 4
0 0 3 13 0 0
0 0 9 9 19 0 0 9 7 0 9
o Infected with race 104Am2,3,GT (avirulent with respect to the Lr20 gene). b See Fig. 1. e Resistance gene carried by cultivar. d Undesignated gene for mature plant resistance.
DISCUSSION These results show that necrosis, characteristic of the expression of the Lr20 gene [S, 71, was spccifklly caused in certain batches of fungicide-fed transplants which had been exposed for only 21 h to certain inoculated leaves. This necrosis was caused by a stimulus from the inoculated leaves. The stimulus was not caused by
311
Leaf rust in wheat
infection of the transplants because extensive necrosis often occurred at sites in transplants where no hyphae had grown. At those sites where hyphae had crossed from the inoculated leaves, the area and depth of necrosis greatly exceeded the part of the tissue contacted by the superficial hyphae. The occurrence and extent of the necrosis were consistent with the diffusion of a toxin. The toxin is seen to be specific in its action on wheat cultivars and lines bearing the Lr20 gene. It does not cause necrosis in cultivars lacking this gene and bearing TABLE 3 Effect of near-isogmk tmq’ht Transplant line”
1
lr20 gene Lr20 gene
32 7
lines on necroti
Necrosis in 32 transplants Ratingb 2 3 :
0 10
4 0 7
o Derived from cv. Kenya 744 and placed in leaves of cv. Thew infected with race 104Anz-2,3,GT (avirulent with respect to the Lr20 gene). bSeeFig. 1.
other genes for resistance against leaf rust. The toxin is suggested to react with a product of the Lr20 gene and thereby to cause necrosis. The specific toxin is also seen to be a product of avirulent hyphae and not of infected wheat cells bearing the Lr20 gene. This is because the toxin is detected similarly regardless of the genotype of the cultivar used to grow the rust. Clearly it can be produced by hyphae avirulent with respect to the Lr20 gene and growing in cultivars lacking this gene. The death of a few cells on the surface of the exposed mesophyll of all transplants would seem to be a general response to the removal of the epidermis. The build-up of deposits on the walls of other cells on the surface of the mesophyll was presumably a reaction to metabolites emanating from infected tissue during the period of contact. The implications of our experiments differ from those of other workers [3, 81 who found that necrosis of several host tissues followed exogenously-induced inhibition of established virulent hyphae. In our present experiments, marked necrosis only occurred when transplants, with or without fungicides, were held in leaves infected with hyphae avirulent to the transplant. Previously we showed that the extensive necrosis characteristic of Lr20-based hypersensitivity differs from the limited necrosis induced by inhibition of virulent hyphae [7]. We also found that in the natural sequence, detectable changes in host cells leading to the Lr20-based hypersensitivity precede a decrease in hyphal growth rate [S]. Earlier workers implicated toxins as a cause of symptoms characteristic of low infection types in interactions between Puccinia graminis f. sp. tritici and wheat. Silverman [16] investigated the cause of necrosis which occurred around developing uredia in the cv. Marquis at 21 “C, but not at 32 “C, after inoculation with race 38. From infected leaves he extracted and partially fractionated a toxin which caused chlorosis in cv. Marquis at 21 “Cl, but not at 32 “C. The toxin did not cause chlorosis in Little Club, a cultivar susceptible to race 38. Olien [13] applied electrodes to
318
D. R. Jones and 6. J. Deverall
leaves of cv. Khapli Emmer which were developing uredia of race 56 within areas of necrosis. He displaced the necrosis towards the anode and suggested that a negatively charged toxin was the cause of this symptom. The concept of a specific toxin as a product of an avirulent rust and active on a host possessing a complementary gene for resistance is consistent with the predictions made by Ellingboe [5j. He suggested that where low infection type is determined by complementary genes for avirulence and resistance in parasite and host, the specific interaction is between these genes and not their alleles for virulence and susceptibility. He implied that a product of the gene for avirulence interacts with a product of the gene for resistance, as has also been suggested by Albersheim & Anderson-Prouty [l]. In the resistance determined by the Lr20 gene, a specific toxin may cause hypersensitivity and thereby failure of the rust to grow normally perhaps because of starvation in an area of dead host cells [a. In Triticum-Puccinia interactions for low infection types unaccompanied by widespread necrosis, for example those involving the Einkorn resistance [4, 17-J controlled by the Sr21 gene [19], specific products may interact in an unknown way to cause cessation of parasite development. Ellingboe [5j contrasted these interactions with relationships such as that between Helminthosporium victoriae and oats bearing the V6 gene, where the most particular genetic interaction is for high infection type and where a host-specific toxin is implicated as a product of the virulence gene [14] and active on a hypothetical product of the susceptibility gene in the host. Other host-specific toxins of this latter type and their receptor sites have been investigated in recent years [18]. We thank Dr R. A. McIntosh for seeds of the near-isogenic lines of cv. Kenya 744, Professor I. A. Watson for the loan of growth cabinets at the Plant Breeding Institute, Castle Hill, and Mr D. Gow for photography. We also thank Mr G. K. Campbell for valuable discussion and development of procedures for leaf-transplant techniques. The senior author was supported by a University of Sydney Postdoctoral Fellowship.
1. ALBERSHEXM,P. & ~~NDRRSON-PR~~TY, A. J. (1975). Carbohydrates, proteins, cell surfaces, and the biochemistry of pathogenesis. Annual Reviewof Pkmt Physiology26,31-52. 2. ATHWAL, D. S. & WATSON, I. A. (1957). Inheritance studies on certain leafrust resistant varieties of T&icum mJgmc Vill. Proceedingsof th.t Linnean So&~ of NW South Wales 82,272-284. 3. BARNA, B., &UUC, T. & ML, S. F. (1974). Hypersensitive reaction of rust-infected wheat in compatible host-parasite relationships. Actu PhytopathologicaAcaakti ScientiarumHungaricae9,
293-300. 4. BROWN, J. F., SHIPTON, W. A. & WHITE, N. H. (1966). The relationship between hypencmitive tissue and resistance in wheat seedlin8s infected with Puminia graminis tdici. Annals of A&Vied
Biology Ss, 279-290. 5. ELLXNGBOE,A. H. (1976). Genetics of has-parasite interactions. In En&@dia of Plant Physiology Jlr,w S&s, Vol. 4, PhysiologicalPlant Pathology,Ed. by R. Heit& & P. H. Williams, pp. 761778. Springer-Verlag, Berlin and New York. 6. JONES, D. R. & DEVERALL, B. J. (1977). The effect of the Lr20 re&stance gene in wheat on the development of leaf rust, Pu&nia rccond&. PhysiologicalPlant Pathology10,275-284. 7. JONES,D. R. & DE~ERALI, B. J. (1977). Experimental manipulation of the hypersensitive response associated with the expression of the L20 gene in wheat following infection by leaf rust,
Puccbdancondita. PhysiologicalPlant Path&p M&285-290. 8. WY, Z., BARNA, B. & ~?,RSEK,T. (1972). Hypersensitivity plant resistance to infection. Nature 239,456-458. 9. LUIQ, N. H. (1977). Personal communication.
as a consequence, not the cause, of
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10. Lure, N. H. (1977). Wh,wt rurt swvcy Z97&77. The University of Sydney Plant Breeding Institute. 11. MCINTOSH, R. A. (1973). A catalogue of gene symbols for wheat. In Rocee&gs of L 4th Zaternational Wheat Gcnctics @n@siw, Ed. by E. R. Sears & L. M. S. Sears, pp. 893-937. Agricultural Experimental Station, College of Agriculture, University of Missouri. 12. MCINTOSH, R. A. (1977). Nature of induced mutations affecting disease reaction in wheat. In Induced Mutations against P&ant ZXscases.International Atomic Energy Agency, Vienna. 13. OLIEN, C. R. (1957). Electrophoretic displacement of the necrotic area from the region of mycelium development in Khapli emmer wheat infected with race 56 of Pucciniu graminti var. nitin’. Phytopathologv41, 26. (Abstr.) 14. SCHEPFER,R. P. & YODER, 0. C. (1972). Host specific toxins and selective toxicity. In Phytotoxins in Plant Diseases, Ed. by R. K. S. Wood, A. Ballio & A. Graniti, pp. 251-272. Academic Press, London and New York. 15. SHARP, E. L. & EYGE, R. G. (1958). A “tissue transplant” technique for obtaining abundant sporulation of races of Puccinia graminis var. tritici on resistant varieties. Phytupathologv 48, 696-697. 16. SILVERMAN, W. (1960). A toxin extracted from Marquis wheat infected by race 38 of the stem rust fimgus. Phyt+thology 50, 130-136. 17. STAKMAN, E. C. (1914). A study in cereal rusts: physiological races. Bulktin of the Minncsotu Agricultural Expsrbncntal Station, No. 138. 18. STROBEL, G. A. (1976). Toxins of plant pathogenic bacteria and fungi. In Biochemical Aspects of Plant-Pumasite l?&tionshi&, Ed. by J. Friend & D. R. Threlfall, pp. 135-139. Academic Press, London and New York. 19. THE, T. T. (1973). Chromosome location of genes conditioning stem rust resistance transferred from diploid to hexaploid wheat. Nature .New Biology 241, 256. 20. WATSON, I. A. & LIJIO, N. H. (1961). Leaf rust on wheat in Australia: a systematic scheme for the classification of strains. Proceedingsof the Linncan Sock(y of New South Waks 86,241-250.
The use of leaf transplants to study the cause of hypersensitivity to leaf rust, Puccinia recondifa, in wheat carrying the Lr20 gene D. R. JoNEst and B. J. DEVERALL Depormzmtof Plant Patho@ and Agricultural EntmoLogy, Uniocrsity of S’dncy, N.S. W. 2006, Australia (Accepedfor jwblication December1977)
Mycelia of virulent and normally avirulent races of leaf rust were established in cultivars carrying the temperature-sensitive Lr20 gene for resistance by incubating inoculated seedlings for 1 day at 20.5 “C and 3 days at 30.5 “C. Inoculated medhngs lacking this gene were incubated for 4 days at 20.5 “C. Epidermis-free transplants fiom uninfected fimgicide-fed leaves were placed under the lower epidermis of infected leaves and the two mesophylls held together for 21 h at 20.5 “C. Transplants were then removed, placed under the epidermis of uninfected leaves and exam&d for necrosis after a further 2 days at 20.5 “C. Widespread necrosis, seen to be free of hyphae, only occurred in transplants carryiq the Lr20 gene which had been held in leaves infected with avirulent mycelia. The genotype of the infected leaves did not affect the necrosis. The results are dkcmsed in relation to an hypothesis about the action of an Lr20 gene-specific toxin produced by rust with the complementary avirulence gene.
INTRODUCTION
In interactions between the wheat cultivar Thew, carrying the Lr20 gene for resistance, and an avirulent race of leaf rust Puccinia reconditaf. sp. t&i, the expression of resistance was closely associated with physiological changes leading to the collapse of host protoplasts [6]. The pattern of this necrosis including its tendency to spread along veins suggested that a phytotoxin was released from infection sites to kill surrounding tissue. Circumstantial evidence for the existence of a toxin was derived from experiments utilizing the temperature sensitivity of interactions involving the Lr20 gene to establish colonies of the avirulent rust in primary leaves of cv. Thew and then administering heat shocks [7l. Widespread necrosis occurred around inhibited avirulent, but not virulent, colonies and it also had the same temperature dependency as that associated with the normal expression of the Lr20 gene. The hypothetical toxin might be either a specific product of avirulent hyphae active only on wheat cells bearing the Lr20 gene or a non-specific product released after a recognition reaction between avirulent haustoria and invaded resistant cells. The experiments reported herein were designed firstly to detect the diffusion of a toxin from the exposed mesophyll of infected leaves of cv. Thew into the exposed t Present address: Plant Pathology Branch, Department Indooroopilly, Brisbane, Queensland 4068, Australia. 0048-4059/78/0501-0311 22
$01.00/O
of Primary
Industries,
Meiers Road,
@ 1978 Academic Press Inc. (London)
Limited
D. R. Jones and B. J. Deverall
312
mesophyll of transplants from uninfected leaves, using a procedure modified from that of Sharp & Emge [15]. Secondly, the source and specificity of the toxin were determined by comparison of the effects of several genotypes of host and transplant cultivars, including near-isogenic lines differing in presence and absence of the Lr20 gene. In the course of these experiments, there was a possibility for hyphae to grow into the transplant from the infected leaf, but this was minimized by incorporating a systemic fungicide, active against leaf rust, in the transplant cultivar. The transplant was held in the inoculated leaf for a pre-determined period and then transferred to an uninfected fungicide-treated leaf for a longer period. The transplant was then examined for necrosis and hyphae. MATERIALS
AND
METHODS
Wheat cultivars and leaf rust isolates Triticum aestivum L. cultivars Brevit (Sydney University Act. no. W972) carrying the Lr2c gene, Chinese Spring (W1806) carrying an undesignated gene for mature plant resistance [2], Kenya 744 (W744) carrying the Lr20 gene, Norka (W578) carrying the Lrl and Lr20 genes, Spica (W2341) carrying the Lrlla gene, Thew (W203) carrying the Lr20 gene, and two near-isogenic lines derived by Dr R. A. McIntosh [12] from Kenya 744 and differing in the presence or absence of the Lr20 gene were selected for this work [II]. Puccinia recondita Rob. & Desm. f. sp. tritici Eriks. & Henn. isolates 76-Anz-0 (Sydney University Act. no. SSSSS), 76-Anz-2,3 (70202)) 104-Anz-2,3,GT (74408)) 104~Anz-2,3,GTS (76694) and 122-Anz-2,3,4 (76348) avirulent on wheat cultivars containing the Lr20 gene and isolates 64-Anz-1,2,3 (70638), 104~Anz-1,2,3,GT (74606), 135-Anz-1,2,3,4,5 (64-L-3) and 162~Anz-1,2 (62408) virulent with respect to the Lr20 gene were used in this study [lo, 201. Avirulent isolates cause a ; or ;l infection type on cv. Thew and virulent isolates a 3+ infection type [9]. Isolate 104-Anz-2,3,GT causes a ; or ;l infection type on cvs Norka and Kenya 744 and a 3+ infection type on cvs Brevit, Chinese Spring and Spica [S, 91. Uredospores were stored in liquid nitrogen before use. Seedling treatmnzts Seedlings were grown and inoculated as previously described [6J and then incubated at 20.5 “C under incandescent and cool-white fluorescent light (12 h photoperiod, 20 000 Ix at soil surface) for 1 day. Colonies of races, avirulent with respect to the Lr20 gene, were established in leaves of cultivars carrying this gene by incubating seedlings for a further 3 days at 30.5 “C [7], a temperature which permits rust development [6]. Seedlings of cv. Thew infected with virulent races were similarly treated although this was not necessary to establish colonies. Seedlings of cultivars lacking the Lr20 gene were infected with race 104~Anz-2,3,GT and incubated at 20.5 “C for a further 3 days. Pots containing uninfected seedlings that were used as a source of transplants and as the final recipients of transplants were placed in a 0.035 g/l solution of a systemic fungicide (4butyl-4H-1,2,4triazole, RH 124, from Rohm & Haas Australia Pty.
Leaf rust in wheat
313
Ltd) 4 days after sowing. Transplants were excised from primary leaves 7 days after sowing. baf-transplant technique The positions of rust colonies in leaves were indicated by faint chlorotic spots 4 days after inoculation. Areas of primary leaves showing moderate to heavy infection densities (70 to 140 colonies/cm2) were selected as recipients for transplants. The transplant procedure was derived from the method of Sharp & Emge [15]. An infected leaf was placed between two cover slips held in a perspex block and cut transversely with a razor blade. Only the upper epidermis and mesophyll were severed as the coverslips prevented the blade from cutting completely through the leaf. Bending the leaf backwards, the lower epidermis was peeled back to expose infected mesophyll. The lower epidermis was removed from an uninfected primary leaf of a cultivar selected as the transplant donor in the same way as above. A 7 to 8 m m section of transplant was then excised and placed on the infected leaf so that the exposed mesophylls of both were in contact with each other. The lower epidermis of the infected leaf was finally placed in its original position covering the transplant. Seedlings with transplants were incubated at 20.5 “C in continuous light (20 000 lx) for 21 h ( f 0.5 h). Each transplant was then removed from the infected leaf and placed under the epidermis of an uninfected leaf of a seedling of the same cultivar as the transplant. After a further 2 days at 20.5 “C under continuous light, segments of leaves containing transplants were cut from seedlings, fixed, cleared and stained with trypan blue in alcoholic lactophenol and de-stained in chloral hydrate solution as previously described [6-j. Transplants were teased from the leaf segments and mounted in 50% glycerine. Assessnzentof necrosis Microscopic observations were limited to 20 to 32 randomly selected transplants for every rust-host-transplant combination. Wheat cells normally stained a uniform pale blue colour [6J. Cells with intensely stained and often collapsed protoplasts were considered to be dying or dead [q. The extent and distribution of this staining in each transplant was assessed visually and rated on a scale from 1 to 4 (Fig. 1). Necrotic cells associated with wounds made by forceps during handling were readily distinguished by their position and appearance and were disregarded in the rating. RESULTS
In early experiments, seedlings used as a source of transplants were not treated with fungicide and the transplants were held in the infected leaves for 2 days before examination. Necrosis occurred in these transplants in the same way as described below, but a high proportion of the necrotic areas were seen to contain small mycelia. However, the large areas of necrosis greatly exceeded the areas occupied by the hyphae. The modifications of feeding fungicide and of holding transplants in infected leaves for only 21 h were introduced to decrease the changes of hyphal growth into the transplants.
314
D. R. Jones and B. J. Deverall
Efect of rust rate on necrosisin tran.$lants Transplants from cv. Thew were put into leaves of cv. Thew infected with avirulent (76-Anz-0, 76-Anz-2,3, 104~Anx-2,3,GTS, 122-Am+2,3,4) and virulent (64~Anx1,2,3, 104~Anz-1,2,3,GT, 135-Anz-1,2,3,4,5, 162~Anx-1,2) races of leaf rust and also
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Fro. 1. Diagrammatic representation of level9 of necrosis in transplants after contact for 2 1 h and uninkcted tissue for 2 days. Rating : 1, with leaf tissue infected with P. recondika scattered and small groups of dead cells in the surface cell layer of the exposed meaophyll; 2, groups of dead cells extending from the surface of the exposed mcsophyll to the upper epidermis-up to 20% of the surface area necrotic; 3, larger groups of dead cells-between 20 and 40% of the surFace area necrotic; 4, very large groups of dead cells-over 40% of the surface arca necrotic.
in uninfected leaves. At the time of the removal of transplants after 21 h contact, necrotic areas were visible to the unaided eye around colonies of the avirulent rusts in the infected leaves. Microscopic examination of a few representative transplants in contact with infected tissue showed scattered dead cells on the surface of the exposed mesophyll and a few groups of cells with thickened walls caused by extracellular deposits. Areas of tissue that stained deeper than usual were noticeable in transplants that had been in contact with mesophyll infected with avirulent races. After incubation of transplants in infected leaves of cv. Thew for a further 2 days, scattered and small groups of necrotic cells were observed on the surface of exposed mesophyll of all transplants. Groups of living cells, some with translucent, wart-like deposits on their walls and others with heavily stained, extracellular deposits, were seen on the exposed mesophyll of transplants that had been in contact with infected leaf tissue (Plate 1). Large groups of necrotic cells were found in most transplants that had been in contact with mesophyll infected with avirulent races of rust (Plate 2). The necrosis in the transplants usually extended from the surface of the exposed mesophyll to the upper epidermis. Often the layer of surface cells appeared normal or had densely stained walls and the necrosis began in the second layer. A closer examination revealed that small hyphal segments, usually devoid of cytoplasm and
PLATES 1 to 4. Surface of exposed mesophyll of transplants in contact with leaf tissue infected with P. recondita for 21 h and uninfected tissue for 2 days. PLATE 1. Living cells of cv. Thew showing heavily stained, wall deposits after contact with cv. Thew infected with race 64-Am-1,2,3, virulent with respect to the Lr20 gene. x 176. PLATE 2. Necrosis (rating 2) in cv. Thew after contact with cv. Thew infected with race 76-Am-O, avirulent with respect to the Lr20 gene. Small fragments of hyphae lacking cytoplasm and haustoria were found on the surface of 500,h of the necrotic areas. x 27.
[facing page 3141
PLATE 3. Absence of extensive necrosis (rating: 1) in cv. Thew after contact with cv. Thew infected with race 64-Anz-1,2,3, virulent with respect to the Lr20 gene. A few small fragments of hyphae lacking cytoplasm and haustoria w-cre found on the surface of the transplant. Arrows indicate areas of living cells with extracellular deposits. x 26. PLATE 4. Necrotic area in cv. Kenya with race 104~Anz-2,3,GT, avirulent with 220 pm in length and lacking cytoplasm necrotic area. Arrows indicate the margin length and 700 pm wide. x 45.
744 after contact with cv. Chinese Spring infected respect to the Lr20 gene. A branched hypha about and haustoria was found on the surface of this of the necrotic area, which was about 2300 pm in
Leaf rust in wheat
315
lacking haustoria, were present on the surface of about 50% of the necrotic areas, distributed through many transplants. No hyphal fragments could be found on the necrotic areas in about 15% of transplants. No large areas of necrotic cells were seen in transplants that had been in contact with mesophyll infected with virulent rusts (Plate 3). Hyphae and sometimes haustoria were seen in a few places on the surface cells of about 75% of these transplants. The rating of necrosis in each transplant examined is presented in Table 1. TABLE 1 Effect of rDccof rust a nmosis in
Rust race infecting cv. Thew 64-Anz-1,2,3 (v) b 76-Anz-0 (a)” 76-Anz-2,3 (a) 104-k~z-1,2,3,GT (v) 104-Anz-2,3,GTS (a) 122~Anz-2,3,4 (a) 135-Anz-1,2,3,4,5 (v) 162~Anz-1,2 (v) Uninfected
I 20 2 2: 2 2: 20 20
tran$lant
Necrosis in 20 transplants from cv. Thew Ratinga 2 3 0 13 11 0 11 12 0 0 0
0 4 4 0 5 2 0 0 0
4 0 1 2 0 2 1 0 0 0
“SeeFig. 1. b Virulent with respect to the Lr20 gene, but carrying other avirulence genes. e Avirulent with respect to the ~920 gene.
Efect of host and tranrplant gem&be on necrosisin tranrp1ant.s Transplants from cvs Brevit, Chinese Spring, Kenya 744, No&a, Spica and Thew were put into leaves of each cultivar infected with the rust 104~Anz-2,3,GT (avirulent with respect to the Lr20 gene). At the end of each experiment, scattered, necrotic cells and groups of living cells with densely stained wall deposits were seen on the mesophyll surface of most transplants. Large areas of necrosis were observed in most transplants bearing the Lr20 gene (Plate 4). The presence or absence of the Lr20 gene in the infected leaves did not influence the necrosis in the transplant (Table 2). Hyphal fragments, lacking haustoria and usually cytoplasm, were associated with the surface cells of about 50% of the necrotic areas. Effect of near-isogenictransplant lines on necrosis Transplants from two near-isogenic lines of wheat derived from cv. Kenya 744 and differing in the presence or absence of the Lr20 gene were put into cv. Thew infected with avirulent race 104Anz-2,3,GT. Scattered necrotic cells and groups of living cells with stained wall deposits were observed on the surface of the exposed mesophyll of transplants of both lines. Large areas of necrotic tissue, however, were only seen in transplants bearing the Lr20 gene (Table 3). As before, empty hnphal segments were seen on the surface cells of about 50% of the necrotic areas.
D. R. Jones and B. J. Deverall
316 TABLE 2
Effid of host and transplant genotvp on m~osis in tr+&nt Host CultivaP Brevit (Lrzc)e
c. spring (MPR)”
Norka (Lrl, Lr20)
Transplant CUltiVar
1
Necrosis in 20 transplants Ratingb 2 3
Brcvit c. spring Kenya 744 Norka Spica Thew
20 20 4
0 0 12
2: 0
11 0 9
Brevit c. spring Kenya 744 No&a Spica
20 20 1
0 0
ThCW
2: 2
1; 0 7
Brevit c. spring Kenya 744 Norka Spica Thew
20 20 5 0 20 4
0 0 7 10 0 9
Brevit C. Spring Kenya 744 No&a Spica The-w
20 20 1 0 20 5
0
Brevit C. Spring Kenya 744 Norka Spica Thew
20 20 5 1 20 1
Brevit c. spring Kenya 744 No&a Spica Thew
20 20 5 2; 3
4
1: 1 1:
0 0 5 6 0 4
0 0 3 13 0 0
0 0 9 9 19 0 0 9 7 0 9
o Infected with race 104Am2,3,GT (avirulent with respect to the Lr20 gene). b See Fig. 1. e Resistance gene carried by cultivar. d Undesignated gene for mature plant resistance.
DISCUSSION These results show that necrosis, characteristic of the expression of the Lr20 gene [S, 71, was spccifklly caused in certain batches of fungicide-fed transplants which had been exposed for only 21 h to certain inoculated leaves. This necrosis was caused by a stimulus from the inoculated leaves. The stimulus was not caused by
311
Leaf rust in wheat
infection of the transplants because extensive necrosis often occurred at sites in transplants where no hyphae had grown. At those sites where hyphae had crossed from the inoculated leaves, the area and depth of necrosis greatly exceeded the part of the tissue contacted by the superficial hyphae. The occurrence and extent of the necrosis were consistent with the diffusion of a toxin. The toxin is seen to be specific in its action on wheat cultivars and lines bearing the Lr20 gene. It does not cause necrosis in cultivars lacking this gene and bearing TABLE 3 Effect of near-isogmk tmq’ht Transplant line”
1
lr20 gene Lr20 gene
32 7
lines on necroti
Necrosis in 32 transplants Ratingb 2 3 :
0 10
4 0 7
o Derived from cv. Kenya 744 and placed in leaves of cv. Thew infected with race 104Anz-2,3,GT (avirulent with respect to the Lr20 gene). bSeeFig. 1.
other genes for resistance against leaf rust. The toxin is suggested to react with a product of the Lr20 gene and thereby to cause necrosis. The specific toxin is also seen to be a product of avirulent hyphae and not of infected wheat cells bearing the Lr20 gene. This is because the toxin is detected similarly regardless of the genotype of the cultivar used to grow the rust. Clearly it can be produced by hyphae avirulent with respect to the Lr20 gene and growing in cultivars lacking this gene. The death of a few cells on the surface of the exposed mesophyll of all transplants would seem to be a general response to the removal of the epidermis. The build-up of deposits on the walls of other cells on the surface of the mesophyll was presumably a reaction to metabolites emanating from infected tissue during the period of contact. The implications of our experiments differ from those of other workers [3, 81 who found that necrosis of several host tissues followed exogenously-induced inhibition of established virulent hyphae. In our present experiments, marked necrosis only occurred when transplants, with or without fungicides, were held in leaves infected with hyphae avirulent to the transplant. Previously we showed that the extensive necrosis characteristic of Lr20-based hypersensitivity differs from the limited necrosis induced by inhibition of virulent hyphae [7]. We also found that in the natural sequence, detectable changes in host cells leading to the Lr20-based hypersensitivity precede a decrease in hyphal growth rate [S]. Earlier workers implicated toxins as a cause of symptoms characteristic of low infection types in interactions between Puccinia graminis f. sp. tritici and wheat. Silverman [16] investigated the cause of necrosis which occurred around developing uredia in the cv. Marquis at 21 “C, but not at 32 “C, after inoculation with race 38. From infected leaves he extracted and partially fractionated a toxin which caused chlorosis in cv. Marquis at 21 “Cl, but not at 32 “C. The toxin did not cause chlorosis in Little Club, a cultivar susceptible to race 38. Olien [13] applied electrodes to
318
D. R. Jones and 6. J. Deverall
leaves of cv. Khapli Emmer which were developing uredia of race 56 within areas of necrosis. He displaced the necrosis towards the anode and suggested that a negatively charged toxin was the cause of this symptom. The concept of a specific toxin as a product of an avirulent rust and active on a host possessing a complementary gene for resistance is consistent with the predictions made by Ellingboe [5j. He suggested that where low infection type is determined by complementary genes for avirulence and resistance in parasite and host, the specific interaction is between these genes and not their alleles for virulence and susceptibility. He implied that a product of the gene for avirulence interacts with a product of the gene for resistance, as has also been suggested by Albersheim & Anderson-Prouty [l]. In the resistance determined by the Lr20 gene, a specific toxin may cause hypersensitivity and thereby failure of the rust to grow normally perhaps because of starvation in an area of dead host cells [a. In Triticum-Puccinia interactions for low infection types unaccompanied by widespread necrosis, for example those involving the Einkorn resistance [4, 17-J controlled by the Sr21 gene [19], specific products may interact in an unknown way to cause cessation of parasite development. Ellingboe [5j contrasted these interactions with relationships such as that between Helminthosporium victoriae and oats bearing the V6 gene, where the most particular genetic interaction is for high infection type and where a host-specific toxin is implicated as a product of the virulence gene [14] and active on a hypothetical product of the susceptibility gene in the host. Other host-specific toxins of this latter type and their receptor sites have been investigated in recent years [18]. We thank Dr R. A. McIntosh for seeds of the near-isogenic lines of cv. Kenya 744, Professor I. A. Watson for the loan of growth cabinets at the Plant Breeding Institute, Castle Hill, and Mr D. Gow for photography. We also thank Mr G. K. Campbell for valuable discussion and development of procedures for leaf-transplant techniques. The senior author was supported by a University of Sydney Postdoctoral Fellowship.
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293-300. 4. BROWN, J. F., SHIPTON, W. A. & WHITE, N. H. (1966). The relationship between hypencmitive tissue and resistance in wheat seedlin8s infected with Puminia graminis tdici. Annals of A&Vied
Biology Ss, 279-290. 5. ELLXNGBOE,A. H. (1976). Genetics of has-parasite interactions. In En&@dia of Plant Physiology Jlr,w S&s, Vol. 4, PhysiologicalPlant Pathology,Ed. by R. Heit& & P. H. Williams, pp. 761778. Springer-Verlag, Berlin and New York. 6. JONES, D. R. & DEVERALL, B. J. (1977). The effect of the Lr20 re&stance gene in wheat on the development of leaf rust, Pu&nia rccond&. PhysiologicalPlant Pathology10,275-284. 7. JONES,D. R. & DE~ERALI, B. J. (1977). Experimental manipulation of the hypersensitive response associated with the expression of the L20 gene in wheat following infection by leaf rust,
Puccbdancondita. PhysiologicalPlant Path&p M&285-290. 8. WY, Z., BARNA, B. & ~?,RSEK,T. (1972). Hypersensitivity plant resistance to infection. Nature 239,456-458. 9. LUIQ, N. H. (1977). Personal communication.
as a consequence, not the cause, of
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10. Lure, N. H. (1977). Wh,wt rurt swvcy Z97&77. The University of Sydney Plant Breeding Institute. 11. MCINTOSH, R. A. (1973). A catalogue of gene symbols for wheat. In Rocee&gs of L 4th Zaternational Wheat Gcnctics @n@siw, Ed. by E. R. Sears & L. M. S. Sears, pp. 893-937. Agricultural Experimental Station, College of Agriculture, University of Missouri. 12. MCINTOSH, R. A. (1977). Nature of induced mutations affecting disease reaction in wheat. In Induced Mutations against P&ant ZXscases.International Atomic Energy Agency, Vienna. 13. OLIEN, C. R. (1957). Electrophoretic displacement of the necrotic area from the region of mycelium development in Khapli emmer wheat infected with race 56 of Pucciniu graminti var. nitin’. Phytopathologv41, 26. (Abstr.) 14. SCHEPFER,R. P. & YODER, 0. C. (1972). Host specific toxins and selective toxicity. In Phytotoxins in Plant Diseases, Ed. by R. K. S. Wood, A. Ballio & A. Graniti, pp. 251-272. Academic Press, London and New York. 15. SHARP, E. L. & EYGE, R. G. (1958). A “tissue transplant” technique for obtaining abundant sporulation of races of Puccinia graminis var. tritici on resistant varieties. Phytupathologv 48, 696-697. 16. SILVERMAN, W. (1960). A toxin extracted from Marquis wheat infected by race 38 of the stem rust fimgus. Phyt+thology 50, 130-136. 17. STAKMAN, E. C. (1914). A study in cereal rusts: physiological races. Bulktin of the Minncsotu Agricultural Expsrbncntal Station, No. 138. 18. STROBEL, G. A. (1976). Toxins of plant pathogenic bacteria and fungi. In Biochemical Aspects of Plant-Pumasite l?&tionshi&, Ed. by J. Friend & D. R. Threlfall, pp. 135-139. Academic Press, London and New York. 19. THE, T. T. (1973). Chromosome location of genes conditioning stem rust resistance transferred from diploid to hexaploid wheat. Nature .New Biology 241, 256. 20. WATSON, I. A. & LIJIO, N. H. (1961). Leaf rust on wheat in Australia: a systematic scheme for the classification of strains. Proceedingsof the Linncan Sock(y of New South Waks 86,241-250.