Physiological and Molecular Plant Pathology (1990) 36, 95-108

95

Resistance of wheat to Puccinia graminis f.sp . tritici :Histological investigation of resistance caused by theSr5 gene

R. TIBURZY,t U . NOLL and H. J . REISENERInstitut für Biologie III (Pflanzenphysiologie), Rheinisch-Westfälische Technische Hochschule Aachen, Worringer Weg,D-5100 Aachen, Federal Republic of Germany

(Accepted for publication March 1989)

Seedlings of six wheat (Triticum aestivum L .) lines containing the Sr5 gene for resistance to wheatstem rust were inoculated with urediospores of Puccinia graminis Esp. tritici, race 32, which carriesthe P5 gene for avirulence, and primary leaves were examined for the temporal relation betweenthe hypersensitive reaction (HR) and fungal growth. In up to 95 % of all infection sites the firsthost cell that was penetrated by a fungal haustorium was an epidermal cell . Infected epidermaland mesophyll cells containing the Sr5 gene reacted hypersensitively with an accumulation ofyellow autofluorescing compounds . Fungal hyphae that were associated with intensely auto-fluorescing epidermal cells developed not more than one, two, or three haustorial mother cells(HMCs) . Significant inhibition of fungal growth in Prelude-Sr5 occurred after formation of thefirst haustorium, at a time when the number of infection sites with a necrotic epidermal cellrapidly increased from 11 to 36 . 5 %. The time interval between formation of the first haustoriumin epidermal cells and appearance of the HR of the first-invaded epidermal cells varied from 4 . 6to 8 . 1 h, depending on the wheat line used . In wheat cvs . Napayo (Sr5, 6, 7a, 11) and Manitou(Sr5, 6, 7a, 9g, 12) more epidermal cells relative to Prelude-Sr5 reacted hypersensitively indicatingthat the expression of the Sr5 gene is modified by additional resistance genes in these cultivars . Thedata demonstrate that resistance controlled by the Sr5 gene is closely associated with the HR ofinfected epidermal cells .

INTRODUCTION

Infection of wheat carrying the Sr5 gene for resistance to wheat stem rust with a raceof Puccinia graminis Esp . tritici (P.g.t .) containing the complementary P5 gene foravirulence results in a type O infection type ; that is, no sign of disease or only

t To whom correspondence should be addressed .Abbreviations used in text : DIMBOA, dihydroxymethoxybenzoxazolinone ; HMC, haustorial mother

cell ; HR, hypersensitive reaction ; P.g.t ., Puccinia graminis Esp. tritici.

0885-5765/90/020095+14 $03 .00/0

© 1990 Academic Press Limited4

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involvement of the latter host cells in resistance conditioned by the Sr5 gene. Beardmoreet al . [2] investigated cellular lignification as a possible factor in the hypersensitiveresistance conditioned by the Sr5 gene. Again, there was no information on theoccurrence of reactions of infected epidermal cells . Skipp et al. [24] found that the hostcell penetrated by the first haustorium of P.g .t . was an epidermal cell at almost 50 per-cent of all infection sites, and Rohringer et al . [23], investigating the Sr5/P5 geneinteraction using near-isogenic wheat lines, demonstrated that penetrated epidermalcells express incompatibility similar to penetrated mesophyll . Incompatibility isindicated by conspicuous yellow autofluorescence, which apparently results froman accumulation of lignin or lignin-like compounds in these cells [2, 20, 25-27] .Autofluorescing wheat cells in the Sr5/P5 interaction are necrotic [23], a conclusionthat has been supported by ultrastructural investigations [11] . The data obtained byRohringer and co-workers show that linear growth of fungal colonies which wereassociated with fluorescing epidermal cells did not significantly increase after 24 hfollowing invasion of the leaves . The occurrence of inhibited necrosis-free colonies,however, led the authors to suggest that inhibition of fungal growth in this system is notcorrelated with host cell necrosis . On the other hand, hypersensitivity conditioned bythe Sr5 gene was seen to be closely associated with resistance and has been describedas `determinant hypersensitivity' emphasizing that fungal growth comes to a completehalt by this response [3] . Eventually, differences in infection types, as found betweenthose resulting from the Sr5/P5 and Sr6/P6 gene interactions, may be an expression ofdifferences in reactivity of the epidermal cells [11] .

From these reports it is difficult to conclude whether the hypersensitive response ofepidermal cells is of any relevance in the Sr5/P5 gene interaction . More detailedinformation appears to be necessary to clarify this point . This paper describes theSr5/P5 gene interaction focussing on the histological characterization of thehypersensitive response, time of its appearance relative to haustorium formation and toinhibition of fungal growth .

A preliminary report of some of the results has been published [20] .

MATERIALS AND METHODS

Plant materials and inoculationThe wheat (Triticum aestivum L .) lines used throughout the experiments were : the near-isogenic pair Reliance x Prelude'/ Prelude (Sr5/sr5), subsequently referred to as Prel-Sr5 and Prel-sr5, M .M.E. x Prelude' (Sr6), cv . Manitou (Sr5, 6, 7a, 9g, 12), cv. Napayo(Sr5, 6, 7a, 11), cv . Feldkrone (Sr5, 31), cv. Neepawa (Sr5, 6, 7a), cv. 417/65 (Sr5, ?), 1-

4-3Na101 x Mq' (Sr7a), cv. Eagle (Sr9g, 26), C . 1. 14171 Ch . Spring x Timstein (Sr11),

BtSrl2Tc (Sr12) . Seedlings were grown under controlled conditions (22 °C, 16 h photo-period, 18000 to 20000 lx, 60% RH) were inoculated with urediospores of Puccinia

graminis (Pers .) Esp . tritici Eriks. & E. Henn., race 32, which contains, among othergenes for avirulence, the P5 gene. The spores were suspended in Freon 113, andsprayed onto 7-day-old plants . After inoculation, the plants were kept in moistchambers for 24 h with an initial dark period of 16 h and then grown under conditionsas described above, with the exception of wheat cvs . Feldkrone, Neepawa and 417/65,which were grown at 20 °C .

Resistance of wheat to stem rust : histology

97Determination offungal growthRust infected primary leaves were collected at 24 h intervals after inoculation over aperiod of 6 days . Leaves were cut into pieces of 1 . 5 cm length, fixed, and stained withthe optical brightener Uvitex 2B (Ciba Geigy AG, Basel, Switzerland) as described byRohringer et al . [22] . Samples were observed with a Zeiss Universal Microscope withepifluorescence equipment (HBO 50, KG 1, BG 38, BP 390-440, FT 460, LP 470) .

At each sampling, about 170 infection sites were examined for three parameters : (1)colony size as estimated by determining the colony diameter parallel to the long axisof the leaf; (2) number of haustorial mother cells (HMCs) ; (3) occurrence of necrosisin infected host cells . Measurements were taken using a calibrated eyepiece micrometer .

In further experiments infected leaves of Prel-Sr5 and Prel-sr5 were collected at 2 hintervals from 20 h to 40 h post-inoculation (p .i .) and stained as described above . Themycelium at about 200 infection sites was evaluated by determining the size of theintercellular fungal structures (substomatal vesicles, infection hyphae including allbranches, HMCs) ; the occurrence of necrosis in penetrated host cells was also recorded .A similar approach was used to determine the time interval from penetration of thehost cell by the first haustorium to the appearance of a visible host cell reaction inwheat cvs . Neepawa, Feldkrone and 417/65 . Colonies deriving from doubly penetratedstomata and merged colonies were excluded . The one-sided Kolmogorov-Smirnovtwo-sample test [4] was used to compare colony size statistically .

RESULTS

Infection typesInfection types recorded 13 days p .i . as described by Stakman et al. (1962 : in [21]) wereas follows : Prel-sr5: 23, Prel-Sr5 : 0 ;, cv . Manitou (Sr5, 6, 7a, 9g, 12) : 0, cv. Napayo (Sr5,6, 7a, 11) : 0, cv . Feldkrone (Sr5, 31) : 0, cv. Neepawa (Sr5, 6, 7a) : 0, cv. 417/65 (Sr5,?) : 0, M.M.E. x Prelude' (Sr6) at 18 °C : 0 ;, 1-4-3Nal01 x Mq' (Sr7a) : 34, cv. Eagle(Sr9g, 26) : 12C, C .I. 14171 Ch. Spring x Timstein (Sr11) : 1 C, BtSrl2Tc (Sr12) : 4 .

Fluorescence microscopyTreatment of rust infected leaf pieces with Uvitex 2B resulted in a bright bluefluorescence of the intercellular fungal structures . HMCs differed from hyphal cells bya more intense fluorescence . Haustoria in susceptible wheat cells usually were notstained unless the samples were treated as described by Kuck et al. [14] . Epidermal andmesophyll cells with a characteristic yellow autofluorescence [23] were observed inresponse to penetration by fungal haustoria in the Sr5/P5 gene interaction . Haustoriain autofluorescing necrotic wheat cells did not stain with the fluorochrome, althoughthey were visible as yellow autofluorescing structures . Although there was a distinctvariation in fluorescence intensities of individual fluorescing epidermal cells of the near-isogenic wheat line Prel-Sr5, necrotic epidermal cells of cvs . Feldkrone, 417/65,Manitou, Napayo and Neepawa fluoresced more intensely with less variation betweenindividual cells .

Growth of rust mycelium in Prelude-Sr5 and Prelude-sr5During the 16 h dark period after inoculation, urediospores germinated on the surfaceof the wheat leaves and germ tubes grew perpendicularly to the long axis of the leaf .

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FIG . 1 . Infection structures of P. graminis Esp. tritici. A = appressorium, E = epidermal cell,G = guard cell, GT = germ tube, H -- haustorium, HMC = haustorium mother cell, IH =infection hypha, SV = substomatal vesicle .

Appressoria were formed above stomata, but leaves were not entered by the fungusuntil penetration was induced by light . Then the substomatal cavities were colonizedsimultaneously by the fungal individuals . Substomatal infection structures wereformed, each consisting of a bipolar substomatal vesicle and a primary HMC . Theprimary HMC was larger than those formed later and occasionally was separated fromthe substomatal vesicle by a short infection hypha . Together the substomatal vesicleand the first HMC formed a straight cigar-shaped body, which was closely attached tothe underside of the guard cells, following their course parallel to the long axis of theleaf (Fig. 1) . The epidermal cell next to the ends of the guard cells of the samelongitudinal cell row was penetrated by the first haustorium . Sometimes a secondHMC was formed at the opposite end of the substomatal vesicle . An infection hyphaarose near the septum which separates the first HMC from the substomatal vesicle .

Incompatibility in the Sr5/P5 gene interaction was expressed by the appearance ofa characteristic yellow autofluorescence of the infected epidermal and mesophyll cells .Haustoria in intensely autofluorescing epidermal cells were visible as yellowautofluorescing spherical structures with a diameter of about 4 gm . Haustoria inautofluorescing mesophyll cells had the same size and form. However, they could notbe detected in most of these cells since the mesophyll cells collapsed and becameirregularly shaped, or the haustoria could not be distinguished from chloroplasts sincethey were similar in size and were usually displaced from their HMCs . Infectedepidermal cells in Prel-sr5 did not react hypersensitively and haustoria in these cellswere found to be bean-shaped or branched with sizes several times larger than thosein necrotic cells. Although there were a few autofluorescing mesophyll cells at infectionsites in the susceptible line, they were a rare exception and appeared primarily in hosttissue colonized by large colonies .

Development of wheat stem rust in Prel-Sr5 and Prel-sr5 is shown in Figs. 2 and 3 .No differences in mean colony size between both lines were detected at 24 h and48 h p .i. Beginning at the third day p.i ., however, growth of hyphae and formationof HMCs in the resistant line more or less ceased whereas the size of the fungal coloniesin the susceptible line increased steadily .

Resistance of wheat to stem rust : histology

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Ftc . 2 . Growth of P. graminis f.sp . tritici, race 32, in (a) the resistant isogenic wheat line Prelude-Sr5 and (b) the susceptible background line Prelude-sr5. Colony sizes were determined bymeasuring their diameters parallel to the longitudinal axis of the leaves .

Within the observation period of 6 days a distribution of the rust population into twodistinct groups of colonies became apparent (Fig . 3) . The first group consisted ofcolonies with only one to three HMCs and the second group consisted of colonies withmore than five HMCs . The majority of the colonies with one to three HMCs wereassociated with intensely fluorescing epidermal cells . Haustoria in these cells remainedsmall and spherical . Fluorescence of infected mesophyll cells occurred in about one-third of these infection sites. Most of the colonies with more than five HMCs wereassociated with fluorescing mesophyll cells and no or only faint fluorescence in infectedepidermal cells . Haustoria in weakly fluorescing epidermal cells were intermediate insize and form between those in intensely fluorescing necrotic epidermal cells and thosein non-fluorescing compatible host cells .

In order to obtain a higher resolution of the time-course of fungal development inboth lines, we investigated development at 2 h intervals from 20 h p .i . to 40 h p .i . (Fig .4) . No significant differences in the size of intercellular fungal structures in either linewere found at 24, 26, 28, and 30 h p .i . Beginning at 32 h p .i . inhibition of fungal growthin Prel-Sr5 compared with that in the susceptible line became apparent . Up to 26 h p .i .only 2-5 % of all infection sites in Prel-Sr5 were associated with autofluorescingepidermal cells (Fig . 4) . This value increased slightly to 11 % during the next 4 h, thenincreased rapidly to 36 . 5 % during the following 2 h (32 to 34 h p .i .) . From this timethe number of infection sites with an intensely autofluorescing epidermal cell increasedat a lower rate and reached about 50 % at 40 h p .i . The number of infection sites withan autofluorescing necrotic mesophyll cell in Prel-Sr5 did not exceed 3 % during theentire observation period (Table 4) . No fluorescence of epidermal or mesophyll cellsoccurred at 20 h p .i . to 40 h p .i . in the susceptible line Prel-sr5 .

Growth of rust mycelium in wheat cvs Manitou and NapayoRust development in wheat cvs Napayo (Sr5, 6, 7a, 11) and Manitou (Sr5, 6, 7a, 9g, 12)was investigated in order to determine the extent of fungal growth in wheat which

0 1 6 7

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R. Tiburzy et al.

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2

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WA

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Time after inoculation (days)

FIG . 3 . Development of P. graminis f.sp . tritici in the susceptible wheat line Prelude-sr5, in theresistant isogenic wheat line Prelude-Sr5 and in wheat cvs Manitou (Sr5, 6, 7a, 9g, 12) and Napayo(Sr5, 6, 7a, 11) from 1-6 days after inoculation . The size of the colonies is characterized by thenumber of HMC . The percentage of infection sites associated with an intensely fluorescingnecrotic epidermal cells, 0, intensely fluorescing necrotic mesophyll cells, ®, or intenselyfluorescing necrotic cells of both cell types, / is shown for colonies with 0 to > 10 HMCs .

contains resistance genes in addition to the Sr5 gene (Fig . 3, Tables 1-3) . Preliminarystudies using wheat lines that carried either the Sr6, Sr7a, Sr9g, Sr11, or Sr12 genewere conducted to determine whether race 32 of P.g .t . contains avirulence genescorresponding to these resistance genes and whether resistance expressed by any ofthese genes is associated with the HR of epidermal cells . Infected plants containingeither the Sr7a gene or the Sr12 gene were susceptible, whereas plants containing theSrll gene or the Sr9g gene were resistant . Resistance was also expressed by thetemperature sensitive Sr6 gene at 18 °C. Resistance conditioned by these genes was notassociated with the necrotic reaction of epidermal cells . Therefore, necrosis of infectedepidermal cells in cvs . Manitou and Napayo is likely to be associated with the Sr5 gene .The fungal population was not distributed into two distinct groups of colonies as

Resistance of wheat to stem rust : histology

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36 38 40

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43 47.5 49 .5

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3 14 36 75 92 94 95 95 95 93

Ftc . 4 . Length of hyphae of P. graminis f.sp . tritici in the susceptible wheat line Prelude-sr5 (a)and in the resistant isogenic wheat line Prelude-Sr5 (b) at 20-40 h after inoculation. Simultaneousfungal penetration into the leaves was induced by light at 16 h after inoculation. +, Significantdifferences in hyphal length in Prelude-Sr5 compared with hyphal length in Prelude-sr5 . -, Nodifferences in hyphal length . Line I : % of infection sites with a fluorescing epidermal cell inPrelude-Sr5. Line II : % of infection sites with a first epidermal haustorium in Prelude-Sr5.

observed in Prel-Sr5 (Fig. 3) . The majority of the rust colonies in cvs . Manitou andNapayo developed only one to three HMCs . The number of infection sites with anecrotic epidermal cell in these lines was higher than in Prel-Sr5 (Table 1) .

The time-course for the formation of the first haustorium and the first microscopicallyvisible host cell reaction in wheat cvs Feldkrone, Neepawa, and 417/65 is shown inFig. 5. The time that it took the host cells to react to haustorial penetration, asgraphically estimated, was 4 . 6 h for cv . Feldkrone, 6 . 4 h for cv . Neepawa, and 8 . 1 h forcv. 417/65 .

DISCUSSION

An answer to the question whether or not fungal growth is inhibited before host cellspenetrated by haustoria have reacted hypersensitively, can help in assessing thesignificance of the hypersensitive reaction in resistance to fungal plant pathogens .Inhibition of prehaustorial growth has been reported for several species of rust fungiincluding the wheat stem rust fungus [12] . In the experiments described here,inhibition of fungal growth in the resistant wheat line Prel-Sr5 was not detected beforehaustoria were formed but occurred after the HR of the host cells that were penetratedby the first haustoria . The sequence of the events : (i) formation of a substomatalinfection structure without visible inhibition ; (ii) penetration of a host cell by the firsthaustorium ; (iii) hypersensitive cell death of penetrated host cells accompanied by

101

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TABLE IOccurrence of necrosis in infected host cells in wheat lines containing the Sr5 gene in response to infection

with Puccinia graminis f.s( . tritici, race 32 .

(a) Percentage of colonies associated with one necrotic epidermal cell only

(b) Percentage of colonies associated with necrotic epidermal and mesophyll cells

Days post-inoculation

1

2

3

4

5

6

Prelude-Sr5 4. 5 17 31 29 . 5Napayo 7 .5 11 26. 5 13 16 . 5Manitou

8

26. 5

28. 5

27. 5

15 . 5

(c) Percentage of colonies associated with necrotic mesophyll cells only

Days post-inoculation

1

2

3

4

5

6

Prelude-Sr5 2 33 . 5 43 . 5 30 . 5 30Napayo 19 18 26 11 28Manitou

0.5

11-5

19. 5

20. 5

14

27

accumulation of lignin or lignin-like compounds [2, 25-27] ; (iv) inhibition of fungalgrowth--demonstrates that the hypersensitive reaction is closely associated withresistance controlled by the Sr5 gene and is possibly the determining factor .

Substomatal infection structures of each rust species have a characteristic morphology[15, 18], a fact that has been used to distinguish different leaf rust pathogens [17] . Thespecies-specific form of a substomatal infection structure predetermines the host cell ortissue type that is preferentially penetrated by the first haustorium . In many rustspecies the first cell penetrated is a mesophyll cell . This does not apply to the wheatstem rust fungus . Skipp et al . [24], studying the Sr6/P6 interaction, found that 34-49 %of all penetrants formed the first haustorium in an epidermal cell . In the presentexperiment with P.g.t ., race 32, epidermal cells were penetrated by the first haustoriumin up to 95 % of all infection sites .

The resistance response of rust infected epidermal cells of Prel-Sr5 was not uniformin all cells as indicated by a variation of the fluorescence intensities of these cells : many

Days post-inoculation

1 2 3 4 5 6

Prelude-Sr5 46 245 30 26 29. 5Napayo 53 60-5 41 56 51 .5Manitou 0-5 55 52-5 44 .5 55 47

Resistance of wheat to stem rust : histology

103

TABLE 2Occurrence of necrosis in infected host cells in wheat lines containing the Sr5 gene in response to infection

with Puccinia graminisfsp. tritici, race 32 : colonies with 1-3 HMCs

(a Percentage of colonies associated with one necrotic epidermal cell that developed1-3 HMCs

(b) Percentage of colonies associated with a necrotic epidermal cell and necrotic mesophyllcells that developed 1-3 HMCs

(c) Percentage of colonies associated with necrotic mesophyll cells only that developed1--3 HMCs

cells exhibited an intense yellow autofluorescence while others revealed only a weakfluorescence. This variation might be due to individual cells of the same tissue beingheterogenous in their physiological capacities . Reduced synthesis of fluorescentcompounds as indicated by a weak fluorescence of some epidermal cells may be causedby a delayed initiation of the resistance response and/or limitations of production andprocessing of precursors .

Inhibition of haustorial development was correlated with the intensity of fluorescenceof necrotic epidermal cells. Intensely fluorescing epidermal cells contained smallspherical haustoria, whereas weakly fluorescing cells contained haustoria that wereintermediate in size between those in intensely fluorescing cells and those insusceptible host cells . Growth of haustoria in intensely fluorescing cells was notterminated before haustorial bodies reached a size of about 4lam in diameter .

Days post-inoculation

1 2 3 4 5 6

Prelude-Sr5 0 0 100 56 53 64Napayo 0 93 91 87 88 94Manitou 0 100 94 70 87 93

Days post-inoculation

1 2 3 4 5 6

Prelude-Sr5 0 100 100 100 100 100Napayo 0 100 100 100 100 100Manitou 100 99 96 94 99 100

Days post-inoculation

1 2 3 4 6

Prelude-Sr5 0 100 34 32 11 20Napayo 0 97 89 85 100 96Manitou 100 100 56 68 96 83

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R . Tiburzy et al.

TABLE 3Occurrence of necrosis in infected host cells in wheat lines containing the Sr5 gene in response to infection

with Puccinia graminisf sp . tritici, race 32 : colonies with more than 5 HMCs

(a) Percentage of colonies associated with one necrotic epidermal cell that developed morethan 5 HMCs

(b) Percentage of colonies associated with a necrotic epidermal cell and necrotic mesophyllcells that developed more than 5 HMCs

(c) Percentage of the colonies associated with necrotic mesophyll cells only that developedmore than 5 HMCo

Apparently, this reflects the minimum time required to develop the resistanceresponse from its induction to its deleterious effect on the haustorium .

Growth of the early fungal hyphae and HMCs was closely correlated with the stateof the first haustorium. Colonies with a medium-sized or a large first haustoriumdeveloped more than five HMCs, whereas hyphae associated with a small firsthaustorium in an intensely fluorescing cell were inhibited after the differentiation ofone, two or three HMCs . Obviously, a rapid and intense reaction of infected epidermalcells does not always immediately arrest fungal growth . Limited further growth of thepathogen may occur on the basis of the endogenous nutrient supply, or of exogenousnutrients absorbed by haustoria which may have been functioning for a short time [13] .Moreover, assimilation of nutrients by intercellular fungal structures from the hostapoplast has to be considered [1, 10] . Cessation of fungal growth may not be primarily

Days post-inoculation

1 2 3 4 5 6

Prelude-Sr5 0 0 0 0 0 0Napayo 0 0 0 0 0 0Manitou 0 0 0 0 0 0

Days post-inoculation

1 2 3 4 5 6

Prelude-Sr5 0 0 30 55 85 75Napayo 0 0 0 4 0 0

Manitou 0 0 18 12 0 11

Days post-inoculation

1 2 3 4 6

Prelude-Sr5 0 0 0 23 37 29Napayo 0 0 0 6 0 0Manitou 0 0 2 7 0 0

- (a)

Resistance of wheat to stem rust : histology

100

80

60

40

20

0100

80

60

40

20

0

20 30

40

50 20

30

40 50

Time after inoculation [h)Fm. 5 . Development of P. graminis f.sp. tritici, race 32, in cv . Feldkrone (a), cv . 417/65 (b), cv .

Neepawa (c) and Prelude-Sr5 (d) . Incidence of the first haustorium, /, and of the hypersensitivereaction, p, in the first-invaded epidermal cell recorded at 2 h intervals . The time fromappearance of the first haustorium at 50 per cent of the infection sites and appearance of the firsthost cell necrosis at 50 per cent of the infection sites was chosen as a representative measure of thetime between haustorial penetration and host cell reaction . It was 4 .6 h for cv . Feldkrone, 6 .4 hfor cv . Neepawa, and 8-1 h for cv . 417/65 . Necrosis of infected epidermal cells of Prelude-Sr5 occurred at less than 50 per cent of the infection sites .

due to shortage of nutrients but may result from effects of antifungal compounds suchas phytoalexins . Also then, the parasite may not be affected immediately since a certaintime is required for antifungal compounds to become effective . The possible involvementof phytoalexins in resistance of Gramineae to fungal pathogens has been the subject ofseveral investigations [16, and references therein] . With wheat there is as yet littleevidence for the existence of such compounds . Derivates of dihydroxymethoxy-benzoxazolinone (DIMBOA) have been recognized as potentially playing a role inresistance of wheat to wheat stem rust [8, 9] . The compounds deriving from DIMBOA,which is thought to be enzymatically released from its glucoside precursor during celldamage [28] or during the HR [7], had an antifungal effect on rust development ondetached leaf cultures [8] . Whether these compounds are present in hypersensitivelyreacting wheat cells in quantities required to inhibit fungal growth completely is notknown .

Cartwright & Russell [5, 6] described the appearance of phytoalexin-like compounds

105

(b)

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, ,

(c) - (d)

o0 0

.

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R . Tiburzy et al.

'TABLE 4Development of Puccinia graminisf sp . tritici, race 32, in Prelude-Sr5 : incidence ofhaustorial mother cells

(HMCs) and necrotic host cells recorded from 20 h p .i . to 40 h p .i . at 2 h intervals

34 h

0

3n = 318

1

542

5 . 53

36 h

0

3n = 194

1

372

133

138 h

0

4- n= 218

1

302 133

3

24

15

0. 5-

40 h

0

7n = 205

1

22. 5 21 . 5- -

2

14. 5 26

13

3. 5

0. 5

1

14

1

0 . 5

Necrotic epidermal cells (E), necrotic mesophyll cells (M), necrotic epidermal andmesophyll cells (M+E) . n = number of infection sites .

in association with durable resistance of wheat to yellow rust . Mesophyll cells whichsurround the substomatal cavities reacted with a hypersensitive-like response whensubstomatal vesicles were formed within the cavities . The HR was followed by therapid degeneration of the substomatal vesicles . Since there was no physical contactbetween mesophyll cells and substomatal vesicles, it was concluded that the HR wasinduced by diffusible fungal compounds and degeneration of the substomatal vesicleswas due to diffusion of antifungal compounds originating from the host cells . Extractsfrom such infected leaves were bioassayed with the result that two compounds withantifungal activity were detected indicating the presence of phytoalexin-likecompounds .The Sr5/P5 interaction greatly differs from the yellow rust/wheat interaction in that

the HR did not occur in cells that had not been penetrated by haustoria and in thatgrowth of substomatal vesicles was not affected . Phytoalexin-like compounds asdescribed above were either not present in Prel-Sr5 or were not effective at the

colonies with colonies withTime necrotic host cells Time necrotic host cellspost- post-inoculation HMC None E M M+E inoculation HMC None E M M+E

20 h 0 89 .5n = 228 1 8 .5 2

22 h 0 89n = 252 1 10 . 5 0 . 5

24 h 0 32n= 227 1 65 2

2 0 .5 0 . 526 h 0 18n = 224 1 78 2

2 1 . 5 0 . 5

28 h 0 11n = 211 1 76 9

2 0 .5 3 0 .5

30 h 0 3 .5n = 197 1 83 8 .5 0 .5

2 2 2 . 5 --

32 h 0 0 .5n = 215 1 57 . 5 33 . 5

2 4 33 14 0 . 5

28. 5 0. 56 0 . 51 0. 5 0 . 5

23 0 . 520 2

0 . 5

22 . 5 1 123

Resistance of wheat to stem rust : histology

107prehaustorial stage of the infection . Bioassays with extracts of infected or uninfectedplants of Prel-Sr5 did not reveal differences in antifungal compounds (unpublishedresults) .

Infected epidermal cells of the cultivars Manitou and Napayo were more likely toreact hypersensitively than those of Prel-Sr5 . The additional resistance genes present incvs . Manitou and Napayo may be responsible for the modified resistance response inepidermal cells controlled by the Sr5 gene. However, in several isogenic lines containingthe Sr5 gene, there were no apparent differences in the reaction of the epidermal cellsof the various lines, indicating no background effect [23] .

We thank R . Rohringer, Research Station, Agriculture Canada, Winnipeg, for thesupply of isogenic wheat lines and wheat cultivars used in this investigation, M .Heisterbaum for technical assistance, and R . C. Staples, Boyce Thompson Institute,Cornell University, Ithaca, for correcting the English in the text . The work wassupported by the Deutsche Forschungsgemeinschaft .

REFERENCES1 . AHMAD, I ., FARRER, J. F. & WHITBREAD, R. (1985) . Membrane integrity in leaves of barley infected by

brown rust : an examination using tracer efflux and in vivo chlorophyll fluorescence . The New Phytologist99, 107-115 .

2 . BEARDMORE, J ., RIDE, J . P. & GRANGER, J. W . (1983) . Cellular lignification as a factor in thehypersensitive resistance of wheat to stem rust . Physiological Plant Pathology 22, 209-220 .

3 . BUSHNELL, W. R . (1982) . Hypersensitivity in rusts and powdery mildews . In Plant Infection : ThePhysiological and Biochemical Basis, Ed. by Y . Asada et al., pp . 97-116 . Japan Science Society Press,Tokyo/Springer-Verlag, Berlin .

4 . CAMPBELL, R . C. (1971) . Statistische Methoden für Biologen and Mediziner. Thieme Verlag, Stuttgart .5 . CARTWRIGHT, D . W. & RUSSELL, G. E . (1980) . Histological and biochemical nature of `durable'

resistance to yellow rust in wheat . Proceedings of the 5th European and Mediterranean Cereal Rusts Conference,pp . 23-26 .

6 . CARTWRIGHT, D . W. & RUSSELL, G. E. (1981) . Possible involvement of phytoalexins in durableresistance of winter wheat to yellow rust . Transactions of the British Mycological Society 76, 323-325 .

7 . DEVERALL, B. J . (1977) . Defence Mechanisms of Plants, pp. 54-55 . Cambridge University Press .8 . ELNAGHY, M. A. & LINKO, P . (1962) . The role of 4-0-glucosyl-2,4-dihydroxy-7-methoxy-1,4-

benzoxazin-3-one in resistance of wheat to stem rust . Physiologia Plantarum 15, 764-771 .9. ELNAGHY, M . A . & SHAW, M. (1966) . Correlation between resistance to stem rust and the concentration

of a glucoside in wheat . Nature 210, 417-418 .10. FARRER, J. F . (1984) . Effects of pathogens on plant transport systems . In Plant Diseases : Infection, Damage

and Loss . Ed. by R . K . S . Wood & G . J. Jellis, pp . 87-104 . Blackwell Scientific Publications, Oxford .11 . HARDER, D . E ., ROHRINGER, R ., SAMBORSKI, D . J ., RIMMER, S . R ., KIM, W. K. & CHONG, J. (1979) .

Electron microscopy of susceptible and resistant near-isogenic (sr6/Sr6) lines of wheat infected byPuccinia graminis tritici. II . Expression of incompatibility in mesophyll and epidermal cells and the effectto temperature on host-parasite interactions in these cells . Canadian journal of Botany 57, 2617-2625 .

12 . HEATH, M. C . (1982) . Host defense mechanisms against infection by rust fungi . In The Rust Fungi. Ed .by K . J . Scott & A . K . Chakravorty, pp. 223-245 . Academic Press, London .

13 . HEATH, M . C. (1982) . Fungal growth, haustorial disorganization, and host necrosis in two cultivars ofcowpea inoculated with an incompatible race of the cowpea rust fungus . Physiological Plant Pathology21, 347-359 .

14 . KUCK, K. H., TIBURZY, R ., HANSLER, G. & REISENER, H .J. (1981) . Visualization of rust haustoria inwheat leaves by using fluorochromes . Physiological Plant Pathology 19, 439-441 .

15 . LITTLEFIELD, L. J . & HEATH, M. C . (1979) . Ultrastructure of Rust Fungi . Academic Press, New York .16 . MAYAMA, S ., TANI, T ., MATSUURA, Y ., UENO, T . & FUKAMI, H. (1981) . The production of phytoalexins

by oat in response to crown rust, Puccinia coronata Esp . avenae. Physiological Plant Pathology 19, 217-226 .17. NIKS, R . E. (1983) . Comparative histology of partial resistance and the nonhost reaction to leaf rust

pathogens in barley and wheat seedlings . Phytopathology 73, 60-64 .

108

R. Tiburzy et al.18 . NIKs, R. E . (1986) . Variation ofmycelial morphology between species and formae speciales of rust fungi

of cereals and grasses . Canadian Journal of Botany 64, 2976-2983 .19 . OGLE, H. J . & BROWN, J . F . (1971) . Quantitative studies of the post-penetration phase of infection by

Puccinia graminis tritici . Annals of Applied Biology 67, 309-319 .20 . REISENER, H. J ., TIBURZY, R., KOGEL, K. H ., MOERSCHBACHER, B . & HECK, B. (1986) . Mechanism of

resistance of wheat against stem rust in the Sr5/P5 interaction . In NATO ASI Series, Volume HlBiology and Molecular Biology of Plant-Pathogen Interactions, Ed. by J . Bailey, pp. 142-148 . Springer-Verlag, Berlin .

21 . ROELFS, A. P . & MCVAY, D. V . (1979) . Low infection types produced by Puccinia graminis Esp . tritici andwheat lines with designated genes for resistance . Phytopathology 69, 722-730 .

22 . ROHRINGER, R ., Kim, W. K ., SAMBORSKI, D. J . & HowES, N. K . (1977) . Calcofluor : An opticalbrightener for fluorescence microscopy of fungal plant parasites in leaves . Phytopathology 67, 808-810 .

23 . ROHRINGER, R ., Kim, W . K . & SAMBORSKI, D. J . (1979) . A histological study of interactions betweenavirulent races of stem rust and wheat containing resistance genes Sr5, Sr6, Sr8, or Sr22 . CanadianJournal of Botany 57, 324-331 .

24 . SKIPP, R . A . & SAMBORSKI, D. J . (1974) . The effect of the Sr6 gene for the host resistance on histologicalevents during the development of stem rust in near-isogenic wheat lines . Canadian Journal of Botany 52,1107-1115 .

25 . TIBURZY . R . (1982) . Fluorescence-microscopical studies on the host-parasite interaction between wheatstem rust and several varieties of wheat . Fitopatologia Brasileira 7, 462-463 (Abstr .) .

26 . TIBCRZY, R . & REISENER, H . J . (1982/83) . Fluoreszenzmikroskopische Beobachtungen am Wei-zenschwarzrost . Zeiss Information 27, 54-58 .

27 . TIBCRZY, R . & REISENER, H. J . (1989) . Resistance of wheat to Puccinia graminis Esp . tritici : Associationof the hypersensitive reaction with the cellular accumulation of lignin-like material and callose .Physiological and Molecular Plant Pathology 36, 111-122 .

28 . VIRTANEN, A . I . (1961) . Über einen neuen Typ von Glucosiden in jungen Roggen-, Weizen- andMaispflanzen . Brauwissenschaft 14, 98-101 .