Histological responses in Hordeum chilense to brown and yellow rust fungi

Plant Pathology (1992) 41, 611-617

Histological responses in Hordeum chilense to brown andyellow rust fungi

D RUBIALESInstituto de Agronomia y Proteccion Vegetal. Consejo Superior de Invest igaciones Cientificas. ApdoCorreos 3048. 14080 Cordoba. Spain

and R. E. NIKSDepartment of Plant Breeding. Wageningen Agricuhural University. POB 386, 6700 AJ Wageningen.The Netherlands

The histological reaction of 16 Hordeum chitense accessions to Puccinia recondita. P. hordei and Pstriiformis was investigated. It is not possible to generalize about the reaction of H. chitense to these rustfungi. Several lines showed a susceptible infection type to a brown rust harvested on H.juhatum. One ofthese lines had an intermediate infection type to the wheat brown rust fungus. The H. chilense reaction tothe yellow rust fungi ranged from susceptible to resistant. The lines differed in their response to the rustfungi, as expressed by the level of early abortion of sporelings, and the frequency of plant-cell necrosisassociated with the infection sites. All the lines caused complete early abortion associated withinfrequent necrosis ofthe rye brown rust fungus. The responses were to some extent rust non-specific.The differences in response between the lines may reflect differences in general defence mechanisms.

INTRODUCTION

Hordeum chilense. a wild perennial barley nativeof South America, has been used extensively inwide crosses in the Triticeae since the hybrid withTriticum aestivum was obtained (Martin & Chap-man, 1977). The high crossability of this specieswith Triticum. Hordeum and Secale (Finch &Bennett, 1980; Martin & Cubero, 1981) maymake H. chilense potentially valuable for breed-ing purposes. Two possible approaches are thedevelopment of amphiploids with wheat to beused as a new crop (Cubero et al.. 1986; Martin,1988), and the development of chromosomeaddition and substitution lines in wheat in orderto transfer desirable traits to wheat and triticale(Miller et al.. 1982; Fernandez & Jouve, 1988).Disease resistance is a valuable trait to be trans-ferred to cultivated species. This prompted us tostudy the resistance of H. chilense to rust fungi,which may be transferred to cereal crop species.

Brown rust is a major cereal disease caused byPuccinia recondita f.sp. tritici (Prt). P. reconditaf.sp. recondita (Prr) and P. hordei (Ph) on culti-vated wheat, rye and barley, respectively. Inseedling tests, Rubiales et a/. (1991) demonstratedthe very high level of resistance of H. chilense towheat and barley brown rust fungi (Prt and Ph)

[infection type (IT)0 on a scale of 0-4]. H. chilensemay be a non-host species to Prt and Ph. The ryebrown rust fungus (Prr) is known to have a verylimited host range (Mains, 1933). It is likely thatH. chilense is also a non-host to Prr. Yellow rust iscaused by P. striiformis ff. spp. tritici (Pst) andhordei {Psh) in cultivated wheat and barle\.respectively, although f.sp. tritici may occasion-ally afTect barley (Stubbs, 1985). H. chilense ishost to both forms (Vallega, 1947; Rubiales era/.,1991).

In plants inoculated with inappropriate speciesor special forms of rust fungi, two mechanisms ofresistance may be found: prehaustorial and post-haustorial resistance (Heath, 1981, 1982). Pre-haustorial resistance is not associated withnecrosis, and is very common in non-host interac-tions (Heath, 1981, 1982; Elmhirsl & Heath,1987). Posthaustorial resistance is usually asso-ciated with necrosis of plant cells after initiationor formation of haustoria. This mechanism ispredominant in some inappropriate combina-tions, e.g. in ryc-Prt (Niks & Dckens, 1987) andrye-Ph (Niks & Dekens, 1991)

Prehaustorial resistance is considered to becaused by general defence mechanisms in theplant (Callow, 1977; Heath, 1985) which may bedifficult to overcome by rust fungi (Heath, 1982,

612 D. Rubiales and R. E. Niks

Table I. Source of Hordeum chilense lines

Line

HIH7H8HIOHllHI2HI3H16H17H34H35H46H47H55H61H74

Source

PBI, UKUSDAUSDAUSDAUSDADr Lange. CPO. NLDr Lange, CPO, NLProf, von Bothmer, SWProf, von Bolhmer, SWDr D. Contreras, ChileDr D. Contreras. ChileDr D. Contreras, ChileDr D Contreras. ChileDr D. Contreras. ChileDr D. Contreras. ChileDr D. Contreras, Chile

Code

CL40I000IPI255753P1255754P1283375PI 283374Line 45Line 158H1814H18I6Proy.H 86-1Proy.H/86-:Proy H 86-13Proy.H X6-I4Proy.H 86-22Proy H 86-28Proy.H 86-40

1985). Hence this type of resistance may bedurable.

The purpose of (he present study was todetermine the histological reaction of H. chilenseto special forms of P. recondita and P. striiformis.and to P. hordei. The study may reveal whetherlines of H. chilense differ in the mechanism ofresistance to the inappropriate rusts, and whetherthe reactions depend on the rust species or specialform applied.

MATERIALS AND METHODS

Plant material

The first experiment consisted of 16 //. chilenselines (Table 1) selected from a previous infectiontype study on 35 lines, and wheat cvs Little Cluband Morocco, ryecv. Rogo, barley cvs Akka andTopper, Hordeum juhatum ssp hreviaristatumand Elymus repens. as a control. Five seedlingsper accession were sown in plant boxes of 37 « 39cm, 10 accessions per box. in two parallel rows.

For Psh isolate F24, three successive experi-ments were performed because of inconsistentresults.

(ii) P. recondita f. sp. recondita (Prr), an isolatecollected at Wageningen;

(iii) 'Jubatum brown rust', isolated at Wagen-ingen on H. jubatum. It was probably P.recondita f. sp. agropyri ('Pra"), because of itspathogenicity on wheat grass (Elymusrepens) (D.R. & R.E.N., unpublished data)and the morphology of the infection struc-tures (Niks, 1986; R.E.N., unpublisheddata);

(iv) P hordei (Ph). isolate 1-2-1 (Parlevliet,1983);

(V) P. striiformis f. sp. tritici (Pst), isolate108E41 (collection number 89523) from DrR. W. Stubbs, Research Institute for PlantProtection (IPO), Wageningen;

(vi) P. striiformis f. sp. hordei (Psh). isolatesBolivia and F24 (collection number 81500)from Dr Stubbs.

The urediospores were multiplied on suscep-tible host cultivars, harvested and stored in adesiccator until used.

Inoculation

After the complete unfolding of the primaryleaves (15 20 days after sowing), the leaves werepinned to the soil in a horizontal position. Theadaxial surface was inoculated in a setthng tower.Each plant box received 3 mg of urediosporesdiluted with Lycopodium spores (1:10 ratio). Thisdose results in about 200 urediospores, cm-. Fourvaseline-greased glass slides were placed in eachflat to check the inoculum density. After inocula-tion the plants were incubated overnight indarkness in a mist chamber at 16 C. They werethen transferred to a greenhouse at 20-16X(day-night range).

Pst and Psh Bolivia were inoculated b\ dustingthe seedlings w ith urediospores diluted in Lycopo-dium in an inoculation cabinet at IPO, incubatedfor 24 h at 6 C with high humidity, and thentransferred to a growth chamber at 15 C. Psh F24was applied in a settling tower at the Departmentof Plant Breeding, incubated for 24 h at IOC, andthen transferred to a greenhouse compartment atabout 20 C

Inoculum

The rust usfd were as follows

( i ) P rceondita f s p tritici (Prt). i s o l a t e " I l i i -mingo" (/iiiloks, 1963);

Sampling

The central part of the leaves inoculated withbrow n rust was sampled 6 days after moculation,and ihosc inoculated with >cllow rust were sam-pled at 4 days.

Histological responses in Hordeum chilense 613

Macroscopic observations

About 17 days after inoculation the infectiontypes on the leaf stubs in the plant boxes weredetermined according to the 0-9 scale of McNealetal.(\91\).

IVIicroscopic observations

The leaf segments collected 6 or 4 days afterinoculation were processed for histological obser-vation as described by Rohringer et al. (1977).Instead of Calcofluor we used Uvitex 2B (Ciba-Geigy). The leaf segments were screened with aNikon epifluorescence microscope (Fluophot) atX 100 or x400 magnification. Some structuresbecame more clearly visible when the whitetransmitted light of the microscope was used.Replacement of filter BP390-440 by H436 facili-tated observations on necrosis of host cells, whichdisplayed a golden yellow autofluorescence.

At least 50 infection units (i.e. sporelings that atleast developed an appressorium) per leaf seg-ment were screened for the brown rusts, unlessotherwise stated. For the yellow rust, few infec-tion units were found due to poor germination, soall available infection units were screened. Thedevelopmental stage of the infection units wasdetermined in a components analysis (Niks &Dekens, 1987). Sporelings were considered to be'early aborted' when they had formed at least one,but not more than six, haustorial mother cells.Infection units that had formed more than sixhaustorial mother cells were considered to beestablished. Their growth and development couldhave been arrested later, due to posthaustorialdefence mechanisms.

RESULTS

Macroscopic observations

Four of the H. chilense lines allowed somesporulation by/>« (Table 2): HIO (IT 6), and H8,Hll and HI6 (IT 1-3). The remaining linesshowed no symptoms. Prr was pathogenic onlyon rye, and did not cause symptoms on the otheraccessions. The H. chilense reaction to Pra rangedfrom susceptible to resistant (Table 2). The E.repens and H. jubatum checks were very suscep-tible. Ph was pathogenic only on barley, and didnot cause symptoms on the other accessions(Table 2). The H. chilense reaction to Pst and Pshranged from susceptible (IT 9) to resistant (IT 0)(Table 3).

Microscopic observations

As indicated in Table 2, on some H. chilense lines(H7, HI7, H47, H55 and H74) very few infectionunits were fuund with the brown rusts. Persegment, less than 50 infection units were avail-able for the components analysis. On the,seaccessions the rust fungi showed frequent failureto differentiate appressoria over stomata. Thisphenomenon was studied in a separate experi-ment (Rubiales & Niks, 1992).

With all the brown rust fungi, early abortioncould be associated with relatively little or withfrequent necrosis. The percentage of early abor-tion of Prt in the H. chilense lines ranged from 22to 100% (Table 2). The lowest early abortion andlowest necrosis was found in the accession withthe highest infection type (HIO).

The percentages of early abortion of Prr in allthe H. chilense accessions, wheat, barley and E.repens were almost 100"/,,. The percentages ofearly abortion of Pra in the H. chilense linesranged from 27 to 98%. The lower percentageswere found on the accessions with the highestinfection types. The percentage of early abortionassociated with plant-cell necrosis ranged from 0to 98%. Line HIO was associated uith the lowestlevel of early abortion and of necrosis.

The early abortion of Ph ranged from 68 to100%. HIO again showed the lowest level of earlyabortion, together with low necrosis. The per-centages of early abortion of Pst in the H. chilenselines ranged from 20 to 57% (Table 3). Nine to84% of these aborted sporelings were associatedwith necrosis. The percentages of establishedcolonies with necrosis ranged from 10 to 85".>.There was no obvious correlation between per-centages for early abortion and necrosis on theone hand, and infection type on the other hand.

The percentages of early abortion of Psh onH. chilense were generally lower for the Boli\iaisolate. However, the results for isolate F24demonstrate that average levels of early abortionmay vary per experimental run. The H. chiienselines differed in the amount of necrosis associatedwith Pst and Psh sporelings. Hll had relativelylow frequencies of necrosis associated with earhabortion and with established colonies for allyellow rust isolates. H8 had relati\el\ high fre-quencies of necrosis (Table 3).

DISCUSSION

The very low mlection types of //. ihilen\c hncswith Prt and Ph. and from low to high with Pst


TaUe 2. Components analysis and infection type (IT) ofbrown rusts on 16 Hordewnchilense lines and wheat, barley, rye, H. jubatum and Elymus repens checks

Line

HIH7*H8HIOHl lHI2H13H16H17*H34H35H46H47*H55*H6IH74*

W-LCB-AkkaR-Rogo

E. repensH. jubatum

IT"

10

1-36

1-3I1

1-30-10-1

10

0-1O-I

I0

90

Prt

EA (N)*

96 (71)93 (48)81 (73)22 (30)60(39)94(54)88 (69)84 (67)

100 (4S)91 (39)87 (70)

100(23)92 (33)

100 (36)98 (69)

100 (67)

1(0)91 (18)ND'

NDND

IT

0-10

0-10

0-10-20-1

0000

000

009

00

Prr

EA(N)

100 (26)100(8)100(16)100(0)100(6)100(19)100 (28)100 (24)100 (20)99(12)

100(54)NDND

100(8)99 (24)

100 (53)

99(16)100(10)19 (25)

100 (28)89(5)

IT

1-31

7-87-6011

6-71-35-6

7

4-5

0-2

00

98

Pra

EA(N)

85 (88)95 (55)55(90)27(0)61 (10)75 (92)82 (88)76 (56)98(40)44(50)65 (62)

NDNDNDND

92 (43)

85 (38)90(17)

ND

15(10)20(0)

IT

10-1

11011

0-10-1

11000

0-10

19

Ph

EA(N)

93 (91)100(67)98(70)68(32)85(20)86(91)93 (91)96(91)

100 (38)100(81)100(87)96(91)

100 (80)97 (51)71(86)95(55)

ND12(0)ND

NDND

* On these lines few appressoria were formed by all brown rusts.' IT, infection type according to McNeal et al. (1971).'' EA (N), percentage of early aborted sporelings; the percentage of early abortedsporelings associated with plant cell necrosis is shown in parentheses.'^ ND, not determined.

and Psh. are in accordance with previous data(Rubiales era/., 1991). The susceptibility of somelines to Pra is interesting. In May 1989 wedetected a 'brown rust' epidemic in field experi-ments at Cordoba, Spain. At that time we did notestablish the identity of the rust. Brown rust wasreported on H. chilense in Chile in 1919 (Arthur,192S). Arthur (192S) listed this rust as Pucciniaclematides (D.C,) Lagerh. Tromso Mus. Aarsh,which may be synonymous with P. reconditaagropyri.

The present study indicates variation amongH. chilense lines in reaction to rust ftingi. In H.chilense lines H7. H17, H47, H55 and H74 therewas apparently some mechanism that hamperedthe formation of appressoria over stomata(Rubiales & Niks, 1992), The present experi-mental design did not allow firm conclusions to bedrawn regarding the magnitude of the pre-

appressorial resistance. The boiling of the leafsegments may have caused (part of) the uredio-spores and germlings to be washed off.

It is clear from Tables 2 and 3 that there is alsoconsiderable variation in the frequencies ofeariyabortion and sporeling-indueed necrosis amongthe H. chilense lines. Even with Prr, which hardlyestablished colonies, there were considerable dif-ferences in the amount of necrosis associated withearly abortion. In diploid wheat (Niks A Dekens,1991) and other plants (Heath, 1977; Elmhirst AHeath, 1987), necrosis associated with eviy abor-tion suggests that resistance became eflective afterthe initiation or formation of haustoria. Similarvariation in levels ofeariy abortion and necrosisto inappropriate rust fungi were observed inTriticum tmmoeoecmn and frt (Niks A Dektns.1991), and have been reported in o t e r inappro-priate plant-palhogm combinatioM (OnNe *


I

C

oSo

I?a..

o

T3CCO

c

I

I

UJ

— Q r*-,Z

^ —

p- r i — (js r^ r-

f, r t 30 —

I '-I 3C

t y O O f ,oor- — r̂ , —

C — OZ

Ic

IIII

3 S' o 'o

^ Z =


Davis, 1977; Heath, 1977; Tosa & Shishiyama,1984; Elmhirst & Heath, 1987),

The present study also allows some observa-tions on the rust specificity ofresistanee reactionsin H. chilense. The mechanism that hampersappressorium formation is apparently effectivefor all the brown rust accessions in this study. Thedata on the effectiveness of the mechanism in thecase of the yellow rusts were inconclusive becauseofthe low overall germination ofthe yellow rusts.

HIO is the only accession with an intermediatereaction to Prt. The percentages ofeariy abortionand the frequencies of necrosis associated withearly abortion were typically lower in H10 than inthe other H. chilense lines when inoculated withPrt. Pra or Ph. This line apparently allowed arelatively large number of brown rust sporelingsto establish (small) colonies. We conclude that thedefence mechanism of HIO to the inappropriatebrown rust fungi Prt. Pra and Ph is relativelyweak. Because of lack of seed, line HIO was notincluded in the tests with Pst and Psh.

The reaction of H11 to the brown rust fungiwas characterized by relatively low frequencies ofcell necrosis, but the level of early abortion washigher than in HIO (Table 2). In HI 1 the yellowrusts also caused relatively infrequent necrosis.The relatively low degree of necrosis in H l lappears to be rather non-specific with regard torust species.

On the other hand, several other lines, e.g. H8,were liable to react with necrosis to attempts atinfection by brown and yellow rusts. With thebrown rusts, these lines arrested the sporelings inearly stages of development, but the relativelyhigh frequency of necrosis suggests that thishappened frequently after the onset of haustor-ium formation. Also this feature was relativelynon-specific with regard to rust species.

The above-mentioned observations suggestthat the type of response in the H. chilense lines isrust-species non-specific and is part of a generaldefence mechanism to inappropriate rust fungi.In HIO, the general defence appears to be rela-tively weak. This view is supported by an obser-vation that HIO seedlings carried trace infectionsof the powdery mildew fungus to which otherH. chilense accessions are completely resistant(D. R., unpublished data).

However, the extent of the defence reactionsdepends on the rust species. The amount of earlyabortion, for example, is much higher for Prr andPh than for Pra. This suggest that the various rustfungi differ in their ability to negate generaldefence mechanisms.

ACKNOWLEDGEMENTS

We are gratefully indebted to Dr R. Johnson forcritical reading of the manuscript. We acknow-ledge the financial support to the senior author bythe FPI programme of the Spanish MEC and bythe C.I.C.Y.T. (Project AGR89-0552).

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Histological responses in Hordeum chilense to brown and yellow rust fungi