Euphytica 50 : 1 5 9-162, 1990.©1990 Kluwer Academic Publishers. Printed in the Netherlands .

Mature plant resistance of barley to barley leaf rust,another type of resistance

G . Smit & J.E . ParlevlietDepartment ofplant Breeding, Agricultural University, P. O. Box 386, 6700 AJ Wageningen, The Netherlands

Received 16 February 1990 ; accepted in revised form 10 May 1990

Key words: Hordeum vulgare, barley, Puccinia hordei, barley leaf rust, adult plant resistane, mature plantresistance, mature tissue resistance, hypersensitive resistance, partial resistance, adult vs . mature plantresistance

Summary

Seedling and flag leaves of three barley cultivars were simultaneously inoculated with urediospores of barleyleaf rust, race 1-2-1, and incubated at the same greenhouse bench . The inoculated leaves were harvestedbefore urediospore formation was initiated . The volume of a large number of colonies was estimated bymeasuring colony area and colony depth by embedding the colony containing leaf segments into paraffin formicrotome cutting. The colony volume was considerably smaller in flag leaves than in seedling leaves even inthe extremely susceptible cultivar . On average the difference was about tenfold .

Introduction

Resistance in crops to biotrophic fungi is describedin various ways, such as seedling, overall, adultplant, field and partial resistance. These terms re-fer to either of two types of resistance ; the hy-persensitive or low infection type of resistance andpartial resistance . The former is governed by majorgenes that are race-specific and non-durable, thelatter by genes with smaller effects where race-specific effects play a minor role if at all . Within thelow infection type resistance many of the majorgenes are expressed from the seedling stage on-ward (seedling or overall resistance), other genescome to expression in the adult plant stages only(adult plant resistance) . Partial resistance tends tobe expressed better in the later plant stages . It issometimes called field resistance . For these resist-ances differences between cultivars have been re-ported frequently (Zadoks, 1961 ; Van der Plank,1968; Russell, 1978; Parlevliet, 1981) .

In barley (Hordeum vulgare L.) against barleyleaf rust (Puccinia hordei Otth .) these two types ofresistance have been reported as well . Resistanceof the major genic, low infection type is typicallynon-durable (Parlevliet, 1983), while the polygenicpartial resistance seems highly durable (Parlevliet,1978; Habgood & Clifford, 1981) . Clear cultivardifferences occur for both types of resistance .

Parlevliet & Kievit (1986) suggested that theremight be a third type of resistance. They comparedthe colony size in six barley cultivars ranging fromexceptionally susceptible to partially resistant . Thesize of the colonies was evaluated 7 days (firstseries) or 8 days (second series) after inoculation inseedlings and in young flag leaves and the leavesbelow the flag leaves . In all cultivars even in theextremely susceptible cultivars the colonies weremuch smaller in the mature plant leaves than in theseedling leaves, the differences being about five-fold. They suggested to call this mature plant resist-ance, but also mentioned that they had assessed the

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colony area only, not its volume . Although notvery likely colonies might have penetrated the hostleaves deeper in the adult plants than in the seed-lings resulting in an apparent, but not real differ-ence in resistance .

To investigate this a study was carried out toassess the colony volume in seedling and flag leavesof three cultivars of barley .

Materials and methods

Three barley genotypes varying in level of partialresistance were chosen, Akka, extremely suscep-tible and Vada and 17-5-16 partially resistant . Forthe seedling tests seeds were sown in flats of 37 x39 x 7 .5 cm. For the adult plant tests seeds wereplanted in square plastic pots of 12 x 12 X 12 cm .Per flat 60 seeds were sown, per pot 2 to 3 of whichtwo plants were kept .

Seedlings and adult plants of the same genotypewere always inoculated on the same day with ure-diospores of barley leaf rust isolate 1-2-1 . Thespores used at a given day were from the samesample. The inoculation was carried out as de-scribed by Niks (1982) in a settling tower construct-ed according to the device of Eyal et al . (1968). Atthe moment of inoculation the seedling leaves wereabout one week old from emergence, the flag leav-es were young but fully developed . After depos-iting the urediospores (for densities see Table 1),the seedlings and adult plants were incubated over-night at 100% relative humidity in the dark as de-scribed by Niks (1982) . Per series the seedlings and

Table 1 . Number of barley leaf rust urediospores deposited per cm' leaf area on, number of colonies assessed in and leaf thickness in µ ofseedling (S) and flag (F) leaves of three barley genotypes in five series

adult plants were kept together in a greenhousesection until the inoculated leaves were harvested .

In total five series were inoculated . Per seriesone genotype was inoculated . The inoculationstook place in the period May to August . Six to ninedays (see Table 2) after inoculation the seedlingand flag leaves were harvested . The harvesting wasalways done before the colonies started to formurediospores .

The harvested leaves were cut in segments of 2 to3 cm long . The segments were prepared for fluores-cence microscopy using the procedure described byParlevliet & Kievit (1986) with only one difference .In stead of 0 .1% blancophor, 0.1% Uvitex 2B wasused. The treated leaf segments were embedded inparaffin and with a microtome the paraffin blockscontaining the leaf segments were cut in thin sec-tions with a thickness of 67 µ . The sections wereglued onto slides in the order of cutting using'Kais-er's' glycerin. By gentle heating the sections werefully flattened onto the slides .

Under a UV-microscope, magnification 100 x ,

the colonies were assessed . Usually a colony rang-ed over a few to several sections . When a colonywas present the following observations were doneper section (Fig . 1) :1 . the tichkness of the leaf at the site of the colony

(T) ;2. the greatest depth of the colony (D);3. the greatest width of the colony (W) .The total area of a colony consists of the colonyareas summed over the sections in which the colonyis present. The colony area in each section is W x67 µ (67 being the thickness of the section in µ) . Foreach section the contribution to the volume of a

Genotype Series No. of spores No. colonies Leaf thickness

S F S F S F

Akka A 27 45 41 38 246 184Akka B 112 148 7 33 286 167Vada C 229 182 34 24 250 20717-5-16 D 71 121 29 44 347 25517-5-16 E 77 82 52 73 260 196

colony is estimated as 0 .75 x D x W x 67. Thefactor 0 .75 is used to correct for the decreasingwidth of the colony with increasing depth of thecolony. The volume of the colony was estimated byadding the section volumes of the colony together .

Results and discussion

The seedling leaves were thicker than the flag leav-es in all three genotypes (Table 1) . The density ofthe spores applied to seedling leaves and to the flagleaves taken over the treatments did not differ(Table 1) . Density dependent effects therefore can-not explain possible differences in colony size be-tween seedling and flag leaves . In another study itappeared that even large differences in colony den-sity did not affect the colony size provided spor-ulation had not yet started (Baart et al ., 1991) .

The colony area was larger in the seedling leavesthan in the flag leaves in all five treatments (Table2) . On average the differences was 6 .5 times, avalue slightly higher than reported by Parlevliet &Kievit (1986) . In Table 3 the depth of the colonies isshown. At the moment of harvest the colonies hadnot reached the underside of the leaves (Tables 1and 3). The colony depth in the flag leaves wasalways less than in the seedling leaves . This resultsin much smaller colony volumes in the flag leaves,as compared to those in seedling leaves . Table 4shows this for all five treatments. the weightedaverage difference being 10.4 times .

Studying the various slides the impression was

Table 2 . Mean area in 1000 µz of barley leaf rust colonies six tonine days (age) after inoculation in seedlings (S) and flag leaves(F) and their ratio of three barley genotypes in five series

' * * * * F-value significantly lower than S-value at P = 0 .05 and0 .001 respectively .

W

Fig . 1 . Schematic representation of a barley leaf rust colony in abarley leaf segment .T = thickness of the leaf at the site of the colony ;D = greatest depth of the colony ;W = greatest width of the colony ;The black area is the colony area per segment .

that the mycelial density of colonies in the seedlingsand flag leaves did not differ greatly .

It is clear that the barley leaf rust colonies in flagleaves are far smaller than those in seedling leaves,which means that flag leaves provide an environ-ment less suitable for barley leaf rust developmentthan seedling leaves . This mature plant resistancetherefore represents a third type of resistance, al-ready mentioned by Parlevliet & Kievit (1986) . Thecultivars studied, eight in total (this study and fromParlevliet & Kievit, 1986), showed no substantialdifferences for this type of resistance although theyvaried greatly for partial resistance and carried nomajor genic, seedling or adult plant resistance .

This type of resistance, present in mature planttissue or in mature plants seems to occur wide-spread. However, little research has been directedtoward it. In literature it is often mentioned as anobservation . In perennial plants mature foliar tis-sus is often highly resistant to pathogens where theyoung leaves are susceptible . Typical examples are

Table 3 . Mean maximum depth in µ of barley leaf rust coloniessix to nine days after inoculation in seedling (S) and flag leaves(F) of three barley genotypes in five series

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' * * * * F-value significantly lower than S-value at P = 0 .05 and0 .001 respectively .

Genotype Series Age S F S/F ratio

Akka A 8 63.8 15 .4***' 4 .1Akka B 8 178 .2 40.1* 4 .4Vada C 6 36.4 8 .0*** 4 .617-5-16 D 9 291 .9 43 .0*** 6 .817-5-16 E 8 93.4 10.1*** 9 .2

Genotypes Series S F

Akka A 162 143Akka B 266 134'Vada C 219 110-17-5-16 D 337 187***17-5-16 E 236 122***

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Table 4. Mean volume in 1000µ3 of barley rust colonies six tonine days after inoculation in seedling (S) and flag leaves (F) andtheir ratio of three barley genotypes in five series

' * * * * F-value significantly lower than S-value at P = 0 .05 and0.001 respectively .

grape-Uncinula necator (Doster & Schnathorst,1985), apple-Venturia inaequalis (Williams & Kuc,1969) and peach, apricot-Taphrina deformans (Ro-berts & Boothroyd, 1972) . In annual crops foliartissue of mature plants often but not always ap-pears less susceptible than the seedling tissues .Such observations can be found frequently in liter-ature. Only a few examples are mentioned here,such as tobacco-Peronospora tabacina (Clayton,1945), Lettuce-Bremia lactucae (Dickinson &Crute, 1974) and wheat-Erysiphe graminis f . sp .tritici (Hyde, 1976) .This resistance of mature tissues or mature

plants emphasized in this investigation is generallyneglected as it is not accesible for breeding purpos-es. All cultivars seem to show this type of resistanceto as far as can be seen all variants at the pathogen .And it is this latter aspect, which makes this type ofresistance so interesting . It is apparently of a race-non-specific nature and highly durable . If themechanisms of it would be known it might be pos-sible to use it in breeding .

References

Baart, P.G.J ., J .E . Parlevliet & H . Limburg, 1991 . Effects ofinfection density on the size of barley and wheat leaf rust

colonies before and on the size of uredia after the start ofsporulation . J . of Phytopath ., in press.

Clayton, E.E ., 1945 . Resistance of tobacco to blue mold (Pero-nospora tabacina) . J . Agric. Res . 70 : 79-87.

Dickinson, C .H. & I.R. Crute, 1974 . The influence of seedlingage and development on the infection of lettuce by Bremialactucae. Ann. Appl . Biol . 76 : 49-61 .

Doster, M.A. & W.C. Schnathorst, 1985 . Effects of leaf maturi-ty and cultivar resistance on development of the powderymildew fungus on grapevines . Phytopathology 75 : 318-321 .

Eyal, Z ., B .C. Clifford & A .M. Caldwell, 1968 . A settlingtower for quantitative inoculation of leaf blades of maturesmall grain plants with urediospores . Phytopathology 58 :530-531 .

Habgood, R.M. & B.C . Clifford, 1981 . Breeding barley fordisease resistance : The essence of compromise . In: J .F. Jen-kyn & R .T. Plumb, (Eds.), Strategies for the control of CerealDisease, Blackwell, Oxford: 15-25 .

Hyde, P.M ., 1976 . Comparative studies of the infection of flagleaves and seedling leaves of wheat by Erysiphe graminis.Phytophat . Z . 85 : 289-297 .

Niks, R.E ., 1982 . Early abortion of colonies of leaf rust, Pucci-nia hordei, in partially resistant barley seedlings . Can . J . Bot .60 : 714-723 .

Parlevliet, J .E .,1978 . Further evidence of polygenic inheritanceof partial resistance in barley to leaf rust, Puccinia hordei .Euphytica 27 : 369-379 .

Parlevliet, J.E ., 1981 . Race-non-specific disease resistance . In :J .F. Jenkyn & R .T. Plumb (Eds), Strategies for the control ofcereal disease. Blackwell, Oxford, 47-54 .

Parlevliet, J .E ., 1983 . Models explaining the specificity anddurability of host resistance derived from the observations onthe barley Puccinia hordei system. In : F. Lamberti, J .M .Waller & N .A. de Graaff (Eds), Durable resistance in crops,Plenum Press, New York and London, 57-80 .

Parlevliet, J .E. & C . Kievit, 1986 . Development of barley leafrust, Puccinia hordei, infections in barley. I . Effect of partialresistance and plant stage . Euphytica 35 : 953-959 .

Roberts, D .A. & C.W. Boothroyd, 1972 . Fundamentals of plantpathology, Freeman and Company, San Francisco, p . 210-212 .

Russell, G.E ., 1978 . Plant breeding for pest and disease resist-ance. Butterworths, London, 485 pp .

Van der Plank, J.E ., 1968. Plant disease : Epidemics and con-trol . Academic Press, New York, 349 pp .

Williams, E .B . & J . Kuc, 1969. Resistance in Malus to Venturiainaequalis . Ann. Rev . Phytopath . 7 : 223-246 .

Zadoks, J.C ., 1961 . Yellow rust on wheat . Studies in epidemiol-ogy and physiological specialization . T. P1 . Ziekten 67 : 69-256 .

Genotype Series S F S/F

Akka A 7810 1820***' 4 .3Akka B 35110 4330* 8 .1Vada C 5890 720*** 8.217-5-16 D 63970 5640*** 11 .317-5-16 E 15810 1010*** 15 .7