j . Phytopathology 128, 55—61 (1990)© 1990 Paul Parey Scientific Publishers, Berlin and HamburgISSN 0931-17K5

Laboratory' of Plant Pathology, College of Agriculture,Okayama University, 1-1-1 TsHshimanaka, Okayama 700 japan

Primary Infection of Barley by Erysipbe graminis f. sp. hordeiin Relation to Leaf-Age Dependent Resistance

and the Roles of the Epidermis and Mesophyll in this Resistance

Hi-NRV Ni-i.soN, ToMONORi SHIKAISHI and H A C H I R O

Authors' address: HENBI ' NLLSON, TOMONORl SHIRAISHI and HACHIRO O K U , Laboratory ot PlantPathology, College of Agriculture, C>kavama University, 1-1-1 T.sushimanaka. Okayama 700 (Japan).

W'nh

Recfiz-cd Oclnbcr 24. 1988; accepted Mar 31, 1989

Abstract

The infection frequency of both compatible and incompatible races of Erysiphe graminh f. sp.bordci decreased gradually with incrca.sing leaf age on undetached primary barley leaves. The length ofsecondary hyphae of the compatible race was approximately the same regardless of age. but secondaryhyphae were slightly longer on younger seedlings than on older seedlings in the case of theincompatible race. Both the inlection frfquern.\' and length of secondary h\phae of the two races weredistinctly different. On compcsiie seciions produced by exchanging the epidermal layers of young andrelatively mature primary leaves, the infection frequency ot the compatible race was higher on theepidermis oi young leaves than on the epidermis of older leaves, regardless ot w hich mesophyil wasunder the epidermis. The epidermis appears to play a maior role m age-dependent resistance, while themeaophyll may act disparately by providing a factor promotive to mildew infection in addition tosupporting the resistance function oi the epidermis.

Zusammenfassung

Primarinfektion der Gerste durch Erysiphe graminh i. sp. hordeiin Zusammcnhan^ mit dcr vom Blattalter abhangigen Resistenz

und die Rollcn der Epidermis und des Mesophylls bei dieser Resistenz

Beohachtet wurde, da(s die Inrektion.shaufigkeit von kompatiblen und inkompatihlen Erysiphegraminis f. sp. hnrdci-Kisscn sich mil zunehmendem Alter der nieht abgetrennten Primarblatter derCerste langsam verringert. Bei der kompatiblen R.isse erreichtcn die Sekundarhyphen ungefahr diegleiche Liinge unabhangig vom Blattalter, bei der inkompatibleti Ra.sse wuiden an iiingeren Samlingenlangere Sekundarhyphen als an aliercn beobaehtet. Die Infektionshaufigkeit und die Lange der

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56 NELSON, SHTRAISHI and OKU

Sekundarhyphen der zwei Rassen waren deutlich unterschiedlich voneinander. An zusammengesetz-ten Sektionen, die durch den Austausch von den epidermalen Schichten von junj en und fastausgereiften Primarblattern ent.standen, wurde eine hohere Infektionshaufigkeit der kompatihlenRasse mit der Epidermis von limgeren als der von alteren Blattern crmittelt, unahhiingig vomMesophyll unterhalb der Epidermis. Daher scheint es, dal5 die Epidermis eine bedeutende Rolle in derahersabhangigen Resiscenz spielt. Das Mesophyll dagegen konnte eine unvercmbare I unktion haben,die die epidermaie Resistenz unierstutzt und zusatzlich die Mehlrauinfektion fordcrr.

Previously we reported that the infection frequency of both compatible andincompatible races of Erysiphe graminis f. sp. hordei was very high on detachedand sectioned infant barle\' seedlings, but that a.s the seedlings matured as afunction of days after sowing, the infection frequency of both races rapidlydecreased (NELSON et al. 1989 a). In an earlier study (LtN and EDVCARDS 1974), thesame phenomenon was found on both susceptible and resistant cultivars of barleywhen age was expressed as the length of primary leaves. There have beennumerous studies concerning the primary infection process of powdery mildew asaffected by major resistance genes (McCov and ELLINGBOL 1966, MARTIN andELLINGBOI. 1976) and adult plant resistance (HWANG and HftTFi uss 1982 b, c), butlittle if any detailed work has been done to characterize the age-dependentresistance expressed by individual leaves. In the present stud\', we in\'estigatedwhether or not leaf-age dependent resistance is exhibited by whole undetachedleaves, and then attempted to determine which is most important to the expres-sion of this resistance, the epidermis or the mesophyll.

Materials and Methods

Primar)- leaves of the harley (Hordeum viilgare L.) cukivar H.E.S. 4 were used throughout thestudy. The seeds were sown on consecutive days in row.s across one long plastic planter (12 day.s toone planter), and grown under conditions reported previously (NLLSON CI al. 19fJ9 a). Two races ofErx'iipht gramma f. sp. hordei were used: race Hh4, which is compatible, and race 1, which isincompatible, on H.E.S. 4. Leaf sections were inoculated, only with race Hh4, hy shaking mildewedbarley seedling.s over them as described previously (NLLSON et al. 19K9 a}. Whole undetached leaveswere inoculated on their adaxial surface, with one or the other of the races, over about 2 cm of then-length by transferring conidia from vigorous colonies with a soft hair hrush. Composite leaf sectionswere prepared by stripping the abaxial epidermis oft of one leat and placing it on ihe exposed ahasialmesophyll of another leaf as descrihed previously (NELSON et al. 19S9 h). These sections wereinoculated on their abaxial surface. Leaf sections were floated on 200 jtjl of distilled water and seated ina plastic case from iust after inoculation until fixation 48 h later.

Three whole undetaehed leaves were inoculated for each day after sowing. For the experimentswith leaf sections, 6 sections were prepared fur each treatment group. Specimens cleared in methanolwere stained with lactophenol-cotton blue. At least 100 conidia at the appressonal stage or beyondwere counted on each specimen under the lii ht microscope. The infection frequency was calculated asa percent of conidia with secondary hyphae to the total of conidia counted, i.e., those with appressoriaonly plus those with secondary hyphae. Data are presented as the mean ± standard deviation of -1—Sexperiments with undetached leaves and 6—8 experiments with leaf sections. The lengths of 10—20individual secondary hyphae were measured for each day after .sowing in each of 2 experiments, .indaveraged. No .secondary hypha less than 20 .Km in length was measured, in order to be sure thai theparasite had established a functioning relationship with the host (SlESINSKI and El.LINGBOl 19(i9).Analyses of variance were performed, followed by Duncan's multiple range test for multiplecotnparisons and Student's t test for paired data.

Primary Infection in Relation to Leaf Age 57

Results

The effect of days after sowing on the infection frequency of both thecompatible and incompatible races on undetached leaves was highly significant,with the infection frequency of both races showing a steady decline withincreasing age of the seedlings (Fig. 1). The difference in the infection frequencybetween the races was highly significant.

The effect of days after sowing had no significant influence on the secondaryhyphal length of the compatible race on undetached leaves, while this effect wassignificant on the hyphal length of the incompatible race (Eig. 2). The differencein the length of secondary hyphae between the two races was highly significant.

The abaxial epidermis stripped from a 6- or 11-day-old leaf was placed onthe exposed mesophyll of another 6- or 11-day-old leaf, and the infectionfrequency of the compatible race on the various combinations of composite leafsections was compared in an attempt to determine the relative importance of theepidermis and mesophyll to age-dependent resistance (Fig. 3). The type of sectionhad a highly significant effect on the infection frequency. The infection frequencywas higher on the 6-day-old epidermis than on the 11-day-old epidermis, nomatter which mesophyll was under the epidermis. This difference was significantbetween 6/6 and 11/6. The infection frequency on an epidermis of either age was

LU

o

60-

50-

40-

30-

20-

10-

S 6 7 8 9 10 11 12 13 14 IS 16

DAYS AFTER SOWINGi ig. t. Changes in the infection frequency of Eiysiphc graminis \. sp. hordei with aging of barleyleaves. First leaves of sequentially planted harley (cv. H.E.S. 4) seedlings inoculated with a compatihle(Hh4, • ) or incompatible (I, I ) race. The inoculated area of the leaf was cut oft and fixed 48 h afterinoculation. Vertical lines indicate the standard deviation in one direction. The .same letter heside these

lines indicates no significance at p = 0.05

J,Phytc>paihiiloi;>.Bii. I?K. llcft I

58 NELSON, SHIRAISHI and OKU

300-

200-

g 100H

8 9 10 11 12 13

DAYS AFTER SOWING14 15 16

Fig. 2. Length of secondary hyphae of Erynphe graminis f, sp. hordei on barley seedlings of variou.sages. First leaves of sequentially planted barley {cw H.E.S, 4) .seedlinjis were inoculated with acompatible (Hh4, • } or incompatible {], • ) race. The inoculated area of the leaf was cut off and fixed48 h after inoculation. Vertical lines indicate ± standard deviation. The same letter beside these lines

indicates no sis-nificance at p - 0,05

50-1

^ 4 0 -

UJ

O- 30-UJQ:

^ 20^

I—LJUJ 10-

6/6 6/11 11/11 11Fit;, 3, Infection frequency of the compatible race ot Erysiphe graminis f, sp, hordei on composiie andintact first leaf sections ot 6- and ll-day-old barley seedlings. Ctjmpositc sections are represented infractional form, where the numerator indicates the inoculated abaxial epidermis, and the denominatorindicates the exposed mesophyll upon which the epidermis was laid. An integer indicates a section noistripped of either epidermis, inoculated on the abaxial surface. Vertical lines indicate ± standard

deviation. The same letter beside these lines indicates no significance at p = 0.05

Primary Infection in Relation to Leaf Age

40-

LU

20H

10-J

6-day-old U-day-old

INTACT STRIPPED INTACT STRIPPEDAd Ab Ad Ab Ad Ab Ad Ab

I'ig. 4. Infection frequency of the compatibte race of Erysiphc graminis f, sp. hordei on tht- adaxial(Ad) and abaxial (Ab) surfact's of sections of intact and .stripped first leaves of 6- and 11-day-otd barteyseedling.s. Stripped means that the epidermis opposite irom the inoculated surface was removed. Imactmeans that the epidermis was not removed from either side. Vertical tines mdicate ± standarddeviation. Asteristis indicate significant difference from the respective 6-day-otd sections: '•, p < 0.05;

''". p < 0.0!

higher when the tnesophyll was from an It-day-old leaf than when it was from a6-day-old leaf, although this difference was significant only between 11/6 and t l /11. The infection frequency was lower on the intact sections than on therespective composite sections with an epidermis and mesophyll of the same age.Smce this difference was highly significant in both cases (6 vs. 6/6 and 11 vs. 11/11), further investigation of the increase in infection frequency due to separationof the epidermis and mesophyll was felt warranted.

The infection frequency on the abaxial and adaxial surfaces of leaf sections,with or without the opposite side stripped off, was examined (Fig. 4). Theinfection frequency was lower on the intact sections than on the correspondingstripped sections, although this difference was significant only on the adaxia!epidermis when data for 6-day-old and 11-day-old leaves were combined. Theinfection frequency was significantly higher on 6-day-old sections than on 11-day-oid sections of the same type.

Discussion

Leaf-age dependent resistance to infection by both compatible and incom-patible races of Er)'siphe graminis f. sp. hordei was maintained on whole leaves ason leaf sectiotis. This resistance was expressed by both the adaxial epidermis andahaxial epidermis of leaf sections. Since the infection frequency was lower on the11-day-old epidermis than on the 6-day-oId epidermis, no matter whichmesophyil was under the epidermis, it seems that the epidermis is most importantto age-dependent resistance. The fact that the expression of this resistance was

60 NELSON. SHIKAISHI and OKU

maintained on sectioned epidermal la\'ers of detached leaves indicates that thistype of resistance may occur at the tissue level.

The mesophyll may play a supportive role in the resistance since theinfection frequency was considerably lower on mtact sections than on sectionswhich were made by placing an epidermal layer on an exposed mesophyll of thesame age, an operation that destroys the natural connection between the twolayers. However., if the mesoph}ll is acting solely in the direction of resistance,one wouid expect composite sections with an 11-day-old mesophyil to have alower infection frequency than sections with a 6-day-old mesophyll, but such wasnot the case. Also, the infection frequency was higher on sections stripped of theepidermis opposite from the inoculated surface than on sections with the epider-mis intact on both sides. These results point to the possibility that damaging themesophyl! by stripping off the epidermis results in the leakage of some substancewhich facilitates mfection by powder)' mildew. In fact, such a substance has beenshown to exist in the homogenate of healthy barley leaves (SHIRAISHI ct at. 1988).Thus, the mesophyll may have the potential to promote mfection in contradictionto its role of supporting the resistance reaction which occurs in the epidermis.

The difference in the infection frequency between the races was highlysignificant on whole undetached leaves. However, this difference was ratherobscure on leaf sections in a previous study (NF.LSON et al. 1989 a). This differencewas highly significant on undetached leaves due to a much higher infectionfrequency of the compatible race on undetached leaves than on leaf sections, anddid not result from a lower infection frequency of the incompatible race. As withthe infection frequency, the h}-phal length was not appreciably different betweenthe compatible and incompatible races on leaf sections (data not shown), but wasshown to be significantly different on undetached leaves in the present study. Thelength of secondary h\-phae was about the same on sections as on undetachedleaves in the case of the compatible race, but it was distinctly shorter onundetached leaves than on sections in the case of the incompatible race. In otherwords, the processing of leaves into sections clearK' decreased the capability' ofthe compatible race to infect, but not to elongate secondary hyphae, whereas itdid not f^urther hamper the ability of the incompatible race to infect, but greatlyfacilitated the development of this race beyond the haustorial stage. The elonga-tion of shorter secondary hyphae on undetached leaves than on leaf sections by anincompatible race could be explained by the existence of a systemic resistancesystem in barley (Hvv ANG and Hi.iTht uss 1982 a, CHO and SMhDEGAARD-Pi-Ti-.RShN1986, FujiwARA et al. 1986). The lower infection frequency of a compatible raceon detached leaf sections than on undetached leaves suggests the intriguingpossibility that a compatible race may condition the host toward susceptibility(HLATH 1981) in a process that requires the whoie plant.

Literature

C H O , B. H . , and V. SMKliFGAAHD-PFTiRsrN, 1986r Induction of resistantic to Erysiphc graminis i, sp.hardei in near-isoj;cnic barley lines. Phytopathology 76, 301—305.

Primary intV'ction in Relation to Leaf Age 61

I-'ujlVi'AKA, M., H. OKU, T . SIIIRALSHI, and S. Oucni, 19M6: Induction of systemic resistance in barleyleaves hy pruning of organs against powdery mildew funf^us. Ann. Pbytopach. Soc. Japan 52,33&—335.

HI:AT1I, M . C , I9S1: A LjenCTalized concept (if liost-parasite specificity. Phytopathology 71,1121—1123.

HWANG, B. K. , and R. Hi;iTi:iuss, 1982 a: Induced resistance of spring barley to En'siphe gramimsf. sp. hordei. Phytopath. Z. 103, 41—47.

— —, and —-—, 1982 b; Characterization of adult plant resistance of spring barley to powderymildew (Erysiphc graminis f. sp. horde!). 1. Race specificity and expression of resistance.Phvtopath.'Z. 104, U>8—178.

, and , 1982 c: Characterization of adult plant resistance of .spring barley to powderymildew [Erysiphc graminii f. sp. hordei). II, Infection process at different leaf stages.Pbytopath. Z. 104, 179—190.

LIN. M . - R . , and H. H. EDWARDS, 1974: Primary penetration process in powdery mildewed barleyrelated to host cell age, cell type, and occurrence of basic stainmg material. New Phytol. 73,131 — 137.

IvlARTi.N', T. J., and A. H. El.LINGBOIi, 1976; Differences between compatible parasite/host genotypesinvolving the Pm4 locus of wheat and the corresponding genes in Erynphe gramims f. sp.tritid. Phytopatholof^y 66, 143S—1438.

Mc;Cov, M. S., and A. H. ELLINGBOF., 1966: Major genes for resistance and the formation ofsecondary hyphae by Erysiphe graminis f. sp. hordei. Phytopathology 56, 683—686.

Nri.StiN, H., T. SlIiRAIsm, and H. O K L , 1989 a: Effect of leaf age and etiolation of barley onsusceptibility to powdery mildew infection. J. Phytopathol. 124, 101—106.

~, — —, and -, 19S9 b: Respective roles ot the epidermis and mesophyll in the resistance ofbarley to powdery mildew. Ann. Phytopath. Soc. japan 55, 156—160.

SlilKAiS}!!. T., T. MHAZAKI. H . OKU, T . YAMADA, and S. OuCHi, 1988: Powdery mildew-infectionenhancing factor in healthy barley leaves. Abstr. II-9, pg. 79 in Abstracts of the JapanesePhvtopathological Society Conference, April 1988, Tokyo.

Sl.lSINSKi, R. S., and A, H. E[ l INGUOI-., 1969: The genetic control of primary infection of wheat byF.rysiphc gramims f. sp. tritici. Phytopathology 59, 1833—1837.