Localized accumulation of fluorescent and u.v.-absorbing compounds at penetration sites in barley leaves infected with Erysiphe graminis hordei

Physiological Plunt Patholqgv (1978)

13,347-354

Localized accumulation of fluorescent and u.v.-absorbing compounds at penetration sites in barley leaves infected with Erysiphe graminis hordei S.

MAYAMA?

and J.

SHISHIYAMA

L&oratory of Plant Pathology, Faculty of Agriculture, Fijvto uni.vclti~, K-to 606, Japan (Accepedfor publication May 1978)

Cytophotometrical stud& of the penetration sites of Erysi~ gmnik hor&i in barley leaf epidermal cells were conducted. Papillae and haloa formed during the primary infection process were detected by autofluorescence in both compatible and incompatible combinations, and hypersensitively fluoresced cells were found in the latter combination by u.v.-fluorescence microscopy. Analysis of fluorescent sites by microapectrophotometry with U.V. light indicated the deposition of u.v.-absorbing substances. The U.V. absorption spectrum showed peaks at about 282 and 325 run in both papillae and cell walb in halo areas; these pcalm are basically similar to those in xylem wall that was positive in h&chemical tests for lignin. Nevertheless, the papilla and halo area did not give a clear positive reaction in the tests, but the sites were stained with aao-reagents. These results suggest that autofluorescent and u.v.-absorbing substances accumulating in the penetration sites could be polyphenol compounds. The need for chemical analysis of the compounds was pointed out in order to establish the role of the substances in resistance.

INTRODUCTION It is known that wall-like depositions sueh as papillae or callosities areformed between the plasma membranes and cell walls at infection sites of fungal parasites in many host plants [I, 3, 5]. Aist [I] h as recently reviewed these depositions and discussed the significance of papilla formation in host cells in response to f&gal attacks in disease resistance. Although many reports suggested that papilla formation might act as a barrier for fungal penetration [S, 8, IS, 201, a sole and decisive fimction of papilla has not been substantiated. In powdery mildew of barley, some electron microscopical studies indicated that formation of papillae was initiated prior to fungal penetration [5, 81. This has been generally confirmed by monitoring primary infection in living epidermal cells of barley coleoptiles with phase-contrast or interference-contrast microscopy [I, 2, 61. Bushnell & Bergquist [6j examined the development of cytoplasmic aggregates, papillae and haustoria in the epidermal cells of several host-parasite combinations, and presented experimental evidence that the papilla is a significant component of t Present address: Laboratory Kagawa 76 l-97, Japan. 0048-4059/78/1101-9347 n5

$02.06/O

of Plant

Pathology,

Faculty

of Agriculture,

@ 1978 Academic

Press Inc.

Kagawa (London)

University, Limited

S. Mayama and J. Shishiyama

348

host resistance to fungi. Aist & Israel [.?I have recently studied extensively the defensive r8le of papillae against mildew penetration but they concluded that papillae in the Olpidium-Brassica system were not responsible for penetration failures. The chemical nature of papillae in the mildewed barley leaves has been found to be callose [17J, a basic staining material [7], some inorganic elements such as Ca and Si [9, II] and autofluorescent substances [12, 133. In this paper, we have examined papillae formed at infection sites of Erysiphe graminis ho&i in barley leaf epidermal cells, using u.v.-fluorescence microscopy, microspectrophotometry in the ultraviolet region and histochemistry in order to provide additional information on the function of papillae in host-parasite interactions. MATERIALS

AND

METHODS

Growth and inoculation of plants Three cultivars of barley (Ho&urn uulgare L.), Trebi I, albino type, Turkey 290 and Goseshikoku, were grown in vermiculite supplied with Hyponex solution every other day at 20 + 1 “C in a growth chamber with a photoperiod of 12 h. Inoculation of the primary leaves was accomplished 7 days after sowing by blowing conidia of Erys$he graminis DC f. sp. /KWU% Marchal, race I, which shows infection type 0; with Trebi I, albino type, type 1 with Turkey 290, and type 4 with Goseshikoku. Inoculated plants were immediately placed in a moist chamber for 1 h and then replaced in the chamber mentioned above. Tissuepreparation The primary leaves collected at various periods after inoculation were immediately boiled in alcoholic lactophenol for 2 min and mounted in lactophenol after several rinsings with water. Stripped epidermal cells were immersed in water and then mounted on quartz slides in glycerol. Both preparations were used for observation of surfaces of infected leaves using fluorescence microscopy or u.v.-microspectrophotometry. Transverse sections of infection sites were made as described below. Primary leaves were cut into approximately 5 x 8 mm segments and immediately fixed in 70% ethanol. The leafsegments were further cut into approximately 2 x 4 mm, dehydrated in alcohol and embedded in an Epon 812 mixture. Sections (1 w thick) were made with a Sorvall MT-l Ultramicrotome using a glass knife. The sections were floated on a drop of water and heated over the flame of an alcohol lamp in order to stretch them out. After removal of the water, the sections were mounted in glycerol and observed under a fluorescence microscope or a u.v.-spectrophotomicroscope. Fluorescencemicroscopy The prepared tissues were examined under a Reichert fluorescence microscope with a Type BG 12 exciter filter and GG9 + OGI barrier filter (transmission range 390448 nm, peak transmission 420 run) or a Nikon fluorescence microscope using a BV exciter filter and BV absorption filter (Y). The emission spectrum and intensity of fluorescence were measured with the Reichert microscope and expressed as a

infection

of barley leaves

with Erysiphe

graminis

hordei

percentage of that emitted by an uranium glass (Hitachi). taken using a fast black and white Kodak Tri-X pan film.

949

Microphotographs

were

Microspectrophotomeby Analysis of the infection microspectrophotometer. thickness were examined done on specimens over were taken using a Fuji

sites under U.V. light was conducted with a Zeiss UMSP I The papilla and halo areas in transverse sections of lprn over the wavelength range 250 to 500 nm. Scanning was 1 (un diameter. The pictures of images absorbing U.V. light panchromatic film.

Histochemical tests Some histochemical stainings were carried out to analyse the nature of fluorescent and u.v.-absorbing substances observed in papillae and halos. The tests used were as follows : phloroglucinol-HCl and chlorine water-sodium sulfite tests for lignin [ 161, lacmoid and aniline blue fluorescence tests for callose [16], diazobenzene sulfonic acid and diazotized pnitroaniline tests for polyphenol compounds [la]. Fresh infected albino leaves or green leaves fixed in 95% alcohol were used for the above tests. If necessary, transverse sections of fresh tissues were made with a freezing microtome. Leaf samples were observed every day up to 4 days after inoculation. RESULTS

Fluorescencemicroscopy A chemical treatment is usually necessary to reveal papillae or halos in plant cells at sites of penetration by fungi. However, u.v.-fluorescence microscopy and u.v.microspectrophotometry were found to detect modified cell walls at infection sites without staining. Bright yellow autofluorescence was detected in papillae and halos at penetration points of the mildew into epidermal cells of barley (Plate 1). Fluorescent epidermal cells and lateral walls were also observed. Fluorescent lateral walls were always found within halos though fluorescence of the wall sometimes extended along the adjacent cell wall beyond a halo area. Papilla-like deposition developed on the lateral wall of a cell adjacent to the one under attack, if the papilla was formed closely to the wall between the twa cells. Although fluorescence of papilla, halo, lateral wall or papilla-like deposition was observed in both compatible and incompatible host-parasite combinations, fluorescent epidermal and mesophyll cells were formed exclusively in the latter combination. The intensely fluorescent epidermal cells in Plate 1 were previously shown to be hypersensitively collapsing by observation of transverse sections; the parasite did not form normal mature haustoria in these cells [12]. The transverse view of the fluorescent papilla is shown in Plates 2 and 3. The cup-shaped papilla developed inward on the inner surface of the cell wall, and fluorescence was emitted from the whole part of the papilla and the cell wall within a radius of halo. The fluorescence was bright yellow in all these sites as well as in guard cells when the infected tissues were also processed in an alcohol series and acetone for fixation and dehydration. However, walls of sclerenchyma in ribs and tracheary elements fluoresced yellowish green. Maximum emission of fluorescence in papillae and cell walls was 545 run regardless of the host-parasite combinations (Fig. 1).

S. Mayama and J. Shiehiyama

350 16,

6

0 500

520

540

560 Wavelength

FIG. 1. Emission spectra of the (b) papillae of powdery-mildewed barley race I; A, Goxshikoku/ race I.

560

600

I nm)

autofluorescent leaves. 0, Turkey

(a) epidennal cell 290/ race I; 0, Trebi

walls and I (albino)/

Since intense fluorescence was emitted from the papillae without using fluorochromes, we thought the fluorescence might be associated with u.v.-absorbing compounds and conducted microspectrophotometry with U.V. light. The papillae and halos revealed by fluorescence in Plate 6 were examined with various monochromatic lights. It is clearly demonstrated that there are substances which absorb U.V. light in the fluorescent papilla and halo areas (Plates 4 and 5). Under light of 500 nm wavelength, these areas were not distinguished from other parts of cells, whereas the areas were clearly imaged with light of 282 nm (Plate 4) or 325 nm (Plate 5). The images of papilla and halo revealed by fluorescence corresponded exactly to those seen under U.V. light. The U.V. absorption at 325 nm was observed especially on the papilla and lateral wall (Plate 5), and that at 282 nm was marked in the halo as well as the papilla and lateral wall (Plate 4). The halo area in a compatible host-parasite combination was usually composed of two rings as shown in Plate 6 in contrast to

Abbreviations mcd in micrograph: Ap, appressorium; cell; H, halo; LW, lateral wall; P. papilla. PLATES 1 to 3. u.v. microphotographs E. graminis hordci, race I. PLATE 1. Detection albino leaves. x 250.

C, conidium;

of incompatible

of penetration

sites with

FEC,

barley

fluorescence,

fluorescent

leaves, 3 days

Trebi after

epidermal

I, infected

with

inoculation

in the

PLATES 2 and 3. Transverse view of the fluorescent papilla and halo area in the green albino leaves, respectively, 25 h after inoculation. x 1300 and x 970, respectively. PLATES 4 to 6. Microspectrophotometric 4 days after inoculation in compatible PLATE 4. The papillae wavelength. PLATE 5. The 325 nm wavelength. PLATE

6.

analysis of the papilla and halo area with u.v. light infected with E. graminis hordei. x 235.

and halos visualized

papillae Note

Fluorescent

barley

by absorption

and halos visualized the absorption of light papillae

and

halos

PLATE 8. albino leaves.

Transverse x 1500.

PLATE 9. Transverse tion of Trebi I albino spectrum was examined PLATE inoculation

view

of the u.v.-absorbing

ofmonochromatic

by absorption in the papillae

detected

7. Fluorescent papillae and halos detected 290, by u.v.-fluorescence microscopy, 24 h after

PLATE

Turkey

and

light of282

of monochromatic and lateral walls.

by u.v.-fluorescence

25 h after

light

of

microscopy.

in the incompatible inoculation. x 220.

papilla,

nm

barley

inoculation

leaves,

of Trebi

I

view of the u.v.-absorbing cell wall in the halo area, 25 h after inoculaleaves. x400. A, B and C show the points where U.V. absorption as shown in Fig. 3.

10. The papilla visualized of an incompatible barley

by staining with diazobenzene leaves, Trebi I. x 450.

sulfonic

acid,

48 h after

lfacina Pace 3.5c

PLATES 1, 2, 3, 4, 5, 6 and

7

PLATES 8,9

and

10

infection

of barley leaves with frysiphe

gramink

horde;

351

that in the incompatible combinations in Plates 1 and 7. The inner area adjoining directly to the papilla was less fluorescent and absorbed less U.V. light than the outer ring area. Plates 8 and 9 show transverse views of the u.v.-absorbing papilla and halo areas, respectively, in the incompatible host-parasite combination 25 h after inoculation. The U.V. absorption spectrum of autofluorescent papilla and epidermal cell wall was examined in transverse sections of 1 v thickness (Figs 2 and 3). Maximum absorp tion was-etected at about 282 nm and 325 nm in both papilla and cell wall, and was

s 6 c f a

Wavelength( nm) Fm. 2. Absorption spectra of autofluorescentpapilla and epidermal cell wall of barley leaves (Trebi I) infected with E. graminis h&i (race I). 1, Papilla; 2, xylem; 3 and 4, fluorescent cell wall; 5, non-fluorescent cell wall; 6, cell wall in the uninoculated plant.

basically similar to that at 280 nm and 325 nm in xylem tissues. However, the 280 : 320 absorbance ratio in the papilla and cell wall was much lower than that in xylem tissue. No absorption of U.V. light was found in uninoculated barley epidermal cells and the non-fluorescent wall of infected cells (Fig. 2). Absorption spectrum of U.V. light was examined at three points in the u.v.-absorbing cell wall of a halo area as shown in Plate 9 and Fig. 3. There were strong U.V. absorptions at both 282 nm and 325 mu in the fluorescent cell walls; absorption at 325 nm was stronger in the central area of the halo than in the peripheral region, where absorption at 282 run was marked.

S. Mayama and J. Shishiyama

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240

280

.A

...: ..’

,-.., .: \I

320 Wavrlrnpth

FIG. 3. Absorption

- 0.8 ‘...

360

400

440

(nm)

spectra and AE curve of the fluorescent halo area in incompatible barley leaves (Trebi I) infected with A, B and C show the different sites in the fluorescent halo area spectrum was measured as in Plate 9.

epidermal

cell wall

E. graminis ha& where

the

u.v.

of the (race I), absorption

Hi.dochmnist~ The tests with phloroglucinol-HCl and chlorine water-sodium sulfite for lignin did not give a clear positive reaction in the papilla and halo areas. However, phloroglucinolHCl showed a red staining in tracheary elements and a faint one in girder sclerenchyma, while chlorine water-sulfite gave a definite positive reaction in both tissues. The papillae gave an orange-red color with the diazobenzene sulfonic acid test and a yellow colour with the nitroaniline test, indicating that polyphenol compounds may be contained in the papillae (Plate 10). Tracheary elements, sclerenchyma and guard cells were also positively stained with diazobenzene sulfonic acid. The lacmoid test for callose was faintly positive in the region of papilla deposition. However, the aniline blue fluorescence test for callose did not intensify fluorescence in papillae much compared with the greenish-yellow fluorescence in background tissues; in contrast, the papilla was easily observed by its bright yellow fluorescence without treatment with aniline blue. DISCUSSION

It was observed in the present study that the papilla and cell wall in the halo strongly absorb U.V. light and it was demonstrated that substances with an aromatic nature

Infection

of barley leaves with Erysiphe graminis hordei

353

were deposited locally at the penetration sites of the fungi in barley leaf epidermal cells. The major compounds deposited in infection sites in the mildewed barley leaves could be different from lignin, the accumulation of which has been clearly demonstrated in other diseases [4, 161, since the sites did not give a definite positive reaction in the histochemical tests for lignin. Although callose has been demonstrated in papillae of powdery-mildewed host cells with the aniline blue fluorescence method [17], the fluorescent compounds demonstrated here should not be callose, since the emission could be observed without using aniline blue [l.?, 131and the polysaccharide, a p-1,3 glucan, does not absorb near-u.v. light. Autofluorescence found in those sites could be associated with the phenolic compounds. The previous observation indicated that fading in fluorescence and browning in the infected cells were highly correlated

[W In powdery mildew of barley, it has been demonstrated that most parasite units in resistant host cells ceased their growth especially during the stage of penetration into host cells [S, 10, 14, 171. Thus, the localized accumulation of phenolic compounds at penetration sites might have a chemical role in controlling the infectivity of the mildew fungus in addition to a role of papillae as a .physical barrier as shown previously [S, 201. Oku et al. [15] recently pointed out that a phytoalexin, pisatin, synthesized in powdery-mildewed pea seedlings has an inhibitory activity on the infectivity of the pathogen in the primary infection process in addition to prevention of the colony expansion. The morphological difference (Plates 6 and 7) and the intensity of fluorescence of the depositions at infection sites are most likely to be correlated with infection establishment; the results will be published elsewhere. Chemical analysis of the fluorescent and u.v.-absorbing compounds, which accumulated locally at the mildew-infected sites, is necessary for elucidation of the role of these compounds in resistance. We thank Dr M. Fukutomi and Dr P. Park for their help and advice in the experimental preparation. We are greatly indebted to Professor U. Hiura, Institute of Agricultural Biology, Okayama University, for his generous supply of barley seeds; Professor K. Takeuchi, Laboratory of Cytology, Faculty of Science, Kyoto University, for his help with the fluorescing microscope; and Professor 0. Midorikawa and Mr M. Fujioka, Department of Pathology, Faculty of Medicine, Kyoto University, for the use of the Zeiss UMSP I microspectrophotometer.

REFERENCES

of

1. AIST, J. R. (1976). Papillae and related wound plugs of plant cells. Annual Rcuicw Phytopnthology 14, 145-163. 2. Arvr, J. R. & ISRAEL, H. W. (1977). Effectv of heat-shock inhibition of papilla formation on compatible host penetration by two obligate parasites. Physiological Plant Pathology 10, 13-20. 3. AKAI, S. (1959). Histology of defence in plants. In Plant Patho&, Ed. by J. G. Horsfall & A. E. Dimond, pp. 391-434. Academic Press, New York. 4. &ADA, Y. & MATWJMOTO, I. (1971). Microspectrophotometric observations on the cell wall of Japanese radish (Ra&au.s sativur) root infected by Pcronospwa parasitica. Physiological Plant Pathology 1, 377-383. 5. BRACKER, C. E. & LIITLEPIELD, L. J. (1973). Structural concepts of host-pathogen interlhces. In Fwtgal Pathogmicity and the Plant’s Rm~onse, Ed. by R. J. W. Byrde & C. V. Cutting, pp. 159317. Academic Prcra, New York.

354

S. Mayama and J. Shishiyama

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