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Induced susceptibility and enhanced resistance at the cellular level in barley coleoptiles. I. The significance of timing of fungal invasion
Physiological
Plant Pathology
( 1985) 27,43%54
Induced susceptibility and enhanced resistance at the cellular level in barley coleoptiles. I. The significance of timing of fungal invasion* HITOSHI KUNOH, AKIO HAYASHIMOTO, Laborato!e
of Plant Pathology,
;Accepted,forpublication
Februav
Facula
of Agriculture,
MASARU HARUI and HIROSHI ISHIZAKI Me
Uniuenity,
Tsu-ciy,
514, Japan
1985)
Growth of Erysiphe graminis hordei and Erysiphe pisi on the same cells of barley coleoptiles was observed in detail by light microscopy to determine significant factors conditioning host cells toward susceptibility. When E. pisi attempted penetration more than 60 min earlier than E. graminis on the same coleoptile cell, E. pisi never succeeded in penetration (0% penetration efficiency) and the penetration efficiency of E. graminis was lowered from 75.0 to 28.6%, suggesting that the resistance to E. graminis invasion might be enhanced under this condition. When both fungi attempted penetration of the coleoptile cell almost simultaneously (within 30 min of each other), the penetration efficiency of E. pisi increased to 11.8%. Moreover, the penetration efficiency of E. graminis was 55.8%. When E. graminis attempted penetration 60 min or more earlier than E. pisi, the mean penetration efficiency of E. pisi was 29.20/, and that of E. graminis to 75,0”,. These observations suggest that the coleoptile cells are conditioned toward susceptibility by prior-penetration of E. graminis or even by its post-penetration if it penetrates after E. pisi. E. pi~i developed haustoria only in coleoptile cells where E. graminis formed haustoria. The induced susceptibility and enhanced resistance states in coleoptile cells had not transferred to the adjacent cells within 3 h.
INTRODUCTION Why some micro-organisms are capable of establishing infection in certain plants, while the majority are not, has been one of the most interesting and basic questions in plant pathology. Clues to the answer to this question have been given in a number of papers concerning susceptibility and resistance enhanced by pathogens and nonpathogens. Since the first report of increased susceptibility to yellow rust in buntinfected wheat, induced susceptibility has been reported in many fungus-plant combinations, especially in diseases caused by obligate parasites [II]. Tsuchiya & Hirata [I91 reported that prior inoculation of barley leaves with a strain of E&& graminis that was virulent on the host cultivar allowed 30 out of 51 infections originally avirulent on that cultivar to produce conidia on the leaves. Ouchi and colleagues [ll, 12, 131 extensively studied induced susceptibility and resistance using the barley and wheat-E. graminis systems and made many interesting observations. (i) Susceptibility to an originally avirulent race of E. graminis or resistance to an originally virulent race *Contribution No. 69 from the Laboratory of Plant Pathology, Mie University. This work was partially supported by Grant-in-Aid for Scientific Research (1980) from the Ministry of Education and Science of -Japan. 0048.-4059/85/010043
+ 12 $03.00/O
0
1985 Academic
Press Inc.
(London)
Limited
H. Kunoh et al.
44
was induced in barley leaves by prior inoculation of the virulent or avirulent race, respectively [12]. (ii) Susceptibility ofbarley leaves induced by prior inoculation with a virulent race of E. graminis was effective in promoting additional development not only of avirulent races of that fungus but also of other powdery mildew fungi and necrotrophic non-pathogenic fungi [13]. (iii) For the completion of cellular conditioning toward susceptibility, 15-18 h were required, while conditioning toward resistance required only 6 h [II]. Based on their results, Ouchi et al. [l2] proposed the term accessibility to connote the acceptance-rejection relations in parasite-plant interaction at levels of tissues, cells and molecules, and reserved the susceptibility for expressing the parasite-host relations at the organ and whole plant levels. However, in the inoculation method used by Ouchi et al. [II, 12, 131 the secondary inoculum (challenger) was inoculated onto the barley leaves after the primary inoculum (inducer) had been wiped off with a wet cotton ball. Therefore, it is hard to observe by their method how inducers and challengers grow on the same host cells, how they interact with each other and how host cells respond to the invaders. In this sense, their method is inadequate to discuss accessibility at the cellular level. If the above terminology of accessibility is applied, the discussion of this phenomenon should be based on detailed observation of the growth of inducers and challengers on single, living host cells and of host cell responses. For cytological studies of powdery mildew, Bushnell et al. [q provided an excellent host cell system, namely, partially dissected barley coleoptiles which consisted of onecell layer of epidermis. The cells which comprise this epidermis are relatively uniform in morphology. Since this system was reported in 1967, it has greatly promoted studies of the events during the infection of barley and wheat by E. gruminis [l-3,5,9,16,20]. In the present study, by employing this system we observed both the growth of E. graminis hordei and Erysiphe pisi fungi on the same barley coleoptile cells and host cell responses, and attempted to determine significant factors conditioning responses of host cells to fungal invasion.
MATERIALS
AND METHODS
Test plant undfungi Barley (Hordeum uulgure L. cv. Kobinkatagi) was used as a host plant. Erysiphegruminis f. sp. hordei race I (abbreviated as E. gruminis below) and Erysiphepisi race 1 (E. pisi below) were employed as a pathogenic and a non-pathogenic fungus, respectively. Erysiphe gruminis was maintained on cultivar Kobinkatagi in a chamber maintained at 20 “C, 70:/, relative humidity (RH) under 4000 lx of fluorescent light for 12 h per day. of pea (Pisum sutiuum L.) in a separate Evsiphepisi was maintained on cultivar “Alaska” but similar chamber.
Specimen preparation Barley was grown from Coleoptiles were excised dermal layers of partially [16]. The coleoptiles were
seed under 4000 lx of fluorescent light for 14 h per day. from barley seedlings 9 days after sowing and single cell epidissected coleoptiles were prepared, as previously described floated on 0.01 M CaCl, solution in Petri dishes.
Cell level susceptibility
and resistance
of barley I
45
E. pisi A E. graminis
p>7-------,v;-, 31*
0
E plsi E grominis
VL--------/.;-, 67
IO
33
1213
D
16
C. pisi E. graminis
VP&
- -Y1415
1------;TT, 10
Observation
I
12 V V -L.
Inoculation Time
FIG. 1. Time courses observation of test conidia
time
of transfer
r---l of E. pisi
conidio
l
Time after
Incubation
inoculation
A-D (time given in hours) of inoculation, ofErysiphepisi and Erysiphegraminic.
of 15 gramhis
incubation,
transfer,
and
Inoculation and transfer of conidia, incubation and observation One pair of coleoptiles was prepared for each experiment. One coleoptile (coleoptile A) was inoculated with 15-20 freshly harvested conidia of E. graminis by a brush. One to eleven hours after inoculation with E. graminis, another coleoptile (coleoptile B) was inoculated similarly with about 100 freshly harvested conidia of E. pisi, following the time schedule in Fig. 1. Such timing of inoculation of the two fungi allowed them to attempt penetration at time intervals mentioned below. Before transfer of germinating conidia of E. pisi, germinating conidia of E. graminis each having an appressorial apex on a single coleoptile celi were selected [Fig. 2 (a)]. Selection of conidia was such that no other germlings were located within five host cells from the selected conidia. Three hours after inoculation of E. pisi, selected conidia of E. pisi which had an appressorial germ tube were each transferred with a micromanipulator from coleoptile B to coleoptile A so that appressoria of transferred E. pisi attempted to penetrate the same cells of coleoptile A as did appressoria of untransferred E. graminis [Fig. 2(b)]. Using separate pairs of coleoptiles, selected conidia of E. pisi on coleoptile B were transferred onto laterally adjacent cells of E. graminis-attacked cells of coleoptile A. In each experiment, five to 10 conidia ofE. pisi were transferred and thus five to 10 pairs of both fungi were prepared on one coleoptile A as the subjects of each experiment. Transfer of E. pisi was completed within 1 h after initiation of the manipulation. Immediately after the transfer was completed, coleoptile A was returned to the Petri dish and incubated for 3 h. Subsequently, coleoptile A was mounted with 0.01 M CaCl, on a glass slide and covered with a cover slip. These specimens were set, cover-slip side down, on a Zeiss inverted interference contrast microscope IM35, and fungal growth and host cell response were observed at x 400 every 15 min. For each pair of both fungi, the timing of initiation of cytoplasmic aggregates induced by the respective fungi was recorded. Coleoptile A was again incubated for a further 26 h, and subsequent haustorium formation was recorded. Data were accumulated from more than 100
l-l. Kunoh et al.
46
FIG. 2. (a) A germinating conidium of E. graminis having an appressorial germ tube b! A germinating conidium of E. pisi, grown on a separate coleoptile, after transfer micromanipulator onto the cell where the E. gralninis appressorium was developing. Bar=
(AGT). with a 10 pm.
separate, but replicated, experiments performed over a period of two years. During manipulation and observation, the microscope room was maintained at 20 f 2 “C and 70° (, RH. RESULTS Growth of E. graminis and E. pisi singly inoculated onto barley coleoptiles, and host cell responses Conidia of E. gramilzis, inoculated onto barley coleoptiles usually produced primary germ tubes between 1 and 2 h after inoculation and appressorial germ tubes between 3 and 4 h after inoculation [9]. When mature appressoria attempted to penetrate host walls between 10 and 12 h after inoculation, host cytoplasmic aggregates were initiated at the sites of attempted penetration. Within 1 to 2 h after initiation of cytoplasmic aggregates, incipient globose haustorial bodies became visible at apices of penetration
Cell level susceptibility
and resistance
FIG. 3. (a) A haustorium ,&r formed in a coleoptile gminis. Bar= 10 pm.
of barley I
(H) ofE. g raminir in a coleoptile cell. (b) A globose haustorium cell where susceptibility has been induced by prior-penetration
of E. by E.
pegs arising from appressorial lobes. They gradually developed to form typical haustoria with branched finger-like projections [Fig. 3 (a)]. When E. graminis alone was inoculated onto coleoptiles, 70-80% of conidia succeeded to produce haustoria. On the other hand, between 1 and 2 h after inoculation, one appressorial germ tube arose from conidia of E. pisi without emergence of short germ tubes corresponding to reported by Kunoh et al. [IO]. Apices of primary germ tubes of E. graminis, as previously the appressorial germ tubes swelled to form unique polymorphic appressoria usually of two to five short lobes. Host responses to appressorial penetration by this fungus was unique in that there were two stages of cytoplasmic aggregates. Between 6 and 7 h after inoculation, small cytoplasmic aggregates occurred beneath one of the short lobes of
48
H. Kunoh et al.
appressoria. Appressorial lobes sometimes interferred with observation of initiation of such small cytoplasmic aggregates. These aggregates lasted generally for 15-45 min, then disappeared. After an additional period ranging from 15 to 60 min, sometimes longer, active cytoplasmic aggregates of larger diameter developed beneath the same lobes of the appressoria. About 30 min after recurrence of cytoplasmic aggregates, clear, circular structures, probably corresponding to penetration pegs or penetration pores in host walls, suddenly formed in the lobes or host walls beneath the lobes. Among 76 inoculated conidia, such a structure was apparently distinguishable in 59 appressoria. In response to penetration by E. PC, papillae were commonly formed under appressoria but sometimes appressorial lobes were too large to distinguish papillae that may have formed underneath them. Single inoculations of E. pisi were never successful in producing haustoria in coleoptile cells. The unique response of barley coleoptile cells to E. pisi will be published elsewhere.
Timing of attemptedpenetrationsby E. graminis and E. pisi on the samecoleoptilecells Initiation of a cytoplasmic aggregate was usually the first visible response of a host cell at the attempted penetration sites from appressoria of both fungi, and thus the time of initiation of the cytoplasmic aggregate was considered to be the first sign of attempted penetration by the respective fungi. As mentioned above, two successive cytoplasmic aggregates occurred under the same appressorium of E. pisi. The second cytoplasmic aggregate was accompanied by the appearance of a penetration-pore-like structure in the lobe but the first one was not. Therefore, it was inferred that it was ,the second cytoplasmic aggregate, not the first, that signalled the time of host wall penetration by E. pisi. Thus, the following description and discussion of the time of E. pisi concerns the second cytoplasmic aggregate. Figure 4 illustrates growth patterns of both E. graminis and E. pisi on the same coleoptile cell and the host cell responses. By 9 h after inoculation of E. graminis, both fungi formed an appressorium which appeared morphologically mature [Fig. 4(a)]. The cytoplasmic aggregate was seen below only the appressorium of E. graminis 10 h 45 min after inoculation of E. graminis [Fig. 4(b)]. Thirty minutes later, a penetration peg was seen to form from a lobe of the appressorium [Fig. 4(c)]. The cytoplasmic aggregate was initiated beneath the appressorium of E. pisi 11 h 30 min after inoculation of E. graminis, as shown at the right of Fig. 4(d). At that time the cytoplasmic aggregate persisted below the appressorium of E. graminis. At 13 h 15 min, a penetration-pore-like structure was distinguished in a lobe of the E. pisi appressorium below which the cytoplasmic aggregate persisted [Fig. 4(e)]. Figure 4(f) illustrates growth patterns of both fungi observed 33 h after inoculation of E. graminis: by this time E. graminis successfully penetrated to form a haustorium followed by elongation of a secondary hypha from the primary lobe of the appressorium, but E. pisi failed to penetrate from the primary lobe and attempted another penetration from the secondary lobe. Thus, Fig. 4 illustrates an example in which E. graminis attempted penetration 45 min earlier than E. pisi, since the cytoplasmic aggregate was first seen below E. graminis and E. pisi, respectively, 10 h 45 min [Fig. 4(b)] and 11 h 30 min [Fig. 4(d)] after inoculation of E. graminis. Similarly, the timing of the initiation of a cytoplasmic aggregate below the respective fungi was determined for each pair of appressoria through successive experiments. If the timing for either member of a pair failed to be
Cell level susceptibility
and resistance
of barley I
FIG. 4. A series of interference contrast micrographs showing successive growth patterns of E. graminis (G) and E. pisi (P) on a single barley coleoptile cell. The number in the upper right corner of each frame indicates the time after inoculation of E. graminis. The bar in frame a indicates IOpm. (a) By 9 h, appressoria (APP) of both fungi had matured. (b) At 10h 45min, a cytoplasmic aggregate (CA) was seen beneath the appressorium ofE. graminis. (c) At 11 h 15 min, a papilla (PA) was observed at the apex of the penetration peg (PP) of E. graminis. (d) At 11 h 30 min, a small cytoplasmic aggregate occurred beneath the appressorium of E. pk. (e) At 13 h 15 min, a circular structure (PP) corresponding to either a penetration peg or a penetration pore was visible in the primary lobe of E. pisi below which the active cytoplasmic aggregate had persisted. (fi By 33 h, the E. graminis appressorium had penetrated, forming a haustorium (H) and elongating secondary hypha (ESH). On the other hand, the E. pisi appressorium had failed to penetrate from the primary lobe (PL) and tried another penetration from a secondary lobe (SL).
49
50
H. Kunoh et al. E. pisi
w
180 w
15 grominis
v
%
‘13
v
O/5
‘15
v
O/IO
v
%‘I
150 w
0.0%
%o
28.6%
120 w 90 v
v 60 WV 30
O/IO
%6
V 0
%
VV 30
I
Z/i/II
v 60 w
v
%O
V
1
Z/9
90
840
1
519
’ 75.0%
29.2%
v
V
2/2
212
‘/3
%
120 v
150
V
FIG. 5. Effects ofrelative time (min) ofinitiation pisi (V) and Erysiphe graminis (v) on haustorium coleoptile cells of barley.
.
ofcytoplasmic aggregates induced by Eysiphe formation by the respective fungi on the same
determined, the datum pair was abandoned. Timing of the initiation of cytoplasmic aggregates and rates of haustorium formation (penetration efficiency) of the respective fungi were successfully determined for 134 appressorial pairs. These results are summarized in Fig. 5, based on time-intervals between the initiation of cytoplasmic aggregates induced by both fungi. When E. pisi attempted penetration more than 60 min earlier than E. gruminis, none of the E. pisi appressoria penetrated successfully. Moreover, penetration efficiency of E. graminis lowered to 28.6% on average, ranging from 20.0 to 40.0%. However, when both fungi attempted penetration almost simultaneously (within 30 min of each other), the penetration efficiency of E. pisi increased to l1*8o/o. Moreover, the penetration efhciency of E. graminis recovered to 55.8% on average. When E. graminis attempted penetration 60 min or more earlier than E. pisi, the mean penetration efficiency of E. pisi was enhanced to 29.2% and that of E. graminis to 75.0%. As indicated in Fig. 3(b), E. pisi produced a globose haustorium in a barley coleoptile cell having induced susceptibility as reported previously [4]. How long does such an effect of prior-attack by E. gruminis on haustorium formation by E. pisi last? As shown in Fig. 6, when E. graminis attempted penetration 300&600 min earlier than E. pisi, the mean penetration efficiency of E. pisi was 36.613, and that ofE. graminis was 71.0%. Thus, the induced susceptibility was effective for at least 5 10 min, probably until 600 min. As mentioned above, E. pisi, which, by itself, was originally avirulent to barley coleoptile cells, became capable of forming haustoria in cells which were conditioned toward susceptibility. However, successful penetration by E. pisi occurred only when the penetration of E. graminis was successful in the same cells.
Cell level susceptibility
and resistance
of barley I
51
E. pisi T
v
E graminis
%5
300 T
V
%5
%5
330 T
v
‘3126
360 T
v
%s
390 T
v
“127
420 'I
v
2/l 4
450 T
v
%I
480 T 'I
FIG.
2/5 0
540
T
I penrtration
v
510
‘12
v
600
%
6. Effect of timing (min) of prior-attack by E. gramlnis (T) on haustorium efficiency) by E. pisi ( 5’ ) on the same coleoptile cells of barley.
2h % v5I
E pisi
E. gramhis
%
% v
T
%
T
Oh
T
%
120 v 90 v
formation
77.4%
O/12
--k--V 0 VW 30 'I v 60 T v 90 T 120
T
150
T
1.0%
O/9
O/b
%
V v B
180 FIG. 7. Effect of the relative E. plsi i V) and E. graminis (v) coleoptile cells of barley.
VI6
3Ll
-
-
-
-
“1,
'/I
' 72.7%
,
time (min) of initiation of cytoplasmic aggregates on penetration efficiency by the respective fungi
induced by on adjacent
The e$ect of prior-attack on interactions with adjacent coleoptile cells ‘To determine whether this susceptibility and enhanced resistance can be transferred to the adjacent cells, E. pisi conidia with a short appressorial germ tube were placed onto cells adjacent to E. graminis attacked cells. As shown in Fig. 7, E. pisi never produced
52
H. Kunoh
etal.
haustoria in adjacent cells either when E.&i attempted penetration 30-180 min earlier than E. graminis or when the latter did so 30-180 min earlier than the former. On the other hand, the penetration efficiency of E. graminis was maintained at the level of 72.7-77.476 in all cases, except that a somewhat lower rate of 59.5% was obtained when both fungi penetrated almost simultaneously (within 30 min of each other).
DISCUSSION
The present results definitely show that barley coleoptile cells are conditioned toward susceptibility by prior-attack by a pathogenic fungus and that the resistance of the cell is enhanced when a prior-attack by a nonpathogenic fungus occurs at least 60 min earlier than attack by a pathogenic fungus. It is apparent from Fig. 5 that the timing of attempted penetration by both fungi is very important in determining the cellular response. When E. pisi attempted penetration more than 60 min earlier than E. graminis, the penetration efficiency of E. graminis was reduced to only 28.6%, indicating that the resistance of coleoptile cells is enhanced under this condition. Conversely, when E. graminis attempted penetration earlier than E. pisi, or when penetration was almost simultaneous, the coleoptile cells were conditioned toward susceptibility to E. pisi. Under these conditions, the resistance which the coleoptile cells possess natively might be disrupted in some way. A low penetration efficiency of E. pisi (11.80/,) and intermediate one of E. graminis (55.874) obtained when both attempted penetration almost simultaneously probably indicates a state of coleoptile cells insufficiently conditioned toward susceptibility. The present observation that the timing of cytoplasmic aggregates occurring near the plasmalemma (and thus traversal of the host wall) is associated with cellular conditioning led us to recall the hypothesis of Ellingboe & Slesinski [fl that the gene in the host will be constitutive in function and confer specificity at the level of the plasmalemma. However, as Tani et al. [17J reported, cellular conditioning of oat leaf tissues toward resistance to incompatible races of Puccinia coronata avenueis irreversibly established between 8 and 12 h after inoculation when the fungus has penetrated only through stomata. Their results suggest that penetration through the plasmalemma might not be a prerequisite for the alteration of the cellular condition of the host. With barley leaves and several races of E. graminis as employed by Ouchi et al. [!I, 12,131, 15-18 h were required to complete cellular conditioning toward susceptibility, while conditioning toward resistance required only 6 h. However, unlike their results, the present data indicate that as little as 60 min may be required to change the coleoptile cells towards susceptibility. It was also suggested that, at the cellular level, enhancement of resistance and induction of susceptibility are gradual processes crossing over & 30 min when either fungus attempts penetration earlier. As indicated in Figs 5 and 6, the penetration efficiency of E. pisi obtained was at most 36.676, even if the coleoptile cells were penetrated first by E. graminis hordei. Using the similar micromanipulation technique, Kunoh [S] transferred conidia of a compatible race of E. graminis onto barley coleoptile cells and observed their growth after inoculation with the purpose to elucidate the effect of prior-attack by primary germ tubes of 6. graminis conidia on the penetration efficiency of their appressoria. In that experiment, the penetration efficiency of transferred E. graminis conidia was at most
Cell level susceptibility
and resistance
53
of barley I
45.8-48.5% in contrast with 7O-8Oo/o of untransferred conidia. Thus, the low penetration efficiency of E. PG obtained in this study might be due to the transfer effect, although we cannot rule out another possibility that a substantial resistance which is not affected by the timing offungal penetration might be involved in suppression of the penetration of E. pisi. Ouchi et al. [14, 15] showed that Sphaerotheca fuliginea colonized the barley cells harbouring a compatible E. graminis haustorium with a very high frequency, but it was incapable of colonizing cells located four cell rows apart from the haustoriumharbouring cells, and emphasized that both the induced resistance and susceptibility were localized in the area where the primary interaction was effectively completed. Their data evidently show that the induced cellular conditions are transferred to iadjacent cells or nearby cells. The data presented here indicate that the induced :susceptibility and enhanced resistance are not transferred within 3 h from affected cells to adjacent cells. Our separate experiments (unpublished) have shown that the time intervals of attempted penetration of both fungi required to transfer the cellular condition from one cell to the adjacent cell is at least 18-20 h. These results raise questions of how the timing of attempted penetration by both fungi is related to the final cellular conditioning of the host or how the phenomena revealed herein are associated with various modes of suppression of host defense mechanisms which have been postulated by many earlier researchers in a variety of parasite-host systems [15, 17, 181. Although Ouchi & Oku [II] emphasized that the phenomena relating to induced susceptibility should be evaluated on a molecular basis, the present study suggests that more detailed cytological observation can be helpful before discussing the genetic or molecular aspects of these phenomena. We are indebted and for correcting
to Dr James the English.
R. Aist, Cornell
University,
for his valuable
discussion
REFERENCES 1. AIST, J. R. & ISRAEL, H. W. (1977). Papilla formation: Timing and significance during penetration of barley coleoptiles by Erysiphe graminis hordei. Phytopathology 67,455-46 1. 2. AIS~, J. R., KUNOH, H. & ISRAEL, H. W. (1979). Challenge appressoria of Erysiphe graminis fail to breach preformed papillae of a compatible barley cultivar. Phytopatholou 69, 1245-1250. 3. BUSHNELL, W. R. & BERGQUIST, S. E. (1975). Aggregation of host cytoplasm and the formation of papillae and haustoria in powdery mildew of barley. Phytopathobu 65, 31G-3 18. 1. BUSHNELL, W. R. & GAY, J. (1978). Accumulation of solutes in relation to the structure and function of haustoria in powdery mildew. In The Powdery Mildews, Ed. by D. M. Spencer, pp. 183-235. Academic Press, London. 5. BUSHNELL, LV. R. & ZEYEN, R. J. (1976). Light and electron microscopy studies of cytoplasmic aggregates formed in barley cells in response to Erysiphe graminis. Canadian Journal of Botany 54, 1647-1655. 6. BUSHNELL, W. R., DUECK, J. & ROWELL, J. B. (1967). Living haustoria and hyphae of Erysiphegraminis f. sp. hordei with intact and partly dissected host cells of Hordeum uulgare. Canadian Journal of Botany 45, 1719-1732. 7. ELLINGBOE, A. H. & SLESINSKI, R. S. (1971). Genetic control of mildew development. In Barley Genetics, F.d. by R. A. Nilan, pp. 472-474. Washington State University Press, Pullman. 8. KCNOH, H. (1982). Primary germ tubes of Erysiphe graminic conidia. In Plant Infection: The Physiological and Biochemical Basis, Ed. by Y. Asada, W. R. Bushnell, S. Ouchi & C. P. Vance, .pp.. 45-59. -Japan . Scientific Societies Press, Tdkyo and Springer-Verlag, Berlin. 9. KI~NOH, H., Am, J. R. & ISRAEL, H. W. (1979). Primary germ tubes and host cell penetrations from appressoria of Erysiphe graminis hordei. Annals of the Phytopathological Society ofJapan 4.5,326-332.
54
H. Kunoh
et al.
IO. KUNOH, H., ITOH, O., KOHNO, M. & ISHIZAKI, H. (1979). Are primary germ tubes of conidia unique to ETsz$he graminis? Annals of the Phytopathological Society ofJapan45,675602. I I. OUCHI, S. & OKU, H. (1982). Physiological basis of susceptibility induced by pathogens. In Plant Infection: The Physiological and Biochemical Basis, Ed. by Y. Asada, W. R. Bushnell, S. Ouchi & C. P.
Vance,
12. 13.
14. 15.
16.
17.
18.
19.
20.
pp. 117-136. Japan Scientific Societies Press, Tokyo and Springer-Verlag, Berlin. S., OKU, H., HIBINO, C. & hIYAMA, 1. (1974). Induction of accessibility and resistance in leaves of barley by some races of Erysiphe graninis. Phytopathologische
Plant Pathology
( 1985) 27,43%54
Induced susceptibility and enhanced resistance at the cellular level in barley coleoptiles. I. The significance of timing of fungal invasion* HITOSHI KUNOH, AKIO HAYASHIMOTO, Laborato!e
of Plant Pathology,
;Accepted,forpublication
Februav
Facula
of Agriculture,
MASARU HARUI and HIROSHI ISHIZAKI Me
Uniuenity,
Tsu-ciy,
514, Japan
1985)
Growth of Erysiphe graminis hordei and Erysiphe pisi on the same cells of barley coleoptiles was observed in detail by light microscopy to determine significant factors conditioning host cells toward susceptibility. When E. pisi attempted penetration more than 60 min earlier than E. graminis on the same coleoptile cell, E. pisi never succeeded in penetration (0% penetration efficiency) and the penetration efficiency of E. graminis was lowered from 75.0 to 28.6%, suggesting that the resistance to E. graminis invasion might be enhanced under this condition. When both fungi attempted penetration of the coleoptile cell almost simultaneously (within 30 min of each other), the penetration efficiency of E. pisi increased to 11.8%. Moreover, the penetration efficiency of E. graminis was 55.8%. When E. graminis attempted penetration 60 min or more earlier than E. pisi, the mean penetration efficiency of E. pisi was 29.20/, and that of E. graminis to 75,0”,. These observations suggest that the coleoptile cells are conditioned toward susceptibility by prior-penetration of E. graminis or even by its post-penetration if it penetrates after E. pisi. E. pi~i developed haustoria only in coleoptile cells where E. graminis formed haustoria. The induced susceptibility and enhanced resistance states in coleoptile cells had not transferred to the adjacent cells within 3 h.
INTRODUCTION Why some micro-organisms are capable of establishing infection in certain plants, while the majority are not, has been one of the most interesting and basic questions in plant pathology. Clues to the answer to this question have been given in a number of papers concerning susceptibility and resistance enhanced by pathogens and nonpathogens. Since the first report of increased susceptibility to yellow rust in buntinfected wheat, induced susceptibility has been reported in many fungus-plant combinations, especially in diseases caused by obligate parasites [II]. Tsuchiya & Hirata [I91 reported that prior inoculation of barley leaves with a strain of E&& graminis that was virulent on the host cultivar allowed 30 out of 51 infections originally avirulent on that cultivar to produce conidia on the leaves. Ouchi and colleagues [ll, 12, 131 extensively studied induced susceptibility and resistance using the barley and wheat-E. graminis systems and made many interesting observations. (i) Susceptibility to an originally avirulent race of E. graminis or resistance to an originally virulent race *Contribution No. 69 from the Laboratory of Plant Pathology, Mie University. This work was partially supported by Grant-in-Aid for Scientific Research (1980) from the Ministry of Education and Science of -Japan. 0048.-4059/85/010043
+ 12 $03.00/O
0
1985 Academic
Press Inc.
(London)
Limited
H. Kunoh et al.
44
was induced in barley leaves by prior inoculation of the virulent or avirulent race, respectively [12]. (ii) Susceptibility ofbarley leaves induced by prior inoculation with a virulent race of E. graminis was effective in promoting additional development not only of avirulent races of that fungus but also of other powdery mildew fungi and necrotrophic non-pathogenic fungi [13]. (iii) For the completion of cellular conditioning toward susceptibility, 15-18 h were required, while conditioning toward resistance required only 6 h [II]. Based on their results, Ouchi et al. [l2] proposed the term accessibility to connote the acceptance-rejection relations in parasite-plant interaction at levels of tissues, cells and molecules, and reserved the susceptibility for expressing the parasite-host relations at the organ and whole plant levels. However, in the inoculation method used by Ouchi et al. [II, 12, 131 the secondary inoculum (challenger) was inoculated onto the barley leaves after the primary inoculum (inducer) had been wiped off with a wet cotton ball. Therefore, it is hard to observe by their method how inducers and challengers grow on the same host cells, how they interact with each other and how host cells respond to the invaders. In this sense, their method is inadequate to discuss accessibility at the cellular level. If the above terminology of accessibility is applied, the discussion of this phenomenon should be based on detailed observation of the growth of inducers and challengers on single, living host cells and of host cell responses. For cytological studies of powdery mildew, Bushnell et al. [q provided an excellent host cell system, namely, partially dissected barley coleoptiles which consisted of onecell layer of epidermis. The cells which comprise this epidermis are relatively uniform in morphology. Since this system was reported in 1967, it has greatly promoted studies of the events during the infection of barley and wheat by E. gruminis [l-3,5,9,16,20]. In the present study, by employing this system we observed both the growth of E. graminis hordei and Erysiphe pisi fungi on the same barley coleoptile cells and host cell responses, and attempted to determine significant factors conditioning responses of host cells to fungal invasion.
MATERIALS
AND METHODS
Test plant undfungi Barley (Hordeum uulgure L. cv. Kobinkatagi) was used as a host plant. Erysiphegruminis f. sp. hordei race I (abbreviated as E. gruminis below) and Erysiphepisi race 1 (E. pisi below) were employed as a pathogenic and a non-pathogenic fungus, respectively. Erysiphe gruminis was maintained on cultivar Kobinkatagi in a chamber maintained at 20 “C, 70:/, relative humidity (RH) under 4000 lx of fluorescent light for 12 h per day. of pea (Pisum sutiuum L.) in a separate Evsiphepisi was maintained on cultivar “Alaska” but similar chamber.
Specimen preparation Barley was grown from Coleoptiles were excised dermal layers of partially [16]. The coleoptiles were
seed under 4000 lx of fluorescent light for 14 h per day. from barley seedlings 9 days after sowing and single cell epidissected coleoptiles were prepared, as previously described floated on 0.01 M CaCl, solution in Petri dishes.
Cell level susceptibility
and resistance
of barley I
45
E. pisi A E. graminis
p>7-------,v;-, 31*
0
E plsi E grominis
VL--------/.;-, 67
IO
33
1213
D
16
C. pisi E. graminis
VP&
- -Y1415
1------;TT, 10
Observation
I
12 V V -L.
Inoculation Time
FIG. 1. Time courses observation of test conidia
time
of transfer
r---l of E. pisi
conidio
l
Time after
Incubation
inoculation
A-D (time given in hours) of inoculation, ofErysiphepisi and Erysiphegraminic.
of 15 gramhis
incubation,
transfer,
and
Inoculation and transfer of conidia, incubation and observation One pair of coleoptiles was prepared for each experiment. One coleoptile (coleoptile A) was inoculated with 15-20 freshly harvested conidia of E. graminis by a brush. One to eleven hours after inoculation with E. graminis, another coleoptile (coleoptile B) was inoculated similarly with about 100 freshly harvested conidia of E. pisi, following the time schedule in Fig. 1. Such timing of inoculation of the two fungi allowed them to attempt penetration at time intervals mentioned below. Before transfer of germinating conidia of E. pisi, germinating conidia of E. graminis each having an appressorial apex on a single coleoptile celi were selected [Fig. 2 (a)]. Selection of conidia was such that no other germlings were located within five host cells from the selected conidia. Three hours after inoculation of E. pisi, selected conidia of E. pisi which had an appressorial germ tube were each transferred with a micromanipulator from coleoptile B to coleoptile A so that appressoria of transferred E. pisi attempted to penetrate the same cells of coleoptile A as did appressoria of untransferred E. graminis [Fig. 2(b)]. Using separate pairs of coleoptiles, selected conidia of E. pisi on coleoptile B were transferred onto laterally adjacent cells of E. graminis-attacked cells of coleoptile A. In each experiment, five to 10 conidia ofE. pisi were transferred and thus five to 10 pairs of both fungi were prepared on one coleoptile A as the subjects of each experiment. Transfer of E. pisi was completed within 1 h after initiation of the manipulation. Immediately after the transfer was completed, coleoptile A was returned to the Petri dish and incubated for 3 h. Subsequently, coleoptile A was mounted with 0.01 M CaCl, on a glass slide and covered with a cover slip. These specimens were set, cover-slip side down, on a Zeiss inverted interference contrast microscope IM35, and fungal growth and host cell response were observed at x 400 every 15 min. For each pair of both fungi, the timing of initiation of cytoplasmic aggregates induced by the respective fungi was recorded. Coleoptile A was again incubated for a further 26 h, and subsequent haustorium formation was recorded. Data were accumulated from more than 100
l-l. Kunoh et al.
46
FIG. 2. (a) A germinating conidium of E. graminis having an appressorial germ tube b! A germinating conidium of E. pisi, grown on a separate coleoptile, after transfer micromanipulator onto the cell where the E. gralninis appressorium was developing. Bar=
(AGT). with a 10 pm.
separate, but replicated, experiments performed over a period of two years. During manipulation and observation, the microscope room was maintained at 20 f 2 “C and 70° (, RH. RESULTS Growth of E. graminis and E. pisi singly inoculated onto barley coleoptiles, and host cell responses Conidia of E. gramilzis, inoculated onto barley coleoptiles usually produced primary germ tubes between 1 and 2 h after inoculation and appressorial germ tubes between 3 and 4 h after inoculation [9]. When mature appressoria attempted to penetrate host walls between 10 and 12 h after inoculation, host cytoplasmic aggregates were initiated at the sites of attempted penetration. Within 1 to 2 h after initiation of cytoplasmic aggregates, incipient globose haustorial bodies became visible at apices of penetration
Cell level susceptibility
and resistance
FIG. 3. (a) A haustorium ,&r formed in a coleoptile gminis. Bar= 10 pm.
of barley I
(H) ofE. g raminir in a coleoptile cell. (b) A globose haustorium cell where susceptibility has been induced by prior-penetration
of E. by E.
pegs arising from appressorial lobes. They gradually developed to form typical haustoria with branched finger-like projections [Fig. 3 (a)]. When E. graminis alone was inoculated onto coleoptiles, 70-80% of conidia succeeded to produce haustoria. On the other hand, between 1 and 2 h after inoculation, one appressorial germ tube arose from conidia of E. pisi without emergence of short germ tubes corresponding to reported by Kunoh et al. [IO]. Apices of primary germ tubes of E. graminis, as previously the appressorial germ tubes swelled to form unique polymorphic appressoria usually of two to five short lobes. Host responses to appressorial penetration by this fungus was unique in that there were two stages of cytoplasmic aggregates. Between 6 and 7 h after inoculation, small cytoplasmic aggregates occurred beneath one of the short lobes of
48
H. Kunoh et al.
appressoria. Appressorial lobes sometimes interferred with observation of initiation of such small cytoplasmic aggregates. These aggregates lasted generally for 15-45 min, then disappeared. After an additional period ranging from 15 to 60 min, sometimes longer, active cytoplasmic aggregates of larger diameter developed beneath the same lobes of the appressoria. About 30 min after recurrence of cytoplasmic aggregates, clear, circular structures, probably corresponding to penetration pegs or penetration pores in host walls, suddenly formed in the lobes or host walls beneath the lobes. Among 76 inoculated conidia, such a structure was apparently distinguishable in 59 appressoria. In response to penetration by E. PC, papillae were commonly formed under appressoria but sometimes appressorial lobes were too large to distinguish papillae that may have formed underneath them. Single inoculations of E. pisi were never successful in producing haustoria in coleoptile cells. The unique response of barley coleoptile cells to E. pisi will be published elsewhere.
Timing of attemptedpenetrationsby E. graminis and E. pisi on the samecoleoptilecells Initiation of a cytoplasmic aggregate was usually the first visible response of a host cell at the attempted penetration sites from appressoria of both fungi, and thus the time of initiation of the cytoplasmic aggregate was considered to be the first sign of attempted penetration by the respective fungi. As mentioned above, two successive cytoplasmic aggregates occurred under the same appressorium of E. pisi. The second cytoplasmic aggregate was accompanied by the appearance of a penetration-pore-like structure in the lobe but the first one was not. Therefore, it was inferred that it was ,the second cytoplasmic aggregate, not the first, that signalled the time of host wall penetration by E. pisi. Thus, the following description and discussion of the time of E. pisi concerns the second cytoplasmic aggregate. Figure 4 illustrates growth patterns of both E. graminis and E. pisi on the same coleoptile cell and the host cell responses. By 9 h after inoculation of E. graminis, both fungi formed an appressorium which appeared morphologically mature [Fig. 4(a)]. The cytoplasmic aggregate was seen below only the appressorium of E. graminis 10 h 45 min after inoculation of E. graminis [Fig. 4(b)]. Thirty minutes later, a penetration peg was seen to form from a lobe of the appressorium [Fig. 4(c)]. The cytoplasmic aggregate was initiated beneath the appressorium of E. pisi 11 h 30 min after inoculation of E. graminis, as shown at the right of Fig. 4(d). At that time the cytoplasmic aggregate persisted below the appressorium of E. graminis. At 13 h 15 min, a penetration-pore-like structure was distinguished in a lobe of the E. pisi appressorium below which the cytoplasmic aggregate persisted [Fig. 4(e)]. Figure 4(f) illustrates growth patterns of both fungi observed 33 h after inoculation of E. graminis: by this time E. graminis successfully penetrated to form a haustorium followed by elongation of a secondary hypha from the primary lobe of the appressorium, but E. pisi failed to penetrate from the primary lobe and attempted another penetration from the secondary lobe. Thus, Fig. 4 illustrates an example in which E. graminis attempted penetration 45 min earlier than E. pisi, since the cytoplasmic aggregate was first seen below E. graminis and E. pisi, respectively, 10 h 45 min [Fig. 4(b)] and 11 h 30 min [Fig. 4(d)] after inoculation of E. graminis. Similarly, the timing of the initiation of a cytoplasmic aggregate below the respective fungi was determined for each pair of appressoria through successive experiments. If the timing for either member of a pair failed to be
Cell level susceptibility
and resistance
of barley I
FIG. 4. A series of interference contrast micrographs showing successive growth patterns of E. graminis (G) and E. pisi (P) on a single barley coleoptile cell. The number in the upper right corner of each frame indicates the time after inoculation of E. graminis. The bar in frame a indicates IOpm. (a) By 9 h, appressoria (APP) of both fungi had matured. (b) At 10h 45min, a cytoplasmic aggregate (CA) was seen beneath the appressorium ofE. graminis. (c) At 11 h 15 min, a papilla (PA) was observed at the apex of the penetration peg (PP) of E. graminis. (d) At 11 h 30 min, a small cytoplasmic aggregate occurred beneath the appressorium of E. pk. (e) At 13 h 15 min, a circular structure (PP) corresponding to either a penetration peg or a penetration pore was visible in the primary lobe of E. pisi below which the active cytoplasmic aggregate had persisted. (fi By 33 h, the E. graminis appressorium had penetrated, forming a haustorium (H) and elongating secondary hypha (ESH). On the other hand, the E. pisi appressorium had failed to penetrate from the primary lobe (PL) and tried another penetration from a secondary lobe (SL).
49
50
H. Kunoh et al. E. pisi
w
180 w
15 grominis
v
%
‘13
v
O/5
‘15
v
O/IO
v
%‘I
150 w
0.0%
%o
28.6%
120 w 90 v
v 60 WV 30
O/IO
%6
V 0
%
VV 30
I
Z/i/II
v 60 w
v
%O
V
1
Z/9
90
840
1
519
’ 75.0%
29.2%
v
V
2/2
212
‘/3
%
120 v
150
V
FIG. 5. Effects ofrelative time (min) ofinitiation pisi (V) and Erysiphe graminis (v) on haustorium coleoptile cells of barley.
.
ofcytoplasmic aggregates induced by Eysiphe formation by the respective fungi on the same
determined, the datum pair was abandoned. Timing of the initiation of cytoplasmic aggregates and rates of haustorium formation (penetration efficiency) of the respective fungi were successfully determined for 134 appressorial pairs. These results are summarized in Fig. 5, based on time-intervals between the initiation of cytoplasmic aggregates induced by both fungi. When E. pisi attempted penetration more than 60 min earlier than E. gruminis, none of the E. pisi appressoria penetrated successfully. Moreover, penetration efficiency of E. graminis lowered to 28.6% on average, ranging from 20.0 to 40.0%. However, when both fungi attempted penetration almost simultaneously (within 30 min of each other), the penetration efficiency of E. pisi increased to l1*8o/o. Moreover, the penetration efhciency of E. graminis recovered to 55.8% on average. When E. graminis attempted penetration 60 min or more earlier than E. pisi, the mean penetration efficiency of E. pisi was enhanced to 29.2% and that of E. graminis to 75.0%. As indicated in Fig. 3(b), E. pisi produced a globose haustorium in a barley coleoptile cell having induced susceptibility as reported previously [4]. How long does such an effect of prior-attack by E. gruminis on haustorium formation by E. pisi last? As shown in Fig. 6, when E. graminis attempted penetration 300&600 min earlier than E. pisi, the mean penetration efficiency of E. pisi was 36.613, and that ofE. graminis was 71.0%. Thus, the induced susceptibility was effective for at least 5 10 min, probably until 600 min. As mentioned above, E. pisi, which, by itself, was originally avirulent to barley coleoptile cells, became capable of forming haustoria in cells which were conditioned toward susceptibility. However, successful penetration by E. pisi occurred only when the penetration of E. graminis was successful in the same cells.
Cell level susceptibility
and resistance
of barley I
51
E. pisi T
v
E graminis
%5
300 T
V
%5
%5
330 T
v
‘3126
360 T
v
%s
390 T
v
“127
420 'I
v
2/l 4
450 T
v
%I
480 T 'I
FIG.
2/5 0
540
T
I penrtration
v
510
‘12
v
600
%
6. Effect of timing (min) of prior-attack by E. gramlnis (T) on haustorium efficiency) by E. pisi ( 5’ ) on the same coleoptile cells of barley.
2h % v5I
E pisi
E. gramhis
%
% v
T
%
T
Oh
T
%
120 v 90 v
formation
77.4%
O/12
--k--V 0 VW 30 'I v 60 T v 90 T 120
T
150
T
1.0%
O/9
O/b
%
V v B
180 FIG. 7. Effect of the relative E. plsi i V) and E. graminis (v) coleoptile cells of barley.
VI6
3Ll
-
-
-
-
“1,
'/I
' 72.7%
,
time (min) of initiation of cytoplasmic aggregates on penetration efficiency by the respective fungi
induced by on adjacent
The e$ect of prior-attack on interactions with adjacent coleoptile cells ‘To determine whether this susceptibility and enhanced resistance can be transferred to the adjacent cells, E. pisi conidia with a short appressorial germ tube were placed onto cells adjacent to E. graminis attacked cells. As shown in Fig. 7, E. pisi never produced
52
H. Kunoh
etal.
haustoria in adjacent cells either when E.&i attempted penetration 30-180 min earlier than E. graminis or when the latter did so 30-180 min earlier than the former. On the other hand, the penetration efficiency of E. graminis was maintained at the level of 72.7-77.476 in all cases, except that a somewhat lower rate of 59.5% was obtained when both fungi penetrated almost simultaneously (within 30 min of each other).
DISCUSSION
The present results definitely show that barley coleoptile cells are conditioned toward susceptibility by prior-attack by a pathogenic fungus and that the resistance of the cell is enhanced when a prior-attack by a nonpathogenic fungus occurs at least 60 min earlier than attack by a pathogenic fungus. It is apparent from Fig. 5 that the timing of attempted penetration by both fungi is very important in determining the cellular response. When E. pisi attempted penetration more than 60 min earlier than E. graminis, the penetration efficiency of E. graminis was reduced to only 28.6%, indicating that the resistance of coleoptile cells is enhanced under this condition. Conversely, when E. graminis attempted penetration earlier than E. pisi, or when penetration was almost simultaneous, the coleoptile cells were conditioned toward susceptibility to E. pisi. Under these conditions, the resistance which the coleoptile cells possess natively might be disrupted in some way. A low penetration efficiency of E. pisi (11.80/,) and intermediate one of E. graminis (55.874) obtained when both attempted penetration almost simultaneously probably indicates a state of coleoptile cells insufficiently conditioned toward susceptibility. The present observation that the timing of cytoplasmic aggregates occurring near the plasmalemma (and thus traversal of the host wall) is associated with cellular conditioning led us to recall the hypothesis of Ellingboe & Slesinski [fl that the gene in the host will be constitutive in function and confer specificity at the level of the plasmalemma. However, as Tani et al. [17J reported, cellular conditioning of oat leaf tissues toward resistance to incompatible races of Puccinia coronata avenueis irreversibly established between 8 and 12 h after inoculation when the fungus has penetrated only through stomata. Their results suggest that penetration through the plasmalemma might not be a prerequisite for the alteration of the cellular condition of the host. With barley leaves and several races of E. graminis as employed by Ouchi et al. [!I, 12,131, 15-18 h were required to complete cellular conditioning toward susceptibility, while conditioning toward resistance required only 6 h. However, unlike their results, the present data indicate that as little as 60 min may be required to change the coleoptile cells towards susceptibility. It was also suggested that, at the cellular level, enhancement of resistance and induction of susceptibility are gradual processes crossing over & 30 min when either fungus attempts penetration earlier. As indicated in Figs 5 and 6, the penetration efficiency of E. pisi obtained was at most 36.676, even if the coleoptile cells were penetrated first by E. graminis hordei. Using the similar micromanipulation technique, Kunoh [S] transferred conidia of a compatible race of E. graminis onto barley coleoptile cells and observed their growth after inoculation with the purpose to elucidate the effect of prior-attack by primary germ tubes of 6. graminis conidia on the penetration efficiency of their appressoria. In that experiment, the penetration efficiency of transferred E. graminis conidia was at most
Cell level susceptibility
and resistance
53
of barley I
45.8-48.5% in contrast with 7O-8Oo/o of untransferred conidia. Thus, the low penetration efficiency of E. PG obtained in this study might be due to the transfer effect, although we cannot rule out another possibility that a substantial resistance which is not affected by the timing offungal penetration might be involved in suppression of the penetration of E. pisi. Ouchi et al. [14, 15] showed that Sphaerotheca fuliginea colonized the barley cells harbouring a compatible E. graminis haustorium with a very high frequency, but it was incapable of colonizing cells located four cell rows apart from the haustoriumharbouring cells, and emphasized that both the induced resistance and susceptibility were localized in the area where the primary interaction was effectively completed. Their data evidently show that the induced cellular conditions are transferred to iadjacent cells or nearby cells. The data presented here indicate that the induced :susceptibility and enhanced resistance are not transferred within 3 h from affected cells to adjacent cells. Our separate experiments (unpublished) have shown that the time intervals of attempted penetration of both fungi required to transfer the cellular condition from one cell to the adjacent cell is at least 18-20 h. These results raise questions of how the timing of attempted penetration by both fungi is related to the final cellular conditioning of the host or how the phenomena revealed herein are associated with various modes of suppression of host defense mechanisms which have been postulated by many earlier researchers in a variety of parasite-host systems [15, 17, 181. Although Ouchi & Oku [II] emphasized that the phenomena relating to induced susceptibility should be evaluated on a molecular basis, the present study suggests that more detailed cytological observation can be helpful before discussing the genetic or molecular aspects of these phenomena. We are indebted and for correcting
to Dr James the English.
R. Aist, Cornell
University,
for his valuable
discussion
REFERENCES 1. AIST, J. R. & ISRAEL, H. W. (1977). Papilla formation: Timing and significance during penetration of barley coleoptiles by Erysiphe graminis hordei. Phytopathology 67,455-46 1. 2. AIS~, J. R., KUNOH, H. & ISRAEL, H. W. (1979). Challenge appressoria of Erysiphe graminis fail to breach preformed papillae of a compatible barley cultivar. Phytopatholou 69, 1245-1250. 3. BUSHNELL, W. R. & BERGQUIST, S. E. (1975). Aggregation of host cytoplasm and the formation of papillae and haustoria in powdery mildew of barley. Phytopathobu 65, 31G-3 18. 1. BUSHNELL, W. R. & GAY, J. (1978). Accumulation of solutes in relation to the structure and function of haustoria in powdery mildew. In The Powdery Mildews, Ed. by D. M. Spencer, pp. 183-235. Academic Press, London. 5. BUSHNELL, LV. R. & ZEYEN, R. J. (1976). Light and electron microscopy studies of cytoplasmic aggregates formed in barley cells in response to Erysiphe graminis. Canadian Journal of Botany 54, 1647-1655. 6. BUSHNELL, W. R., DUECK, J. & ROWELL, J. B. (1967). Living haustoria and hyphae of Erysiphegraminis f. sp. hordei with intact and partly dissected host cells of Hordeum uulgare. Canadian Journal of Botany 45, 1719-1732. 7. ELLINGBOE, A. H. & SLESINSKI, R. S. (1971). Genetic control of mildew development. In Barley Genetics, F.d. by R. A. Nilan, pp. 472-474. Washington State University Press, Pullman. 8. KCNOH, H. (1982). Primary germ tubes of Erysiphe graminic conidia. In Plant Infection: The Physiological and Biochemical Basis, Ed. by Y. Asada, W. R. Bushnell, S. Ouchi & C. P. Vance, .pp.. 45-59. -Japan . Scientific Societies Press, Tdkyo and Springer-Verlag, Berlin. 9. KI~NOH, H., Am, J. R. & ISRAEL, H. W. (1979). Primary germ tubes and host cell penetrations from appressoria of Erysiphe graminis hordei. Annals of the Phytopathological Society ofJapan 4.5,326-332.
54
H. Kunoh
et al.
IO. KUNOH, H., ITOH, O., KOHNO, M. & ISHIZAKI, H. (1979). Are primary germ tubes of conidia unique to ETsz$he graminis? Annals of the Phytopathological Society ofJapan45,675602. I I. OUCHI, S. & OKU, H. (1982). Physiological basis of susceptibility induced by pathogens. In Plant Infection: The Physiological and Biochemical Basis, Ed. by Y. Asada, W. R. Bushnell, S. Ouchi & C. P.
Vance,
12. 13.
14. 15.
16.
17.
18.
19.
20.
pp. 117-136. Japan Scientific Societies Press, Tokyo and Springer-Verlag, Berlin. S., OKU, H., HIBINO, C. & hIYAMA, 1. (1974). Induction of accessibility and resistance in leaves of barley by some races of Erysiphe graninis. Phytopathologische