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Cytological studies of early stages of powdery mildew in barley and wheat leaves: (II) significance of the primary germ tube of Erysiphe graminis on barley leaves
Physiological
Plant
Pathology
(1977)
10, 191-199
Cytological studies of early stages of powdery mildew in barley and wheat leaves: (II) significance of the primary germ tube of Erysiphe graminis on barley leaves? HITOSHI KUNOH,
HIROSHI
ISHIZAKI
Laboratory of Plant Pathology, Faculty Mie University, l&city, 514, Japan (Accepted
for publication
January
and KYOKO NAKAYA
of Agriculture,
1977)
Early stages of the infection processes of Erysiphe graminis hordei, race I (compatible with barley cv. Kobinkatagi) and of Epsijhe graminis tritici, race ts (incompatible with barley cv. Kobinkatagi) were observed on barley (Hurdeum vulgare L., cv. Kobinkatagi) by scanning electron microscopy. Behavior of both fungi was similar and nearly 99% of the germinated conidia of both races had more than one germ tube 12 h after inoculation. Among the germ tubes, one, designated as the primary germ tube, induced a host papilla or cytoplasmic aggregate below it 4 to 6 h after inoculation, and another swelled to become an appressorium. Almost every conidium which had induced a papilla below an appressorium had earlier induced a papilla below the primary germ tube. The results suggest that the induction of a papilla by the primary germ tube may be critical to attempted penetration by the appressorium from the Micromanipulation indicated that the papilla below the epidermal cell wall same conidium. was continuous with or attached firmly to the inner host wall layer, and also that the wax crystals on the leaves were dissolved by the fungal structures which attempted to penetrate, i.e. the primary germ tube and appressorium.
INTRODUCTION
Many earlier studies [S, 7-91 on conidial germination of Epsi~he graminis have shown that several germ tubes appear on each conidium, but that only one of them grows extensively and forms a typical appressorium. Kunoh & Ishizaki [9] noted a small hemispherical papilla just beneath the tip of short, non-appressorial germ tubes by micromanipulation in a scanning electron microscope @EM), and reported that silicon accumulation occurred in abundance below some of such germ tubes, Most of the earlier investigations concerning the infection process of E. graminis [l-3, 5, 8, 11, 15, 171 have focussed on appressorial function and have paid no attention to nonappressorial germ tubes. Our study was undertaken to investigate, by means of micromanipulation in the SEM [lo], whether or not non-appressorial germ tubes of E. graminis conidia have a r81e in the infection process. For comparison, the infection processes of compatible and incompatible races of E. graminis were investigated on one cultivar of barley. MATERIALS
AND
METHODS
Barley (Hordeum uulgare L., cv. Kobinkatagi) was grown from seed in an illuminated chamber in 20 cm glass flats containing vermiculite. The abaxial epidermis of t Contribution
no. 31.
H. Kunoh, H. lshizaki and K. Nakaya
19!2
IO-day-old leaves was inoculated with conidia of Epciphe gram&is hordei, race I (compatible with cv. Kobinkatagi) or E. graminis tritici, race t, (incompatible with cv. Kobinkatagi) and incubated in a chamber illuminated by fluorescent lamps (c. 4000 lx) for 12 h/day. The chamber was maintained at 20 “C and 90% r.h. Inoculated leaves were cut into small pieces, 1.0 x 0.5 cm, with a razor blade at 4,6,9, 12,24, 30 and 36 h after inoculation and were then treated by the modified tannic acid fixation method [13]. The specimens were fixed with 2% unbuffered glutaraldehyde containing 0.2% tannic acid at room temperature (RT) for 12 h, followed by further fixation in 2% unbuffered glutaraldehyde containing 2% tannic acid at RT for 12 h. After being washed in deionized water for 2 h, the specimens were treated in 1y. unbuffered aqueous 0~0, at RT for 12 h and washed in deionized water for 1 h. They were dehydrated in a graded ethanol series, then in a graded ethanol-iso-amyl acetate series and finally placed into lOOo/oiso-amyl acetate, They were then dried in a critical-point dryer and observed with a Hitachi scanning electron microscope HHS-2X at 20 kV accelerating voltage, except as cited. The specimens were dissected with a micromanipulator set in the SEM in order to observe any papillae induced by the fungi [ 101. Three experiments were replicated at each sampling time and the details of approximately 110 germinating conidia were recorded in each experiment. In order to observe the wax crystals on leaves, small pieces of fresh, inoculated barley leaves were observed in the SEM without fixation or coating [3]. RESULTS ( 1) Conidial
germination
to appressorium formation
In the present study, only germinated conidia were selected as test conidia. About 30% of conidia of race I and race t, germinated 4 h after inoculation. The germination rate of both races increased thereafter and reached 50 to 60% at 12 h. In most cases, two to several germ tubes appeared on each conidium of race I and race t, (Plates 4 and 10). Sometimes only one germ tube appeared to emerge from any one conidium, if over-viewed by SEM. However, if the conidium was moved by the microneedle, another germ tube could usually be seen just beneath the conidium. As shown in Table 1, at 4 h, nearly 30% of the test conidia of both races had one germ tube ; nearly 60% had two germ tubes; and 10% had more than two germ tubes. At 6 h, about 14% of race I conidia and 22% of race t, conidia had one germ tube, and 75 and 70%, respectively, had two germ tubes. Between 9 and 24 h after inoculation, nearly 85% of both races had two germ tubes. At 24 h, fewer than 1 y. of the test conidia of both races had one germ tube. Table 1 indicates that the frequency of the conidia with more than two germ tubes was almost consistently 9 to 16% between 4 and 24 h. Whether conidia are to have more than two germ tubes seems to be determined at the early stage of germination, probably prior to 4 h after inoculation. Only one of the multiple germ tubes from each conidium ceased elongation and attached firmly to the host surface (Plate 1). It induced a papilla in the host epidermal cell (Plates 2 and 3). We designated such a germ tube as the primary germ tube. With time after inoculation, another germ tube swelled, especially at the tip, to become an appressorium (Plates 4, 7 and 10). Even if conidia had more than two germ tubes, only one germ tube developed an appressorium (Plate IO). None of
Key to abbrerkztions g, germ
tube;
ha,
(Plates halo;
1 to 13).
ap, appressorium;
N, microneedle;
PLATE 1. A conidium barley, cv. Kobinkatagi,
p, papilla;
of E. graminis hordei, 4 h after inoculation.
c, conidium; pg, primary
race I, with x 2300.
germ
cy, epidermal
cytoplasm;
tube.
a primary
germ
tube
on
a leaf
PLATE 2. After removal of the conidium of Plate 1 by the microneedle, a bright (arrow) became visible at the site where the primary germ tube had been. x 2300.
of
region
PLATE 3. The bright region of Plate 2 was turned inside out by the microneedle. A papilla initial covered with fibrous materials is now visible on the reverse side of the cell wall. x 2300. PLATE 4. A conidium of E. graminis tritici, race t2, with a primary germ sorium 30 h after inoculation. The penetration sites of both the primary appressorium are surrounded by a halo. x 1200. PLATE 5. The A papilla is present x 1200.
halo region around the primary at the center of the halo region.
tube and an apprwgerm tube and the
germ tube of Plate 4 was turned The halo region appears somewhat
PLATE 6. The epidermal cell wall of the halo region around the appressorium reversed. A papilla is present on the reversed halo region. x 1200.
inside out. depressed.
of Plate
4 was
PLATE conidium cytoplasm. aggregate.
7. A papilla on the reverse side of the cell wall below an appressorium of a race I 24 h after inoculation. The epidermal cell wall was clearly separated from the A hole (arrow) in which the papilla was embedded remained in the cytoplasmic x 1000.
PLATE cell wall.
8. A papiila x 2500.
formed
PLATE 9. The papilla appeared on the reversed PLATE 10. another germ x 2300.
6 h after
of race
I on the reverse
of Plate 8 was removed by the microneedle. cell wall. The cell wall was never hollowed
A germinated conidium of race I having a primary tube 9 h after inoculation. Wax crystals cover
PLATE 11. The conidium of Plate characteristically shaped appressorium penetration site. x 2300. PLATE inoculation
inoculation
12. The imprint of race t,.
in
the
An outline of the papilla out. x 2300.
germ tube, appressorium the whole surface of the
10 was removed by the microneedle. remains in the wax crystals. An
wax
PLATE 13. The imprint of an appressorial The appressorial arm did not leave an Observed at 15 kV accelerating voltage.
crystals
lobe imprint.
of
a displaced
side of an epidermal
primary
on a fresh leaf observed An arrow indicates
The arrow
germ
and leaf.
imprint of the indicates the
tube
4 h after
24 h after inoculation. the penetration site.
[facing
page I92 I
PLATES
1, 2, 3 and 4
PLATES
5, 6, 7, 8 and
9
PLATES
10, 11, 12 and
13
193
Primary germ tube of Erydphe graminis .Numbers
TABLE 1 tubes (including appressoriu) emerging from coni&a of E. graminis (race I) and E. graminis tritici (race ts) on barley leavefl
of germ
Time after inoculation 04
Fungal race Race
I
4
t,
4 6
::
tubes More than2
2
4.3 10.3 1.6 0.9 1.6
27.2 4 13-5 21.94 6.6 2.7f 1.5 0.54 0.5 0.34 o-5
9
of germ
1 30.24 13.94 2-82 0.6* 0.94
9” 12 24 Race
Number
60*5+ 75.4+ 86-l + 84.3-1 86.22 62.9+ 70.5+ 86.15 84-i’+ 83*4+
hordei
5.0 8.2 l-7 5.6 7-l
9.1 k5.1 10+6*5*3 11*1&1*5 15.2 f 5.8 12*8+6-O
11.3 7.9 4.3 5.9 2.7
9.9&2-6 7.5&l-6 II-O&1*9 14.8 + 5.7 16~2k3.7
Q Expressed as percentages of the number of corresponding conidia to the total number of conidia observed. Variation is expressed as standard deviation. Based on three experiments with approximately 110 germinated conidia scored in each.
the conidia had more than one appressorium. In the case of conidia with more than two germ tubes, they had a primary germ tube and an appressorium; the other germ tubes did not induce papillae. We scored germ tubes which were more than 1.5 times broader at the tip than at the base as appressoria. As indicated in Table 2, nearly 60% of the test conidia of
Percentages
of aj$ressorium
TABLE 2 formation by conidia of E. graminis hordei tritici (race tJ on barley haves@ Time after inoculation 04 4 ii 12 24
(1 Based
on three
Percentage
experiments
of appressorium
with
Race
Fungal I
0 61*6+6*6 92.2 + 4.4 98.2 + l-4 98.2 f O-9
race Race
(race I) and E. graminis
ts
0 50.2 & 9-9 91*7+2*5 96-l&1*8 99-l&0*9
approximately 110 germinated conidia scored No. of conidia haying an appressorium formation = Total no. of conidia observed
in each. x loo
race I had an appressorium 6 h after inoculation and more than 90% did at 9 h. Similarly, nearly 50% of the test conidia of race ts had an appressorium 6 h after inoculation and more than 90% did at 9 h. With both races, a lobe of the appressorium became conspicuous by 9 h after inoculation. A second lobe often formed midway along the side of the appressorium (Plate 7). The second lobe occurred more frequently with race t, than race I 24 h after inoculation.
194
H. Kunoh, H. lshizaki and K. Nakaya
(2) Papillae indqction by the primary germ tube and apliressorium As mentioned previously, the primary germ tube emerged from the conidium and induced a papilla in the epidermal cell by 4 h after inoculation (Plate 1). When the conidium with the primary germ tube was moved by the microneedle, a small circular region around the penetration site appeared bright (Plate 2). Furthermore, when the epidermal cell wall around the bright region was turned inside out by the microneedle, a small papilla initial was revealed at the penetration site. Careful observations of the reversed primary germ tubes revealed that a minute hole, about O-3 to O-5 pm in diameter, existed on the tip surface of some primary germ tubes which had contacted the host cuticle, suggesting attempted penetration by the primary germ tube. However, we failed to find such a hole on some primary germ tubes. Some of the papilla initials found at 4 h were covered by a cytoplasmic aggregate [Z] with a fibrous nature (Plate 3). These initials usually could not be identified with certainty until the cytoplasmic aggregate was removed by the microneedle. The host response at 4 h was difficult to over-view by light microscopy, because the response area was usually narrower than 5 pm in diameter, and the primary germ tube over the response area blocked the view. The papilla was well defined on the reverse side of the cell wall by 6 h after inoculation (Plates 5 to 9). Of approximately 110 germinated conidia of race I, the proportion of primary germ tubes which had induced a papilla or cytoplasmic aggregate was 50% at 4 h after inoculation, about 7.5% at 6 h and 97% at 12 h (Fig. 1). No haustorium was
+A
4
’
6
I
I
I
9
12
24 Time
Fro. of (a) E. Primary Frequency No.
1. Papillae or cytoplasmic gramti /w&i (race I) germ tube; (~0 = of primary germ tubes Total no. of
after
/ t+m
4
inoculation
’
’
6
9
I
24
12
(h 1
aggregate induction by primary germ tubes and and (b) E. grutiinb titici (race ta) on barley leaves; appressorium. or appressoria primary germ
inducing a papilla tubes or appressoria
or cytoplasmic observed
appressoria (A-A)
aggregate
.
produced by the primary germ tube during the observation period. Germinated conidia of race t, showed a similar tendency concerning papilla induction (Fig. 1). About 52% of the primary germ tubes induced a papilla or cytoplasmic aggregate by 4 h after inoculation, about 86% by 6 h and 92 to 98% by 9 h.
Primary germ tube of Erysiphe gramink
195
Other germ tubes from conidia with primary germ tubes swelled to become appressoria by 6 h (Table 2; Plates 4, 7 and 10). In race I, about 27% of the test appressoria induced a papilla below the epidermal cell wall by 6 h after inoculation, about 78% did by 12 h and 96.5% did by 24 h. On the other hand, 19% of the test appressoria of race t, induced a papilla by 6 h and more than 90% did by 12 h after inoculation. In both races, more than 95% of the test appressoria induced a papilla in the epidermis by 24 h (Fig. 1). Figure 1 indicates that papillae induction by primary germ tubes occurs 5 to 6 h earlier than that by appressoria. As shown in Fig. 2, almost all conidia which induced a papilla below their appressorium had induced a papilla also below their primary germ tube (Plates 4
fimr
after
inoculation
(h)
FIG. 2. Papillae or cytoplasmic ag$egate induction by the primary germ tube and appressorium from the same conidium of (a) E. grmninis hordei (race I) and (b) E. graminis Mici A papilla or cytoplarsmic aggregate was observed below (race tz) on barley leaves. ( l -a) both the primary germ tube and the appressorium from the same conidium; (0- - -0) a papilla or cytoplasmic aggregate was observed only below the primary germ tube.
to 6). During the 24 h after inoculation, fewer than 1 Oh of the test conidia induced a papilla below their appressorium without first inducing a papilla below the primary germ tube. Fig. 2 indicates that the frequency of conidia inducing a papilla below the primary germ tube, but not one below the appressorium, decreased with time and, conversely, that of the conidia inducing papillae below both of the fungal structures increased. These observations suggest that the induction of a papilla by the primary germ tube may be involved in the process of attempted penetration by the appressorium produced by the same comdium. Frequently, some of the appressoria had two appressorial lobes at 24 h, especially in the case of race t, (Plate 7). A papilla was usually produced below each of the lobes. (3) Association of papillae with the host cell walls Sometimes micromanipulation the host cytoplasm, although
succeeded in separating the epidermal cell wall Corn some cytoplasmic vesicles were still attached to the
196
H. Kunoh, H. lshizaki and K. Nakaya
reverse side of the cell wall (Plates 7 to 9). As the result of micromanipulation, a hemispherical papilla was observed on the reverse side of the cell wall at the penetration site (Plates 7 and 8), leaving a hole in the cytoplasmic aggregate in which the papilla had been embedded (Plate 7, arrow). Papillae showed an intense resistance to microneedle pressure when attempts were made to remove them from the reverse side of cell walls. When further force of the microneedle succeeded in removing the papilla, a trace of the papilla remained on the reverse side of the cell wall; the cell wall was never hollowed out (Plate 9, arrow). These results suggest that the papilla, or at least a part of it, is continuous with or attached firmly to the inner layer of the cell wall. (4) Degradation of leaf wax by fungal structures As viewed by SEM, a halo surrounded the penetration sites of both primary germ tubes and appressoria (Plate 4). Observations at a magnification of x 5000 revealed that wax over the halo region remained undissolved. The halo region was difficult to see at first because its brightness was similar to that of other regions. The halo region was somewhat brighter than other areas of the leaf between 9 and 12 h after inoculation and, furthermore, it turned darker by 24 h. These darker halo regions appeared somewhat depressed when viewed from the reverse side (Plates 5 and 6). During the present observations it became evident that wax crystals covered the entire surface of barley primary leaves (Plates 10, 11 and 13). Where conidia with the primary germ tube and appressorium were removed by the microneedle, tracks of these fungal structures, except the conidium itself, appeared on the cuticular surface (Plates 10 to 12). In some cases, such tracks appeared only at the appressorial lobes and not below the appressorial arm (Plate 13). When conidia have several other germ tubes besides the primary germ tube and appressorium, a track never appeared below these germ tubes. These observations suggest that the wax crystals were degraded only by the primary germ tube and appressorium. Another possibility is that tracks of the primary germ tube and appressorium may result from the fibrillar matrix of germ tubes and appressoria pulling wax off, but the reversed surface of both iimgal structures which had contacted the host surface was quite smooth. Thus, these observations are considered to support the possibility of chemical degradation of wax crystals by the primary germ tube and appressorium. DISCUSSION
The present observations on primary germ tubes indicated that a papilla was induced by almost all primary germ tubes regardless of the compatibility of the host-parasite combinations. Furthermore, they showed that the induction of a papilla by the primary germ tube might be critical to attempted penetration by the appressorium and thus to survival of E. graminis. No other fungi known to the authors have such characters. Ellingboe [5] reviewed the infection process in powdery mildew and summarized that the process of primary infection consisted of six morphologically identifiable stages : spore germination, formation of appressorial initials, maturation of appressoria, formation of penetration pegs, formation of haustoria and formation of secondary
Primary germ tube of Erysiphe
graminis
197
hyphae. As shown clearly in this study, the host cell responds to the primary germ tube earlier than to the appressorium. Thus we consider that the formation of, and penetration by, the primary germ tube is important in the infection process of E. graminis. Oku et al. [14] found that there were two phases of phytoalexin production in barley leaves infected with E. graminis. The first phase was detected at 12 h after inoculation. They concluded that this stage just coincides with the time of initial direct contact between cells of the host and the parasite. The present observations, however, suggest that the first direct contact of host cells and primary germ tubes occurs by 4 to 6 h after inoculation. Accordingly, we cannot rule out the possibility that the host responses, including phytoalexin production, may proceed soon after penetration of the primary germ tube and, as a result, the primary germ tube may influence the subsequent infection process, including compatibility. Further observation of specimens prepared at 30 and 36 h after inoculation revealed a haustorium in the epidermis attacked by some of the appressoria of race I, while haustoria were not observed below the primary germ tubes. However, it is uncertain at present whether or not papillae form early enough to present a mechanical barrier to penetration by the primary germ tubes. Lin & Edwards [II] investigated the mechanisms which determined whether penetration would occur in barley infected by E. graminis and concluded that it was the presence of a basic staining material(s) (BSM) in the papilla which correlated with unsuccessful penetration attempts. In preliminary experiments, we observed that some of the papillae induced by primary germ tubes contained BSM, but others did not, regardless of host-parasite compatibility. Thus, BSM cannot determine whether penetration will occur at the site of primary germ tubes. Furthermore, these observations suggest that papillae induced by primary germ tubes may be somewhat different in chemical composition and quantity from those induced by appressoria. The present micromanipulation study indicated that the papillae, or at least part of it, is continuous with or attached firmly to the inner layer of the host cell wall. Bracker [I] studied the ultrastructure of the haustorial apparatus of E. graminis and reported that the transition between the host wall and the papilla (collar) was abrupt and an interface, characterized by a thin electron-dense zone, often separated the two structures after both 0~0, and KMnO, fixations. He concluded that the papilla was not a swelling or ingrowth of the cell wall but a deposit upon the wall. Kunoh [S] also reported that barley papillae induced by E. graminis did not have a cellulosic nature and that the border line between the host wall and the papilla could be readily observed in electron micrographs. Sherwood & Vance [16] investigated the histochemical nature of reed canarygrass papillae and concluded that cellulose was not the major component of the papillae. On the basis of these ultrastructural and hiitochemical observations, it is likely that .the papilla, a deposit upon the wall, attaches firmly to the inner layer of the host cell wall. The substance which makes the attachment is unknown. Edwards & Allen [4] and McKeen & Rimmer [IZ] presented evidence obtained by electron microscopy that the papilla could be formed before the host wall was penetrated .by the fungus. On the other hand, Bushnell & Bergquist [,?I suggested, on the basis of the closeness in time between the appearance of the cytoplasmic aggregate and the appearance of the haustorium, that the fungus might have started
198
H. Kunoh, H. lshizaki and K. Nakaya
to penetrate before papilla formation. In the present study, a minute hole suggesting the site of a penetration peg was observed on the reversed surface of some primary germ tubes which had contacted the host surface. However, we are not completely confident about our attempts to determine the timing of emergence of penetration pegs from the primary germ tubes, since we failed to observe such a minute hole on some other primary germ tubes which had induced papillae or cytoplasmic aggregates in epidermal cells. This problem should be studied in detail by micromanipulation or other techniques. The present micromanipulation studies revealed the imprints of the characteristically shaped primary germ tubes, appressoria and appressorial lobes in wax crystals on barley leaf surfaces. By removal of the overlying mycelium of E. graminis on barley leaves with adhesive cellulose tape, Day & Scott [3] observed hyphal tracks throughout the wax crystals on the cuticle of barley leaves. Similarly, Schwinn & Dahmen [15] and Staub et al. [17] reported the dissolution of wax crystals on barley leaves under appressoria, germ tubes and hyphae of E. graminis. Staub et al. [17] concluded that the dissolution of the wax crystals, as was observed under the germ tube of E. graminis, might be a critical step in the process of direct penetration of barley leaves. The present observations, that only the primary germ tube and appressorium which attempted penetration could dissolve the wax crystals and that the other germ tubes never induced any response of the host cell, seem to support Staub et al.3 conclusion. As they also suggested, close contact of germ tubes other than the primary germ tube and appressorium with the cuticular surface may not be possible, and penetration may be prevented because they cannot dissolve the wax crystals. Since the behavior of primary germ tubes of race I and race t, were similar on the same cultivar of barley, the compatibility of host and parasite may not be determined at the primary germ tube stage. Although the present results may suggest that the primary germ tube is essential for subsequent appressorial penetration, we must wait until we have radioisotopic data on the absorption of host material through the primary germ tube before we speculate further on the physiologicalroleof the primary germ tube. Correction of the English in the text by Dr J. R. Aist, Cornell appreciated.
University,
is
REFERENCES 1. BRACKER, C. E. (1968). Ultrastrncture of the hanstorial apparatus of E&rhe gramiais and its relationship to the epidermal cell of barley. Phytojwthdup 58, 12-30. 2. BUSHNELL, W. R. & BERGQUIST, S. E. (1975). Aggregation of host cytoplasm and the formation of papillae and haustoria in powdery mildew of barley. Phytopathology65, 310-318. 3. DAY, P. R. & SCOTT, K. J. (1973). Scanning electron microscopy of fresh material of Epi@e graminis f. sp hvraki. Phyxiological Plant Pathology 3,433-435. 4. EDWARDS, H. H. & ALLEN, P. J. (1970). A fine-structure study of the primary infection process during infection of barley by Erysipe graminis f. sp. hordei. Pl@o~atholvgv 60, 15041509. 5. ELLINGBOE, ,A. H. (1972). Genetics and physiology of primary infection by Epi)%e gramink
Phytvpathvlogv 62,401-406. 6. HIRATA, K. (1942). On the shape Hvriiculture 5,34-49.
of the germ
tubes
of
Epsiphae. Bulletin of the Chiba College of
Primary germ tube of Erysiphe 7.
8.
9.
10.
11.
12. 13. 14. 15. 16. 17.
199
graminis
HIRATA, K.
(1955). Some observations on the relation between the penetration hypha and haustorium of the barley mildew (E@~si@e graminti DC.) and the host cell (I). Annals of the Phytojathological Society of Japan 19, 104-108. KUNOH, H. (1972). Morphological studies of host-parasite interaction in powdery mildew ofbarley, with special reference to affinity between host and parasite. Bulletin of the Faculty of Agriculture, Mie University 44, 141-224. KUNOH, H. & ISHIZAIU, H. (1976). Accumulation of chemical elements around the penetration sites of &&he graminis hodei on barley leaf epidermis: (III) micromanipulation and X-ray microanalysis. ihysiologieal Plant Pathol&y 8,9f-96. KUNOH. H.. ISHIZAIU. H.. WATANABE. T., YAMADA. M. & NAGATANI. T. (1976). A micromaninula- 1 ting method to observe the inner structure of diseased leaves by scanning electron microscopy. Plant Disease Reporter 60, 95-97. LIN, M. R. & EDWARDS, H. H. (1974). Primary penetration process in powdery mildewed barley related to host cell age, cell type, and occurrence of basic staining material. .New Phytologkt 73, 131-137. MCKEEN, W. E. & RIMMFX, S. R. (1973). Initial penetration process in powdery mildew infection of susceptible barley leaves. Phytopathology 63, 1049-1053. MZUHIRA, V. & FUTAESAICU, Y. (1972). New fixation method for biological membranes using tanmc acids. Acta histochimica $nch&ica 5, 233-235. OKU, H., OUCHI, S., SHIRAISHI, T., KOMOTO, Y. & OKI, K. (1975). Phytoalcxin activity in barley mildew. Annals of the Phytopathological So&y of Japan 41, 185-191. SCHWINN, F. .J. & DAHMEN, H. (1973). Beobachtungen zum Infektionsvorgang bei Erysighe _ _ graminis DC. Phytopathologische &it.sd& 77, 89-92. SHERWOOD. R. T. & VANCE. C. P. (1976). Histocbemistrv of uanillae formed in reed canarvgrass leaves in response to non’mfecting pathogenic fungi. Ph;rtopk&lo~ 66,503-510. ’STAUB, T., DAHMEN, H. & SCH~XNN, E. J. (1974). Light and scanning electron microscopy of cucumber and barley powdery mildew on host and nonhost plants. Phytopathologv 64,364-372. I
I
L
I
I
I
Plant
Pathology
(1977)
10, 191-199
Cytological studies of early stages of powdery mildew in barley and wheat leaves: (II) significance of the primary germ tube of Erysiphe graminis on barley leaves? HITOSHI KUNOH,
HIROSHI
ISHIZAKI
Laboratory of Plant Pathology, Faculty Mie University, l&city, 514, Japan (Accepted
for publication
January
and KYOKO NAKAYA
of Agriculture,
1977)
Early stages of the infection processes of Erysiphe graminis hordei, race I (compatible with barley cv. Kobinkatagi) and of Epsijhe graminis tritici, race ts (incompatible with barley cv. Kobinkatagi) were observed on barley (Hurdeum vulgare L., cv. Kobinkatagi) by scanning electron microscopy. Behavior of both fungi was similar and nearly 99% of the germinated conidia of both races had more than one germ tube 12 h after inoculation. Among the germ tubes, one, designated as the primary germ tube, induced a host papilla or cytoplasmic aggregate below it 4 to 6 h after inoculation, and another swelled to become an appressorium. Almost every conidium which had induced a papilla below an appressorium had earlier induced a papilla below the primary germ tube. The results suggest that the induction of a papilla by the primary germ tube may be critical to attempted penetration by the appressorium from the Micromanipulation indicated that the papilla below the epidermal cell wall same conidium. was continuous with or attached firmly to the inner host wall layer, and also that the wax crystals on the leaves were dissolved by the fungal structures which attempted to penetrate, i.e. the primary germ tube and appressorium.
INTRODUCTION
Many earlier studies [S, 7-91 on conidial germination of Epsi~he graminis have shown that several germ tubes appear on each conidium, but that only one of them grows extensively and forms a typical appressorium. Kunoh & Ishizaki [9] noted a small hemispherical papilla just beneath the tip of short, non-appressorial germ tubes by micromanipulation in a scanning electron microscope @EM), and reported that silicon accumulation occurred in abundance below some of such germ tubes, Most of the earlier investigations concerning the infection process of E. graminis [l-3, 5, 8, 11, 15, 171 have focussed on appressorial function and have paid no attention to nonappressorial germ tubes. Our study was undertaken to investigate, by means of micromanipulation in the SEM [lo], whether or not non-appressorial germ tubes of E. graminis conidia have a r81e in the infection process. For comparison, the infection processes of compatible and incompatible races of E. graminis were investigated on one cultivar of barley. MATERIALS
AND
METHODS
Barley (Hordeum uulgare L., cv. Kobinkatagi) was grown from seed in an illuminated chamber in 20 cm glass flats containing vermiculite. The abaxial epidermis of t Contribution
no. 31.
H. Kunoh, H. lshizaki and K. Nakaya
19!2
IO-day-old leaves was inoculated with conidia of Epciphe gram&is hordei, race I (compatible with cv. Kobinkatagi) or E. graminis tritici, race t, (incompatible with cv. Kobinkatagi) and incubated in a chamber illuminated by fluorescent lamps (c. 4000 lx) for 12 h/day. The chamber was maintained at 20 “C and 90% r.h. Inoculated leaves were cut into small pieces, 1.0 x 0.5 cm, with a razor blade at 4,6,9, 12,24, 30 and 36 h after inoculation and were then treated by the modified tannic acid fixation method [13]. The specimens were fixed with 2% unbuffered glutaraldehyde containing 0.2% tannic acid at room temperature (RT) for 12 h, followed by further fixation in 2% unbuffered glutaraldehyde containing 2% tannic acid at RT for 12 h. After being washed in deionized water for 2 h, the specimens were treated in 1y. unbuffered aqueous 0~0, at RT for 12 h and washed in deionized water for 1 h. They were dehydrated in a graded ethanol series, then in a graded ethanol-iso-amyl acetate series and finally placed into lOOo/oiso-amyl acetate, They were then dried in a critical-point dryer and observed with a Hitachi scanning electron microscope HHS-2X at 20 kV accelerating voltage, except as cited. The specimens were dissected with a micromanipulator set in the SEM in order to observe any papillae induced by the fungi [ 101. Three experiments were replicated at each sampling time and the details of approximately 110 germinating conidia were recorded in each experiment. In order to observe the wax crystals on leaves, small pieces of fresh, inoculated barley leaves were observed in the SEM without fixation or coating [3]. RESULTS ( 1) Conidial
germination
to appressorium formation
In the present study, only germinated conidia were selected as test conidia. About 30% of conidia of race I and race t, germinated 4 h after inoculation. The germination rate of both races increased thereafter and reached 50 to 60% at 12 h. In most cases, two to several germ tubes appeared on each conidium of race I and race t, (Plates 4 and 10). Sometimes only one germ tube appeared to emerge from any one conidium, if over-viewed by SEM. However, if the conidium was moved by the microneedle, another germ tube could usually be seen just beneath the conidium. As shown in Table 1, at 4 h, nearly 30% of the test conidia of both races had one germ tube ; nearly 60% had two germ tubes; and 10% had more than two germ tubes. At 6 h, about 14% of race I conidia and 22% of race t, conidia had one germ tube, and 75 and 70%, respectively, had two germ tubes. Between 9 and 24 h after inoculation, nearly 85% of both races had two germ tubes. At 24 h, fewer than 1 y. of the test conidia of both races had one germ tube. Table 1 indicates that the frequency of the conidia with more than two germ tubes was almost consistently 9 to 16% between 4 and 24 h. Whether conidia are to have more than two germ tubes seems to be determined at the early stage of germination, probably prior to 4 h after inoculation. Only one of the multiple germ tubes from each conidium ceased elongation and attached firmly to the host surface (Plate 1). It induced a papilla in the host epidermal cell (Plates 2 and 3). We designated such a germ tube as the primary germ tube. With time after inoculation, another germ tube swelled, especially at the tip, to become an appressorium (Plates 4, 7 and 10). Even if conidia had more than two germ tubes, only one germ tube developed an appressorium (Plate IO). None of
Key to abbrerkztions g, germ
tube;
ha,
(Plates halo;
1 to 13).
ap, appressorium;
N, microneedle;
PLATE 1. A conidium barley, cv. Kobinkatagi,
p, papilla;
of E. graminis hordei, 4 h after inoculation.
c, conidium; pg, primary
race I, with x 2300.
germ
cy, epidermal
cytoplasm;
tube.
a primary
germ
tube
on
a leaf
PLATE 2. After removal of the conidium of Plate 1 by the microneedle, a bright (arrow) became visible at the site where the primary germ tube had been. x 2300.
of
region
PLATE 3. The bright region of Plate 2 was turned inside out by the microneedle. A papilla initial covered with fibrous materials is now visible on the reverse side of the cell wall. x 2300. PLATE 4. A conidium of E. graminis tritici, race t2, with a primary germ sorium 30 h after inoculation. The penetration sites of both the primary appressorium are surrounded by a halo. x 1200. PLATE 5. The A papilla is present x 1200.
halo region around the primary at the center of the halo region.
tube and an apprwgerm tube and the
germ tube of Plate 4 was turned The halo region appears somewhat
PLATE 6. The epidermal cell wall of the halo region around the appressorium reversed. A papilla is present on the reversed halo region. x 1200.
inside out. depressed.
of Plate
4 was
PLATE conidium cytoplasm. aggregate.
7. A papilla on the reverse side of the cell wall below an appressorium of a race I 24 h after inoculation. The epidermal cell wall was clearly separated from the A hole (arrow) in which the papilla was embedded remained in the cytoplasmic x 1000.
PLATE cell wall.
8. A papiila x 2500.
formed
PLATE 9. The papilla appeared on the reversed PLATE 10. another germ x 2300.
6 h after
of race
I on the reverse
of Plate 8 was removed by the microneedle. cell wall. The cell wall was never hollowed
A germinated conidium of race I having a primary tube 9 h after inoculation. Wax crystals cover
PLATE 11. The conidium of Plate characteristically shaped appressorium penetration site. x 2300. PLATE inoculation
inoculation
12. The imprint of race t,.
in
the
An outline of the papilla out. x 2300.
germ tube, appressorium the whole surface of the
10 was removed by the microneedle. remains in the wax crystals. An
wax
PLATE 13. The imprint of an appressorial The appressorial arm did not leave an Observed at 15 kV accelerating voltage.
crystals
lobe imprint.
of
a displaced
side of an epidermal
primary
on a fresh leaf observed An arrow indicates
The arrow
germ
and leaf.
imprint of the indicates the
tube
4 h after
24 h after inoculation. the penetration site.
[facing
page I92 I
PLATES
1, 2, 3 and 4
PLATES
5, 6, 7, 8 and
9
PLATES
10, 11, 12 and
13
193
Primary germ tube of Erydphe graminis .Numbers
TABLE 1 tubes (including appressoriu) emerging from coni&a of E. graminis (race I) and E. graminis tritici (race ts) on barley leavefl
of germ
Time after inoculation 04
Fungal race Race
I
4
t,
4 6
::
tubes More than2
2
4.3 10.3 1.6 0.9 1.6
27.2 4 13-5 21.94 6.6 2.7f 1.5 0.54 0.5 0.34 o-5
9
of germ
1 30.24 13.94 2-82 0.6* 0.94
9” 12 24 Race
Number
60*5+ 75.4+ 86-l + 84.3-1 86.22 62.9+ 70.5+ 86.15 84-i’+ 83*4+
hordei
5.0 8.2 l-7 5.6 7-l
9.1 k5.1 10+6*5*3 11*1&1*5 15.2 f 5.8 12*8+6-O
11.3 7.9 4.3 5.9 2.7
9.9&2-6 7.5&l-6 II-O&1*9 14.8 + 5.7 16~2k3.7
Q Expressed as percentages of the number of corresponding conidia to the total number of conidia observed. Variation is expressed as standard deviation. Based on three experiments with approximately 110 germinated conidia scored in each.
the conidia had more than one appressorium. In the case of conidia with more than two germ tubes, they had a primary germ tube and an appressorium; the other germ tubes did not induce papillae. We scored germ tubes which were more than 1.5 times broader at the tip than at the base as appressoria. As indicated in Table 2, nearly 60% of the test conidia of
Percentages
of aj$ressorium
TABLE 2 formation by conidia of E. graminis hordei tritici (race tJ on barley haves@ Time after inoculation 04 4 ii 12 24
(1 Based
on three
Percentage
experiments
of appressorium
with
Race
Fungal I
0 61*6+6*6 92.2 + 4.4 98.2 + l-4 98.2 f O-9
race Race
(race I) and E. graminis
ts
0 50.2 & 9-9 91*7+2*5 96-l&1*8 99-l&0*9
approximately 110 germinated conidia scored No. of conidia haying an appressorium formation = Total no. of conidia observed
in each. x loo
race I had an appressorium 6 h after inoculation and more than 90% did at 9 h. Similarly, nearly 50% of the test conidia of race ts had an appressorium 6 h after inoculation and more than 90% did at 9 h. With both races, a lobe of the appressorium became conspicuous by 9 h after inoculation. A second lobe often formed midway along the side of the appressorium (Plate 7). The second lobe occurred more frequently with race t, than race I 24 h after inoculation.
194
H. Kunoh, H. lshizaki and K. Nakaya
(2) Papillae indqction by the primary germ tube and apliressorium As mentioned previously, the primary germ tube emerged from the conidium and induced a papilla in the epidermal cell by 4 h after inoculation (Plate 1). When the conidium with the primary germ tube was moved by the microneedle, a small circular region around the penetration site appeared bright (Plate 2). Furthermore, when the epidermal cell wall around the bright region was turned inside out by the microneedle, a small papilla initial was revealed at the penetration site. Careful observations of the reversed primary germ tubes revealed that a minute hole, about O-3 to O-5 pm in diameter, existed on the tip surface of some primary germ tubes which had contacted the host cuticle, suggesting attempted penetration by the primary germ tube. However, we failed to find such a hole on some primary germ tubes. Some of the papilla initials found at 4 h were covered by a cytoplasmic aggregate [Z] with a fibrous nature (Plate 3). These initials usually could not be identified with certainty until the cytoplasmic aggregate was removed by the microneedle. The host response at 4 h was difficult to over-view by light microscopy, because the response area was usually narrower than 5 pm in diameter, and the primary germ tube over the response area blocked the view. The papilla was well defined on the reverse side of the cell wall by 6 h after inoculation (Plates 5 to 9). Of approximately 110 germinated conidia of race I, the proportion of primary germ tubes which had induced a papilla or cytoplasmic aggregate was 50% at 4 h after inoculation, about 7.5% at 6 h and 97% at 12 h (Fig. 1). No haustorium was
+A
4
’
6
I
I
I
9
12
24 Time
Fro. of (a) E. Primary Frequency No.
1. Papillae or cytoplasmic gramti /w&i (race I) germ tube; (~0 = of primary germ tubes Total no. of
after
/ t+m
4
inoculation
’
’
6
9
I
24
12
(h 1
aggregate induction by primary germ tubes and and (b) E. grutiinb titici (race ta) on barley leaves; appressorium. or appressoria primary germ
inducing a papilla tubes or appressoria
or cytoplasmic observed
appressoria (A-A)
aggregate
.
produced by the primary germ tube during the observation period. Germinated conidia of race t, showed a similar tendency concerning papilla induction (Fig. 1). About 52% of the primary germ tubes induced a papilla or cytoplasmic aggregate by 4 h after inoculation, about 86% by 6 h and 92 to 98% by 9 h.
Primary germ tube of Erysiphe gramink
195
Other germ tubes from conidia with primary germ tubes swelled to become appressoria by 6 h (Table 2; Plates 4, 7 and 10). In race I, about 27% of the test appressoria induced a papilla below the epidermal cell wall by 6 h after inoculation, about 78% did by 12 h and 96.5% did by 24 h. On the other hand, 19% of the test appressoria of race t, induced a papilla by 6 h and more than 90% did by 12 h after inoculation. In both races, more than 95% of the test appressoria induced a papilla in the epidermis by 24 h (Fig. 1). Figure 1 indicates that papillae induction by primary germ tubes occurs 5 to 6 h earlier than that by appressoria. As shown in Fig. 2, almost all conidia which induced a papilla below their appressorium had induced a papilla also below their primary germ tube (Plates 4
fimr
after
inoculation
(h)
FIG. 2. Papillae or cytoplasmic ag$egate induction by the primary germ tube and appressorium from the same conidium of (a) E. grmninis hordei (race I) and (b) E. graminis Mici A papilla or cytoplarsmic aggregate was observed below (race tz) on barley leaves. ( l -a) both the primary germ tube and the appressorium from the same conidium; (0- - -0) a papilla or cytoplasmic aggregate was observed only below the primary germ tube.
to 6). During the 24 h after inoculation, fewer than 1 Oh of the test conidia induced a papilla below their appressorium without first inducing a papilla below the primary germ tube. Fig. 2 indicates that the frequency of conidia inducing a papilla below the primary germ tube, but not one below the appressorium, decreased with time and, conversely, that of the conidia inducing papillae below both of the fungal structures increased. These observations suggest that the induction of a papilla by the primary germ tube may be involved in the process of attempted penetration by the appressorium produced by the same comdium. Frequently, some of the appressoria had two appressorial lobes at 24 h, especially in the case of race t, (Plate 7). A papilla was usually produced below each of the lobes. (3) Association of papillae with the host cell walls Sometimes micromanipulation the host cytoplasm, although
succeeded in separating the epidermal cell wall Corn some cytoplasmic vesicles were still attached to the
196
H. Kunoh, H. lshizaki and K. Nakaya
reverse side of the cell wall (Plates 7 to 9). As the result of micromanipulation, a hemispherical papilla was observed on the reverse side of the cell wall at the penetration site (Plates 7 and 8), leaving a hole in the cytoplasmic aggregate in which the papilla had been embedded (Plate 7, arrow). Papillae showed an intense resistance to microneedle pressure when attempts were made to remove them from the reverse side of cell walls. When further force of the microneedle succeeded in removing the papilla, a trace of the papilla remained on the reverse side of the cell wall; the cell wall was never hollowed out (Plate 9, arrow). These results suggest that the papilla, or at least a part of it, is continuous with or attached firmly to the inner layer of the cell wall. (4) Degradation of leaf wax by fungal structures As viewed by SEM, a halo surrounded the penetration sites of both primary germ tubes and appressoria (Plate 4). Observations at a magnification of x 5000 revealed that wax over the halo region remained undissolved. The halo region was difficult to see at first because its brightness was similar to that of other regions. The halo region was somewhat brighter than other areas of the leaf between 9 and 12 h after inoculation and, furthermore, it turned darker by 24 h. These darker halo regions appeared somewhat depressed when viewed from the reverse side (Plates 5 and 6). During the present observations it became evident that wax crystals covered the entire surface of barley primary leaves (Plates 10, 11 and 13). Where conidia with the primary germ tube and appressorium were removed by the microneedle, tracks of these fungal structures, except the conidium itself, appeared on the cuticular surface (Plates 10 to 12). In some cases, such tracks appeared only at the appressorial lobes and not below the appressorial arm (Plate 13). When conidia have several other germ tubes besides the primary germ tube and appressorium, a track never appeared below these germ tubes. These observations suggest that the wax crystals were degraded only by the primary germ tube and appressorium. Another possibility is that tracks of the primary germ tube and appressorium may result from the fibrillar matrix of germ tubes and appressoria pulling wax off, but the reversed surface of both iimgal structures which had contacted the host surface was quite smooth. Thus, these observations are considered to support the possibility of chemical degradation of wax crystals by the primary germ tube and appressorium. DISCUSSION
The present observations on primary germ tubes indicated that a papilla was induced by almost all primary germ tubes regardless of the compatibility of the host-parasite combinations. Furthermore, they showed that the induction of a papilla by the primary germ tube might be critical to attempted penetration by the appressorium and thus to survival of E. graminis. No other fungi known to the authors have such characters. Ellingboe [5] reviewed the infection process in powdery mildew and summarized that the process of primary infection consisted of six morphologically identifiable stages : spore germination, formation of appressorial initials, maturation of appressoria, formation of penetration pegs, formation of haustoria and formation of secondary
Primary germ tube of Erysiphe
graminis
197
hyphae. As shown clearly in this study, the host cell responds to the primary germ tube earlier than to the appressorium. Thus we consider that the formation of, and penetration by, the primary germ tube is important in the infection process of E. graminis. Oku et al. [14] found that there were two phases of phytoalexin production in barley leaves infected with E. graminis. The first phase was detected at 12 h after inoculation. They concluded that this stage just coincides with the time of initial direct contact between cells of the host and the parasite. The present observations, however, suggest that the first direct contact of host cells and primary germ tubes occurs by 4 to 6 h after inoculation. Accordingly, we cannot rule out the possibility that the host responses, including phytoalexin production, may proceed soon after penetration of the primary germ tube and, as a result, the primary germ tube may influence the subsequent infection process, including compatibility. Further observation of specimens prepared at 30 and 36 h after inoculation revealed a haustorium in the epidermis attacked by some of the appressoria of race I, while haustoria were not observed below the primary germ tubes. However, it is uncertain at present whether or not papillae form early enough to present a mechanical barrier to penetration by the primary germ tubes. Lin & Edwards [II] investigated the mechanisms which determined whether penetration would occur in barley infected by E. graminis and concluded that it was the presence of a basic staining material(s) (BSM) in the papilla which correlated with unsuccessful penetration attempts. In preliminary experiments, we observed that some of the papillae induced by primary germ tubes contained BSM, but others did not, regardless of host-parasite compatibility. Thus, BSM cannot determine whether penetration will occur at the site of primary germ tubes. Furthermore, these observations suggest that papillae induced by primary germ tubes may be somewhat different in chemical composition and quantity from those induced by appressoria. The present micromanipulation study indicated that the papillae, or at least part of it, is continuous with or attached firmly to the inner layer of the host cell wall. Bracker [I] studied the ultrastructure of the haustorial apparatus of E. graminis and reported that the transition between the host wall and the papilla (collar) was abrupt and an interface, characterized by a thin electron-dense zone, often separated the two structures after both 0~0, and KMnO, fixations. He concluded that the papilla was not a swelling or ingrowth of the cell wall but a deposit upon the wall. Kunoh [S] also reported that barley papillae induced by E. graminis did not have a cellulosic nature and that the border line between the host wall and the papilla could be readily observed in electron micrographs. Sherwood & Vance [16] investigated the histochemical nature of reed canarygrass papillae and concluded that cellulose was not the major component of the papillae. On the basis of these ultrastructural and hiitochemical observations, it is likely that .the papilla, a deposit upon the wall, attaches firmly to the inner layer of the host cell wall. The substance which makes the attachment is unknown. Edwards & Allen [4] and McKeen & Rimmer [IZ] presented evidence obtained by electron microscopy that the papilla could be formed before the host wall was penetrated .by the fungus. On the other hand, Bushnell & Bergquist [,?I suggested, on the basis of the closeness in time between the appearance of the cytoplasmic aggregate and the appearance of the haustorium, that the fungus might have started
198
H. Kunoh, H. lshizaki and K. Nakaya
to penetrate before papilla formation. In the present study, a minute hole suggesting the site of a penetration peg was observed on the reversed surface of some primary germ tubes which had contacted the host surface. However, we are not completely confident about our attempts to determine the timing of emergence of penetration pegs from the primary germ tubes, since we failed to observe such a minute hole on some other primary germ tubes which had induced papillae or cytoplasmic aggregates in epidermal cells. This problem should be studied in detail by micromanipulation or other techniques. The present micromanipulation studies revealed the imprints of the characteristically shaped primary germ tubes, appressoria and appressorial lobes in wax crystals on barley leaf surfaces. By removal of the overlying mycelium of E. graminis on barley leaves with adhesive cellulose tape, Day & Scott [3] observed hyphal tracks throughout the wax crystals on the cuticle of barley leaves. Similarly, Schwinn & Dahmen [15] and Staub et al. [17] reported the dissolution of wax crystals on barley leaves under appressoria, germ tubes and hyphae of E. graminis. Staub et al. [17] concluded that the dissolution of the wax crystals, as was observed under the germ tube of E. graminis, might be a critical step in the process of direct penetration of barley leaves. The present observations, that only the primary germ tube and appressorium which attempted penetration could dissolve the wax crystals and that the other germ tubes never induced any response of the host cell, seem to support Staub et al.3 conclusion. As they also suggested, close contact of germ tubes other than the primary germ tube and appressorium with the cuticular surface may not be possible, and penetration may be prevented because they cannot dissolve the wax crystals. Since the behavior of primary germ tubes of race I and race t, were similar on the same cultivar of barley, the compatibility of host and parasite may not be determined at the primary germ tube stage. Although the present results may suggest that the primary germ tube is essential for subsequent appressorial penetration, we must wait until we have radioisotopic data on the absorption of host material through the primary germ tube before we speculate further on the physiologicalroleof the primary germ tube. Correction of the English in the text by Dr J. R. Aist, Cornell appreciated.
University,
is
REFERENCES 1. BRACKER, C. E. (1968). Ultrastrncture of the hanstorial apparatus of E&rhe gramiais and its relationship to the epidermal cell of barley. Phytojwthdup 58, 12-30. 2. BUSHNELL, W. R. & BERGQUIST, S. E. (1975). Aggregation of host cytoplasm and the formation of papillae and haustoria in powdery mildew of barley. Phytopathology65, 310-318. 3. DAY, P. R. & SCOTT, K. J. (1973). Scanning electron microscopy of fresh material of Epi@e graminis f. sp hvraki. Phyxiological Plant Pathology 3,433-435. 4. EDWARDS, H. H. & ALLEN, P. J. (1970). A fine-structure study of the primary infection process during infection of barley by Erysipe graminis f. sp. hordei. Pl@o~atholvgv 60, 15041509. 5. ELLINGBOE, ,A. H. (1972). Genetics and physiology of primary infection by Epi)%e gramink
Phytvpathvlogv 62,401-406. 6. HIRATA, K. (1942). On the shape Hvriiculture 5,34-49.
of the germ
tubes
of
Epsiphae. Bulletin of the Chiba College of
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8.
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10.
11.
12. 13. 14. 15. 16. 17.
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(1955). Some observations on the relation between the penetration hypha and haustorium of the barley mildew (E@~si@e graminti DC.) and the host cell (I). Annals of the Phytojathological Society of Japan 19, 104-108. KUNOH, H. (1972). Morphological studies of host-parasite interaction in powdery mildew ofbarley, with special reference to affinity between host and parasite. Bulletin of the Faculty of Agriculture, Mie University 44, 141-224. KUNOH, H. & ISHIZAIU, H. (1976). Accumulation of chemical elements around the penetration sites of &&he graminis hodei on barley leaf epidermis: (III) micromanipulation and X-ray microanalysis. ihysiologieal Plant Pathol&y 8,9f-96. KUNOH. H.. ISHIZAIU. H.. WATANABE. T., YAMADA. M. & NAGATANI. T. (1976). A micromaninula- 1 ting method to observe the inner structure of diseased leaves by scanning electron microscopy. Plant Disease Reporter 60, 95-97. LIN, M. R. & EDWARDS, H. H. (1974). Primary penetration process in powdery mildewed barley related to host cell age, cell type, and occurrence of basic staining material. .New Phytologkt 73, 131-137. MCKEEN, W. E. & RIMMFX, S. R. (1973). Initial penetration process in powdery mildew infection of susceptible barley leaves. Phytopathology 63, 1049-1053. MZUHIRA, V. & FUTAESAICU, Y. (1972). New fixation method for biological membranes using tanmc acids. Acta histochimica $nch&ica 5, 233-235. OKU, H., OUCHI, S., SHIRAISHI, T., KOMOTO, Y. & OKI, K. (1975). Phytoalcxin activity in barley mildew. Annals of the Phytopathological So&y of Japan 41, 185-191. SCHWINN, F. .J. & DAHMEN, H. (1973). Beobachtungen zum Infektionsvorgang bei Erysighe _ _ graminis DC. Phytopathologische &it.sd& 77, 89-92. SHERWOOD. R. T. & VANCE. C. P. (1976). Histocbemistrv of uanillae formed in reed canarvgrass leaves in response to non’mfecting pathogenic fungi. Ph;rtopk&lo~ 66,503-510. ’STAUB, T., DAHMEN, H. & SCH~XNN, E. J. (1974). Light and scanning electron microscopy of cucumber and barley powdery mildew on host and nonhost plants. Phytopathologv 64,364-372. I
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