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Ultrastructure of the Colletotrichum trifolii-Medicago sativa pathosystem. I. Pre-penetration events
Phyiological and Molecular Plant Pathology (1991), 38, 179-194
1 79
Ultrastructure of the Colletotrichum trifolii-Medicago sativa pathosystem. I. Pre-penetration events ~[ICHAELJ. R. ~IOULD*, G.J. BOLAND'~and JANE ROBB*+ + Departments of Molecular Biology and Genetics* and Environmental Biology,t Unh'ersity of Guelph, Guelph, Ontario ~7G 211V, Canada (Acceptedfor publication February 1991)
Light and electron microscopy were used to study the early stages in the development of the Colletotrichum trifolii-Medicago satira pathosystem, including compatible and incompatible interactions involving races 1 and 2 oftlae pathogen. Neither the fungal race nor the host genotype influenced tile percentage or timing of conidial germination, appressorial development or penetration peg formation. Host cells initiated a response only after contact was made between the fungal penetration peg and the alfalfa cuticle. The response, which was immediate, and localized to the epidermal cell beneath the point of ingress, included increased staining of the wall and cuticle around the penetration peg, the formation of a papilla and vacuolar fragmentation. The initial compatible and incompatible host responses were indistinguishable.
INTRODUCTION A n t h r a c n o s e o f alfalfa (Medicago saliva L.) is caused by tile fungus Collelotrichum lrifolii Bain a n d Essary. A t least two races o f the p a t h o g e n exist [24,35] and, correspondingly, resistance to each race seems to be controlled by an i n d e p e n d e n t d o m i n a n t , tetrasomically inherited gene [9,10]. P r i m a r i l y a stem disease, lesions can be observed within 120 h post-inoculation in susceptible plants [7]. T h e events leading to lesion d e v e l o p m e n t include, in chronological order, eonidial g e r m i n a t i o n , formation o f g e r m tubes a n d appressoria, p e n e t r a t i o n peg d e v e l o p m e n t , g r o w t h o f h y p h a e within the host tissues a n d , lastly, the e r u p t i o n o f acervuli. W h i l e numerous u l t r a s t r u c t u r a l studies have been carried out on related Colletotrichum interactions [2, 5,15,16, 20,21, 26, 27, 38], only two cytological studies have been published on the C. trifolii-alfalfa p a t h o s y s t e m : first, a light and electron microscopic study o f a c o m p a t i b l e interaction involving race 1 [28] and second, a light microscopic study o f the c o m p a t i b l e and i n c o m p a t i b l e responses involving both races 1 a n d 2 [7]. W h i l e both studies emphasized tile early stages o f the interaction (i.e. 0 - 4 8 h postinoculation), the p l a n t a n d fungal structures a n d the i n t e r a c t i n g events which are involved in the initial p e n e t r a t i o n o f the host r e m a i n relatively u n d o c u m e n t e d . In the present study, we used both light and transmission electron microscopy to investigate the p r e - p e n e t r a t i o n events involved in establishing c o m p a t i b l e or .+Author to reprint requests should be sent. 0885-5765/91/030179+ 16 S03.00/0 13
© 1991 Academic Press Limited M P P 38
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incompatible relationships with both races of C. trifolii. T h e two primary objectives were: (I) to investigate the ultrastructure of the appressorium and penetration peg; and (2) to determine the timing and cytological nature of the initial host response. MATERIALS AND METHODS
Culture of the fimgus Cultures of races 1 and 2 of C. trifolii were obtained from the American T y p e Culture Collection (ATCC 41=42881 and ~42041 respectively). Conidial suspensions were prepared from cultures of each race and stored at --70 °C in 25 % aqueous glycerol [14]. Single droplets from the frozen stock cultures were pipetted onto a 10 % (v/v) V8 Juice/water agar medium and the suspensions were spread evenly using a bent glass rod. After 6 to 8 days of growth, suspensions of I-0 x t0 s conidia m1-1 were prepared in sterile distilled water. Fractions of 0-2 to 0"3 ml of each suspension were pipetted onto individual V8 Juice agar plates and spread evenly over the agar. Cultures were maintained at 22 °C under fluorescent lights. Virulence of both races was maintained by periodically infecting and re-isolating the pathogen from a susceptible host plant.
Selection and growth of host plants Ninety-three alfalfa plants of the breeding line Regen-S [30] were screened for resistance to races 1 and 2 ofC. trifolii using a needle inoculation method [23]. Seedlings were selected from an anthracnose disease survey of Ontario-grown cultivars [1] that had shown resistance to race 2 ofthe pathogen ; they were grown to maturity and tested using the same method. For each evaluation, two alfalfa stems were inoculated with each race and one stem per plant was injected with sterile distilled H 2 0 as a control treatment. Plants were rated for disease response at 7, 10 and 14 days post-lnoculation using a 1 to 5 scale [22]: 1 -- resistant (R), no lesion or small water-soaked spots; 5 = susceptible (S), dead stems. Plants that were rated at 1:1 ( R / R ) , 1:5 (R/S) and 5:5 (S/S) to races 1 and 2, respectively, were kept for further study. A minimum of two plants per genotype was used. Shoot cuttings were taken from each plant and rooted in vermiculite. Once these cuttings had achieved adequate maturity, they were repotted into 15 cm diameter plastic pots using an autoclaved 1 : 1 mixture of Turface: Promix (Premier Brands Inc., New Rochelle, N.Y. 10801 U.S.A.). All plants were grown in a Conviron growth chamber (Model 4eE8VH) with 14 h daylight and a maximum photosynthetically active light intensity of 240 g E m -2 s-1. Night and day temperatures were 18 °C and 22 °C respectively. Plants were watered daily with deionized water and fertilized weekly with a 2-4 g 1-1 solution of fertilizer (20:20:20 N : P : K ) . Plants were cut regularly after flowering to ensure succulent growth for subsequent cytological studies.
Preliminary experiments T o establish the timing of conidial germination, appressorium formation, host penetration, disease development and resistance responses, several preliminary inoculations were performed. Stems and leaves of resistant and susceptible plants were inoculated with a conidial suspension (1-0 x 10 Gconidia m1-1 in sterile distilled water) of either race 1 or 2 of C. trifolii by spraying the plant until runoff. Inoculated plants
C. trifolii-alfalfa pathosystem. Part I
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were placed in a mist chamber that maintained continuous plant surface wetness for 72 h at 24 °C. Inoculated stems were harvested at 24, 48, 60, 72, 96 and 120 h postinoculation (h.p.i.). Two stems were inoculated per sampling time and the experiment was repeated three times. Control stems, one for each plant, were sprayed with water. Whole mounts of inoculated leaf tissues (adaxial side up) and freehand radial longitudinal and cross sections of inoculated stems were placed on glass mici-oscope slides. Tissue was stained with two or three drops of0-01% cotton blue in lactophenol (1 lactic acid: 1 ptlenol: 1 water: 2 glycerin) at 60 °C for 20 rain. Alternatively, stem tissues were stained with 0-1% chlorazol black E in lactoglycerol (1 lactic acid: 1 glyercol: 1 water) [4]. Material stained with chlorazol black E was destained in lactoglycerol and mounted in this solution. The percentages of germinated conidia , germinated conidia which formed appressoria and appressoria which formed a penetration pore were recorded (based on measurements of 100 conidia and appressoria per trial). All tissue was examined using brightfield microscopy (Nikon Labophot equipped with a Nikon FX-35 camera).
Cytological studies of pre-penetration events Conidial suspensions of 5"0x10 ~ conidia m1-1 in 0"083% Tween 20 [17] were prepared from 7-day-old cultures ofrace 1 or 2 ofC. trifolii. To ensure uniform coverage of the inoculated stem, each spore suspension was poured into a sterile Petri dish and the upper succulent region of the stem and the uppermost leaves )vere immersed in the suspension. For each experiment, only one race was inoculated onto each plant and incubated in a mist chamber. For each inoculation, three stems per time per race/genotype interaction were inoculated. One stem per plant was immersed in 0-083 % Tween 20 as a control. Material was sampled at 48 h.p.i, and processed for electron microscopy. Stem pieces were trimmed to a length of approximately 2-0 cm, slit lengthways in a Petri dish containing 1% glutaraldehyde in 0"07 M Sorenson's phosphate buffer (pH 6"8) and further trimmed to a length of approximately 1"0 cm. Individual pieces were then transferred to separate vials containing 3 % glutaraldehyde and 1-5 % acrolein in the same buffer amended with 1"0 mM CaC12 and 16 drops of 1% Tween 20 per 500 nal. The tissue was fixed for 3 h at room temperature in the same buffer, post-fixed for 2 h at room temperature with 2 % OsO 4 in the same buffer, dehydrated through a graded acetone series and embedded in Spurr's low-viscosity resin [32]. Tissue was flat-embedded with the stem surface facing upwards. Sections were cut on an LKB Ultratome using a diamond knife (Diatome). Semi-thin (0"25 pm) sections were dried onto slides coated with a subbing agent [25]. Sections from infected stems were taken from an area 5"0 mm from the edge of the lesion. Sections were stained with 0-1% toluidine blue 0 in 1% sodium tetraborate and examined with brightfield microscopy. Thin sections of silver to gold interference colour were collected on acetone-washed 200 mesh copper-rhodamine grids. Sections were stained with a saturated solution of uranyl acetate in 70% methanol (30 min) followed by lead citrate (22 min) [29]. Thin sections were examined with a J E O L 100 CX electron microscope operating at 60 kV. Individual appressoria were located on a stem surface for thin sectioning. Since the thickness of the embedded material was less than 3 mm, the surface of the embedded 13-2
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stem could be viewed at low magnification using a light microscope. The stem surface was scanned and desired spots marked for sectioning by nicking the edge of the resin with a razor blade. Excess stem tissue was trimmed under a binocular microscope until the desired appressoria lay 3 to 41am from the trimmed end. Sections of 0"2 ~tm thickness were initially cut on the ultramicrotome and every fifth section was mounted on a slide and stained with toluidine blue 0 to determine when the site was reached. Thin sections (700-900 nm) were then cut, collected, stained and viewed. A total of 25 appressoria and infection sites were sectioned and used in the studies of the prepenetration events. Five appressoria were reconstructed from serial sections. RESULTS
Preliminary experiments Conidia from races 1 and 2 are cylindrical with rounded ends, averaging 12"5 lain x 5"8 pm (based on measurements of 100 conidia per race). The timing ofpre- and postpenetration events are summarized in Table 1. Neither the fungal race nor host genotype influenced the frequency of conidial germination or appressorial formation [Fig. 1 (a), (b)]. By 24 h.p.i., germination was complete. The spores remained singlecelled and upon germination each formed a short (I-2 lam) germ tube which by 48 h.p.i, ended in a spherical, dark-walled appressorium [average diameter 64 pm; Figs 2, 3 (b)]. Results are similar on both stem and leaves. Appressoria of each race formed over the junctions of the anticlinal cells walls between adjacent epidermal cells [Figs 2, 3(a), (b)]. Appressorium maturation culminated with the migration of the conidial cytoplasm from the conidium to the appressorium via the germ tube [Figs 2, 3 (b)]. A septum developed between the germ tube and the conidium [Fig. 3 (b)]. When devoid of cytoplasm, conidia appeared as non-staining 'ghost cells' which subsequently collapsed [Figs 2, 3(b)]. Electron microscope analysis revealed that these 'ghost cells' were empty of identifiable cytoplasm. By 60 h.p.i appressoria formed penetration pegs and by 72 h.p.i, the epidermal ceils layers of both resistant and suceptible plants were colonized.
Structure of the appressorium and penetration peg formation Individual appressoria of both races of C. trifolii contain large numbers of free ribosomes, lipid bodies, mhochondria, and a single nucleus with a prominent nucleolus [Fig. 4 (a), (b)]. Retraction of the cytoplasm from the cell wall (Fig. 4) in appressoria forming penetration pegs allowed a close examination of the wall (Fig. 5). The threelayered appressorial wall has an overall thickness of 160 to 170 nm. The innermost layer is thin (!5 nm) and highly electron-dense while the second, thicker (70 nm) layer is electron-lucent. The third layer is electron-dense and thicker (80 nm) than the second layer. An electron-lucent, mucilaginous coat, which is continuous with the germ tube wall, is approximately 100 nm thick and contains numerous small, electron-dense particles. Where the appressorium contacts the host cuticle, the mucilaginous coating spreads over the host cuticle (Fig. 5). Close examination of the appressorium in Fig. 4(a) revealed a penetration pore approximately 200 nm in diameter and a small penetration peg [Fig. 6 (a)]. The peg does not contact the host cuticle at any point, the appressorium being attached to the
C. trifolii-alfalfa pathosystem. Part I
183 TABLE 1
Summary of the data from preliminary experiments to determine the timing of the interaction between 12. trifolii and M. sativa Host genotype Time (h.p.i.) 24 % conidia germinated 48 % germinated conidia forming appressorium 60. % appressoria forming penetration peg 72
96
120
S:S =
R:S b
R:R e
Rid-92 R2e-96
RI-94 RS-88
RI-93 R2-87
R1-85 R2-88
R1-90 R2-93
R1-91 R2-86
R1-51 R2-53
R1-53 R2-56
R1-50 R2-51
Initial penetration of the cutlcle, by both races, complete Penetration up to fourth cell layer by both races
Initial penetration of the cuticle, by both races, cgmplete RI restricted to single epidermal cell R2 penetration to 4th cell layer RI infected epidermal cell darkened R2 lesion development
Initial penetration of the cuticle, by both races, complete Restriction to single epidermal cell both races
Lesion development in both races
Infected epidermal ceils darkened in both races
aS:S plants are susceptible to races 1 and 2 ofC. trifolii (i.e. 5:5). bR:S plants are resistant to race 1 and susceptible to race 2 ofC. trlfolii (i.e. 1:5). e R : R plants are resistant to both races ofC. trifolii (i.e. 1:1). dR1 race 1 ofC. trifolii. eR2 race 2 of C. trifolii. host
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r e c o n s t r u c t i o n o f t h e p o r e c o m p l e x [ F i g . 6 ( a ) - ( c ) ] r e v e a l e d it to b e a v e r y i n t r i c a t e s t r u c t u r e ( F i g . 7). T h e w a l l s o f t h e p o r e e x t e n d i n t o t h e a p p r e s s o r i a l c y t o p l a s m t o a p p r o x i m a t e l y o n e - q u a r t e r t h e d i a m e t e r o f t h e a p p r e s s o r i u m ( F i g . 7), f o r m i n g a c o n e . FIo. 1. Longitudinal sections showing chlorazol black E-stained appressoria (A) of C. trifolff distributed over the stem surface at 48 h.p.i. × 800. (a) Race 1-resistant/race 2-resistant ( R / R ) alfalfa plant v. race 2 ofC. trifolii. × 800. (b) Race l-susceptible/race 2-susceptible (S/S) alfalfa plant v. race I of G. trifolii, x 800. FIG. 2. Appressorium (A) of G. trifolli race 2 with attached conidial ghost cell (C). Appressorium has formed over the cell wall junction between two epidermal cells (arrowhead). Chlorazol black E-staining, 48 h.p.i, x 1100. FIo. 3. Toluidine blue 0-stained, uhramicrotome thick sections showing appressoria (A) of C. trifolii formed over the junctions between the anticlinal cell walls between adjacent epidermal cells (i, ii) at 48 h.p.i. Epidermal cell walls and cuticle are evenly stained and host cell cytoplasm exhibits no signs of any host response. × 1200. (a) R / R plant v. race 2. (b) S/S plant v. race 1. Appressorium is attached to conidial ghost cell (C) by a short germ tube (GT). A septum (S) is visible between the conidium and germ tube.
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FIG. 7. Three-dimensional diagrammatic representation of an appressorium ofG. trifolii based upon serial reconstruction from Figs. 5 and 6(a)-(c). Layers a-c and the mucilaginous coat (d) correspond to those in Fig. 5. The penetration peg (pp) forms at the base of the appressorium over the anticlinal wall junction between the epidermal cells. The elaborate cone-shaped pore complex ((2) extends into the appressorium approximately one quarter of the appressorial diameter. N o n e o f the appressorial wall layers a r e c o n t i g u o u s with the cone, a l t h o u g h the second layer projects ihto the appressorial c y t o p l a s m , f o r m i n g a t h i c k e n e d collar a r o u n d the p e n e t r a t i o n pore [Figs 6 (a), (b), 7]. T h e wall of the c o n e e x t e n d s d o w n t h r o u g h the pore to form the e l e c t r o n - l u c e n t wall o f the p e n e t r a t i o n peg. At the tip o f the p e n e t r a t i o n peg the cell wall is very thin [Fig. 6 (a)].
Timing and ultrastructure of the early host responses C o n t r o l p l a n t tissue is well preserved w h e n fixed in 3 % / 1 " 5 % g l u t a r a l d e h y d e / a c r o l e i n . T h e cuticle a n d e p i d e r m a l cell wall s t a i n e d u n i f o r m l y with t o l u i d i n e b l u e 0 (Fig. 8). Fresh free h a n d stem cross sections did n o t stain with chlorazol black E (Fig. 9), facilitating the analysis o f f u n g a l spores, g e r m tubes a n d appressoria o n the l e a f s u r f a c e [Figs l ( a ) , (b), 2]. U h r a s t r u c t u r a l analysis showed that, w i t h i n the e p i d e r m a l a n d cortical cells of n o n i n o c u l a t e d alfalfa stems, a thin layer of c y t o p l a s m s u r r o u n d s a large c e n t r a l v a c u o l e FIo. 5. High magnification of an area of the appressorium seen in Fig. 4 (a). The appressorial cell wall consists of three layers: an innermost, electron-dense layer (a), a second, electron-lucent layer (b), and a third, electron-dense layer (c). A mucilaginous coating (d) containing numerous small, electron-dense particles (arrowhead) spreads over the stem surface. × 67500. FIo. 6. TEMs from a group of serial sections taken in order to form a three-dimensional reconstruction of the penetration peg and cone complex seen in the appressorium of Fig. 4 (a). All magnifications × 48600. (a) Near-median section through the developing penetration peg (pp). An elaborate cone-shaped wall complex (C) extends into the appressorial cytoplasm. (b) Two seetioqs serial to Fig. 6(a) showing walls of the cone (C). (c) Four sections serial to Fig. 6(a) showing walls of the cone (C).
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Flo. 8. Toluidine blue 0-stained uhramicrotome thick section from a HzO-injected control plant. Epidermal cells (i) are devoid of any material in their vacuoles, and cell walls and cuticle are evenly stained, x 800. FIG. 9. Chlorazol black E-stained freehand section from H20-injected control plant. Epidermal (i) and cortical (ii) cells do not react with the stain. × 800. Fro. 10. (a) TEM from control plant. A thin layer of cytoplasm (arrow) surrounds a large, central vacuole (V). Epidermal cell wall (HCW) and cuticle (Cu) are uniform over the periclinal surface of the stem. × 3000. (b) TEM of bracketed area of Fig. 10 (a). Nucleus (N), chloroplasts (Ch), mitochondria (mt) and rough cndoplasmic reticulum are contained within the cortical cell's cytoplasm. × 3900. [Fig. 10 (a)]. T h e thin l a y e r o f c y t o p l a s m c o n t a i n s a single nucleus, chloroplasts w i t h starch grains, m i t o c h o n d r i a , a n d e n d o p l a s m i c r e t i c u l u m [Fig. 10 (b)]. H o s t responses b e g i n w h e n the f u n g a l p e n e t r a t i o n peg c o n t a c t s the host cuticle [Fig.
190
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C. trifolii-alfalfa pathosystem. Part I
191
11 (a)]. Serial section analysis of the cell depicted in Fig. 11 (a) and (b) confirmed that, in this case, the short penetration peg had not breeched tile cuticle. Neither enzymatic degradation nor inward bending of the cuticle and cell wall were evident around the penetration peg although the staining of these structures was altered around the point ofingress. In Fig. 11 (a) and (b), and in other similar cases involving both resistant and susceptible plants, small vacuoles a p p e a r in the host cytoplasm directly beneath tile point of pathogen ingress. Typically, papillae are just beginning to form and, at such an early stage, are homogeneous and electron-lucent [Fig. 11 (a)]. Whether race 1 or 2 of the pathogen is involved, compatible and incompatible reactions are indistinguishable until this stage of development. No host response has ever been observed prior to actual contact of the penetration peg with the host cuticle [Fig. 4 (a)]. Appressoria that do not form a penetration peg do not elicit a host response [Fig. 4 (b)]. DISCUSSION
Conidial and appressorial dimensions are in agreement with those previously reported [34]. However, previous studies have shown that C. trifolii completes conidial germination and appressorial formation by 16 and 24 h.p.i, respectively [7,28] which is faster than in the present study. Differences in tile timing of these events m a y be attributable to isolate, cultivar, inoculation a n d / o r incubation conditions. Neither the fungal race nor host genotype influenced the a m o u n t and timing of conidial germination or appressorial development. T h e formation of appressoria over the anticlinal wall junctions between epidermal cells is typical of Coltetotrichum species [16, 21]. T h e ultrastructure ofappressoria is also normal for such fungal cells,, although the cell wails are a p p a r e n t l y more complex than those previously described, consisting of three wall layers rather than two [3,16,27]. It is possible that the techniques used in earlier studies did not resolve the two innermost wall layers (i.e. Fig. 5, layers a and b). If the structural differences are real, the additional layer in C. trifolii m a y aid the survival of the fungus under adverse conditions [36]. T h e outermost wall layer and the mucilaginous coat a p p a r e n t l y correspond to those previously reported. Darkening of the wall is p r o b a b l y due to the deposition of melanin in the electron-dense layer (i.e. Fig. 5, layer c) and m a y be required for normal infection to occur [6,12,37]. T h e mucilaginous coat (i.e. Fig. 5, layer d) m a y also play several roles in the pathogenic process. With C. graminicola, the mucilage is composed of hemicellulose and serves to anchor the appressorium to the leaf surface [13]. Furthermore, with C. graminicola, this coat which is continuous over the germ tube and conidium, is known to contain proline-rich proteins that can bind phenolic exudates Fro. II. (a) TEM of a median section through a fungal penetration peg (pp) which has penetrated into, but not through, the host cuticle (Cu) of a susceptible plant. Bracketed area, shown at higher magnification in the insert ( x 23600), shows altered staining (arrowhead) of the cuticle and host cell wall (HCW) around the penetration peg. A small papilla (P) is developing between the host plasmalemma and cell wall and numerous small vacuoles (V) are evident in the epidermal cell's cytoplasm, x 16600. (b) TEM ofsame infection site seen in Fig. 11 (a) only one section serial. The penetration peg is no longer visible. Staining of the host cuticle and cell wall is uniform beneath the appressorium (A). The papilla (P) is just visible, while the small vacuoles (V) are still evident within the epidermal cell. x 10000.
192
M . J . R . Mould et al.
from the host, thus protecting the appressorium and conidium [19] from host-derived, toxic phenolic compounds. The development and dimensions of the penetration peg and associated cone-shaped pore complex are similar to those of C. lindemuthianum [16]. Elaborate cone structures also have been reported to be formed by other phytopattaogenie fungi such as C. lagenari,lm [37] and Gloeosporium plalani [31]. With C. trifolii the wall of the penetration peg is continuous with that of the cone. Since tile growing end of the penetration peg is essentially a hyphal tip, the tenuous nature of the cell wall at the apex is not unexpected [11]. It has been reported that disruption of the host cuticle precedes contact between C. trifolii and the alfalfa plant [28]. This was not found to be tile case in the present study and, nor was a host response initiated in resistant or susceptible plants until the penetration peg actually touched the cuticIe. The earliest plant responses include alterations in the staining properties of the host cuticle and epidermal cell wail, tile deposition of papillae and vacuolar fragmentation. The increase in the staining intensity of the host cuticle and cell wall in the immediate area of penetration is suggestive of a chemical, probably enzymatic, interaction between the host and patbogen [16, 21]. The absence of inward bending and mechanical damage to the cuticle suggests that little mechanical pressure is exerted by the peg at the infection point. According to the work of Dickman et al. [8] on C. gloeosporioides a cutinase enzyme present in the wall of the infection peg may cause highly localized degradation of the host cuticle, thereby allowing the infection peg to enter without causing mechanical damage to the host cuticle. No direct chemical evidence for cutinase involvement is available for the C. trifofii-alfalfa pathosystem. The development of papillae in the epidermal cells of the alfalfa plant is localized directly beneath the invading penetration pegs. The presence of numerous small vacuoles in the host cytoplasm adjacent to these wall appositions suggests that some components of the deposit m a y be conducted to the infection site by such vacuoles. In barley coleoptile cells responding to E. graminis hordei, small vacuoles in the cytoplasm appear to actively transport the osmiophillic precursor material to the infection site by vacuolar fragmentation [38]. The origin of the small vacuoles has not been determined. Unfortunately, no published observations of the very earliest host response between bean epidermal cells and C. lindemuthianum are available; however, no small vacuoles are observed in the host cytoplasm during papilla formation in corn leafepidermal cells responding to infection by C. graminicola [27]. T h e initial alfalfa response to C. lrifolii appears to be the same in resistant and sueeptible plants infected by either race of the pathogen. Cytological differences in responding plant cells only develop after the cuticle is actually breeched [18]. This work was supported by grants from tile Natural Sciences and Engineering Research Council of Canada. We would like to thank Anna Schajnoha for help in typing the manuscript. REFERENCES
1. BOLAND,G.J. & BROCIIU,L.D. (1989). Collelolrichumdestructirum on alfalfa in Ontario and cultivar responses to anthracnose. CanadianJournal of Plant Pathology11,303-307.
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2. Bnow.~, G. E. (1978). Hypersensitive response of orange-colored Robinson tangerines to Colletotrichum gloeosporioides after ethylene treatment. Phytopathology 68, 700-706. 3. BRow.~, G.E. (1977). Uhrastructure of penetration of ethylene-degreened Robinson tangerines by Colletotrichum gloeosporioides. PIp'topathology 67, 315-320. 4. BRCXDRE'r'r,.M.C., P:cn~, Y. & PETERSOY, R. L. (1984). A new method for observing the morphology of vesicular-arbuscular mycorrhizae. Canadian Journal of Botany 62, 2128-22134. 5. ClIAtr, K.F. & AI.VAREZ, A.M. (1983). A histological study of anthracnose on Carica papaya. Phytopathology 73, 1113-1116. 6. CmDA T. & SISt.ER, H. D. (1987). Restoration ofappressorial penetration ability by melanin precursors in Pyricularia oryzae treated with antipenetrants and in melanin-deficient mutants. Journal of Pesticide Science 12, 49-55. 7. CnVRClHLL, A. C. L., BAKER, C.J., O'NEILL, N.R. & E[-cIx, J. H. jR (1988). Development of Colletotrichum trifolii races 1 and 2 on alfalfa clones resistant and susceptible to anthracnose. Canadian Journal of Botany 66, 75-81. 8. DICK.~XA.~,M. B., PATIL, S. S. & KOtAT'rtrKCn',', P. E. (1982). Purification, characterization and role in infection of an extracellular cutinolytic enzyme from Colletotrichumgloeosporioides Penz. on Caricapapaya L. Physiological Plant Pathology 20, 333-347. 9. ELoxx, J. H. jR & OSTAZESKX,S. A. (1982). Evaluation of selected alfalfa cuhivars and related Medicago species for resistance to race 1 and race 2 anthracnose. Crop Science 22, 39-42. 10. Et.oL~, J. H. JR & Os'rAzEsKt, S. A. (1985). Inheritance of resistance to race 1 and race 2 anthracnose in Arc and Saranac AR alfalfa. Crop Science 25, 861-865. 1 I. GROVE, S. N., BRACXER, C. E. & MooRE, D.J. (1970). An ultrastructural basis for hyphal tip growth in P.ythium ultimum. American Journal of Botany 57, 245-266. 12. KvBo, Y., SuzuKI, K., FURUSAWA,I. & YAStA.',tOXA,31. (1982). Effect of tricyclazole on appressorial pigmentation and penetration from apprcssoria from Colletotrichum lagenarium. Phytopathology 72, 1198-1200.
13. LAei', *1. S., & SKOROr'AI~, W.P. (1978). Nature of adhesive material of Colletotrichum graminicola appressoria. Transactions of the British Mycological Society 70, 221-223. 14. ~IANIATIS,T., FRITSC}I, E.F. & SASmV.OOK,J. (1982). Molecular Cloning--A Laboratory Manual. Cold Spring Harbor, Cold Spring Harbor, New York, p. 62. 15..\IERcER, P. C., Woon, R. K. S. & GREE.~WOOD, A. D. (1974). Resistance to anthracnose of French bean. Physiological Plant Pathology 4, 291-306. 16. MERCER, P. C., WOOD, R. K. S. & GREE.XWOOD, A.D. (1975). Uhrastructure of the parasitism of Phaseolus rulgaris by Colletotr#hum lindemuthianum. PIo'siological Plant Pathology 5, 203-214. 17. ,\hmlLE, B. R. & LuKEzlc, F. L. (1972). Studies on conidial germination and appressorlum formation by Colletotrichum trifolff Bain and Essary. Canadian Journal of Alicrobiology 18, 1263-1269. 18. MOULD, M . J . R . , BOLAND, G. & ROBB, J. (1990). Uhrastructure of the Colletotr#hum trifolii-alfalfa pathosystem. I I. Post-penetration events. Physiological and Molecular Plant Pathology 38, 179-194. 19. NICIIOLSON,R. L., HtPSKIXO,J. & ttANAU, R..~l. (1989). Protection against phenol toxicity by the spore mucilage of Colletotrichum graminicola, an aid to secondary spread. Physiological and Molecular Plant Pathology 35, 243-252. 20. O'CoxxELt, R.J. (I 987). Absence of a specialized interface between intracellular hyphae of Colletotrichum lindemuthianum and cells of Phaseolus rulgaris. New Phytologist 107, 725-734. 21. O'CoxxELL, R.J., BAILV.V,J. A. & Rtcn.,,zoxo, D. V. (1985). Cytology and physiology of infection of Phaseolus rulgaris by Colletotrichum lindemuthianurn. Pfp'siologkal Plant Pathology 27, 75-98. 22. OSTAZES:
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30. SAUNDERS,J. ~V. & BINGIIAM,E. T. (1972). Production of alfalfa plants from callus tissue. CropScience 12, 804-808. 31. SE1FERS, D. & A.~MON, V. (1980). Mode of penetration of sycamore leaves by Gloeosporium platani. Ph.)'topathology 70, 1050-1055. 32. SPURR, A. R. (1969). A low-vlscosity epoxy resin embedding medium for electron microscopy, jTournal of UltrastructureResearch 26, 3143. 33. TIFFANY, L. H. & GIL.~mN,j . C. (1954). Species of Colletotrichum from legumes. Mycologia 46, 52-75. 34. WEt.TY, R. E. & MUELLER,J. P. (1979). Occurrence of a highly virulent isolate of Colletotrichnm trifolii on alfalfa in North Carolina. Plant Disease Reporter 63, 666-670. 35. WELTY, R.E. & RAWLtNGS,J . O . (1985). Effects of inoculum concentration and temperature on anthracnose severity in alfalfa. Ph.)'topathology75, 593-598. 36. WoLxow, P. M., SISLER, H. D. & VtGtL, E. L. (1983)- Effect of inhibitors of melanin biosynthesis on structure and function of appressoria of Colletotrichum lindemuthianum. Physiological Plant Pathology 22, 55-71. 37. XuH, X. L.,JXRLFORS, U. & Kt;d,J. (1988). Uhrastructural changes associated with induced systemic resistance of cucumber to disease: host response and development of Colletotrichum lagenarium in systemically protected Ieaves. Canadian ffournat of Botany 66, 1028-1038. 38. ZEYEN, R.J. & BrCSHNELL,W. R. (1979). Papilla response of barley epidermal cells caused by Eusiphe graminis: rate and method of deposition determined by microcinematography and transmission electron microscopy. Canadian ffournal of Botany 57, 898-913.
1 79
Ultrastructure of the Colletotrichum trifolii-Medicago sativa pathosystem. I. Pre-penetration events ~[ICHAELJ. R. ~IOULD*, G.J. BOLAND'~and JANE ROBB*+ + Departments of Molecular Biology and Genetics* and Environmental Biology,t Unh'ersity of Guelph, Guelph, Ontario ~7G 211V, Canada (Acceptedfor publication February 1991)
Light and electron microscopy were used to study the early stages in the development of the Colletotrichum trifolii-Medicago satira pathosystem, including compatible and incompatible interactions involving races 1 and 2 oftlae pathogen. Neither the fungal race nor the host genotype influenced tile percentage or timing of conidial germination, appressorial development or penetration peg formation. Host cells initiated a response only after contact was made between the fungal penetration peg and the alfalfa cuticle. The response, which was immediate, and localized to the epidermal cell beneath the point of ingress, included increased staining of the wall and cuticle around the penetration peg, the formation of a papilla and vacuolar fragmentation. The initial compatible and incompatible host responses were indistinguishable.
INTRODUCTION A n t h r a c n o s e o f alfalfa (Medicago saliva L.) is caused by tile fungus Collelotrichum lrifolii Bain a n d Essary. A t least two races o f the p a t h o g e n exist [24,35] and, correspondingly, resistance to each race seems to be controlled by an i n d e p e n d e n t d o m i n a n t , tetrasomically inherited gene [9,10]. P r i m a r i l y a stem disease, lesions can be observed within 120 h post-inoculation in susceptible plants [7]. T h e events leading to lesion d e v e l o p m e n t include, in chronological order, eonidial g e r m i n a t i o n , formation o f g e r m tubes a n d appressoria, p e n e t r a t i o n peg d e v e l o p m e n t , g r o w t h o f h y p h a e within the host tissues a n d , lastly, the e r u p t i o n o f acervuli. W h i l e numerous u l t r a s t r u c t u r a l studies have been carried out on related Colletotrichum interactions [2, 5,15,16, 20,21, 26, 27, 38], only two cytological studies have been published on the C. trifolii-alfalfa p a t h o s y s t e m : first, a light and electron microscopic study o f a c o m p a t i b l e interaction involving race 1 [28] and second, a light microscopic study o f the c o m p a t i b l e and i n c o m p a t i b l e responses involving both races 1 a n d 2 [7]. W h i l e both studies emphasized tile early stages o f the interaction (i.e. 0 - 4 8 h postinoculation), the p l a n t a n d fungal structures a n d the i n t e r a c t i n g events which are involved in the initial p e n e t r a t i o n o f the host r e m a i n relatively u n d o c u m e n t e d . In the present study, we used both light and transmission electron microscopy to investigate the p r e - p e n e t r a t i o n events involved in establishing c o m p a t i b l e or .+Author to reprint requests should be sent. 0885-5765/91/030179+ 16 S03.00/0 13
© 1991 Academic Press Limited M P P 38
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incompatible relationships with both races of C. trifolii. T h e two primary objectives were: (I) to investigate the ultrastructure of the appressorium and penetration peg; and (2) to determine the timing and cytological nature of the initial host response. MATERIALS AND METHODS
Culture of the fimgus Cultures of races 1 and 2 of C. trifolii were obtained from the American T y p e Culture Collection (ATCC 41=42881 and ~42041 respectively). Conidial suspensions were prepared from cultures of each race and stored at --70 °C in 25 % aqueous glycerol [14]. Single droplets from the frozen stock cultures were pipetted onto a 10 % (v/v) V8 Juice/water agar medium and the suspensions were spread evenly using a bent glass rod. After 6 to 8 days of growth, suspensions of I-0 x t0 s conidia m1-1 were prepared in sterile distilled water. Fractions of 0-2 to 0"3 ml of each suspension were pipetted onto individual V8 Juice agar plates and spread evenly over the agar. Cultures were maintained at 22 °C under fluorescent lights. Virulence of both races was maintained by periodically infecting and re-isolating the pathogen from a susceptible host plant.
Selection and growth of host plants Ninety-three alfalfa plants of the breeding line Regen-S [30] were screened for resistance to races 1 and 2 ofC. trifolii using a needle inoculation method [23]. Seedlings were selected from an anthracnose disease survey of Ontario-grown cultivars [1] that had shown resistance to race 2 ofthe pathogen ; they were grown to maturity and tested using the same method. For each evaluation, two alfalfa stems were inoculated with each race and one stem per plant was injected with sterile distilled H 2 0 as a control treatment. Plants were rated for disease response at 7, 10 and 14 days post-lnoculation using a 1 to 5 scale [22]: 1 -- resistant (R), no lesion or small water-soaked spots; 5 = susceptible (S), dead stems. Plants that were rated at 1:1 ( R / R ) , 1:5 (R/S) and 5:5 (S/S) to races 1 and 2, respectively, were kept for further study. A minimum of two plants per genotype was used. Shoot cuttings were taken from each plant and rooted in vermiculite. Once these cuttings had achieved adequate maturity, they were repotted into 15 cm diameter plastic pots using an autoclaved 1 : 1 mixture of Turface: Promix (Premier Brands Inc., New Rochelle, N.Y. 10801 U.S.A.). All plants were grown in a Conviron growth chamber (Model 4eE8VH) with 14 h daylight and a maximum photosynthetically active light intensity of 240 g E m -2 s-1. Night and day temperatures were 18 °C and 22 °C respectively. Plants were watered daily with deionized water and fertilized weekly with a 2-4 g 1-1 solution of fertilizer (20:20:20 N : P : K ) . Plants were cut regularly after flowering to ensure succulent growth for subsequent cytological studies.
Preliminary experiments T o establish the timing of conidial germination, appressorium formation, host penetration, disease development and resistance responses, several preliminary inoculations were performed. Stems and leaves of resistant and susceptible plants were inoculated with a conidial suspension (1-0 x 10 Gconidia m1-1 in sterile distilled water) of either race 1 or 2 of C. trifolii by spraying the plant until runoff. Inoculated plants
C. trifolii-alfalfa pathosystem. Part I
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were placed in a mist chamber that maintained continuous plant surface wetness for 72 h at 24 °C. Inoculated stems were harvested at 24, 48, 60, 72, 96 and 120 h postinoculation (h.p.i.). Two stems were inoculated per sampling time and the experiment was repeated three times. Control stems, one for each plant, were sprayed with water. Whole mounts of inoculated leaf tissues (adaxial side up) and freehand radial longitudinal and cross sections of inoculated stems were placed on glass mici-oscope slides. Tissue was stained with two or three drops of0-01% cotton blue in lactophenol (1 lactic acid: 1 ptlenol: 1 water: 2 glycerin) at 60 °C for 20 rain. Alternatively, stem tissues were stained with 0-1% chlorazol black E in lactoglycerol (1 lactic acid: 1 glyercol: 1 water) [4]. Material stained with chlorazol black E was destained in lactoglycerol and mounted in this solution. The percentages of germinated conidia , germinated conidia which formed appressoria and appressoria which formed a penetration pore were recorded (based on measurements of 100 conidia and appressoria per trial). All tissue was examined using brightfield microscopy (Nikon Labophot equipped with a Nikon FX-35 camera).
Cytological studies of pre-penetration events Conidial suspensions of 5"0x10 ~ conidia m1-1 in 0"083% Tween 20 [17] were prepared from 7-day-old cultures ofrace 1 or 2 ofC. trifolii. To ensure uniform coverage of the inoculated stem, each spore suspension was poured into a sterile Petri dish and the upper succulent region of the stem and the uppermost leaves )vere immersed in the suspension. For each experiment, only one race was inoculated onto each plant and incubated in a mist chamber. For each inoculation, three stems per time per race/genotype interaction were inoculated. One stem per plant was immersed in 0-083 % Tween 20 as a control. Material was sampled at 48 h.p.i, and processed for electron microscopy. Stem pieces were trimmed to a length of approximately 2-0 cm, slit lengthways in a Petri dish containing 1% glutaraldehyde in 0"07 M Sorenson's phosphate buffer (pH 6"8) and further trimmed to a length of approximately 1"0 cm. Individual pieces were then transferred to separate vials containing 3 % glutaraldehyde and 1-5 % acrolein in the same buffer amended with 1"0 mM CaC12 and 16 drops of 1% Tween 20 per 500 nal. The tissue was fixed for 3 h at room temperature in the same buffer, post-fixed for 2 h at room temperature with 2 % OsO 4 in the same buffer, dehydrated through a graded acetone series and embedded in Spurr's low-viscosity resin [32]. Tissue was flat-embedded with the stem surface facing upwards. Sections were cut on an LKB Ultratome using a diamond knife (Diatome). Semi-thin (0"25 pm) sections were dried onto slides coated with a subbing agent [25]. Sections from infected stems were taken from an area 5"0 mm from the edge of the lesion. Sections were stained with 0-1% toluidine blue 0 in 1% sodium tetraborate and examined with brightfield microscopy. Thin sections of silver to gold interference colour were collected on acetone-washed 200 mesh copper-rhodamine grids. Sections were stained with a saturated solution of uranyl acetate in 70% methanol (30 min) followed by lead citrate (22 min) [29]. Thin sections were examined with a J E O L 100 CX electron microscope operating at 60 kV. Individual appressoria were located on a stem surface for thin sectioning. Since the thickness of the embedded material was less than 3 mm, the surface of the embedded 13-2
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stem could be viewed at low magnification using a light microscope. The stem surface was scanned and desired spots marked for sectioning by nicking the edge of the resin with a razor blade. Excess stem tissue was trimmed under a binocular microscope until the desired appressoria lay 3 to 41am from the trimmed end. Sections of 0"2 ~tm thickness were initially cut on the ultramicrotome and every fifth section was mounted on a slide and stained with toluidine blue 0 to determine when the site was reached. Thin sections (700-900 nm) were then cut, collected, stained and viewed. A total of 25 appressoria and infection sites were sectioned and used in the studies of the prepenetration events. Five appressoria were reconstructed from serial sections. RESULTS
Preliminary experiments Conidia from races 1 and 2 are cylindrical with rounded ends, averaging 12"5 lain x 5"8 pm (based on measurements of 100 conidia per race). The timing ofpre- and postpenetration events are summarized in Table 1. Neither the fungal race nor host genotype influenced the frequency of conidial germination or appressorial formation [Fig. 1 (a), (b)]. By 24 h.p.i., germination was complete. The spores remained singlecelled and upon germination each formed a short (I-2 lam) germ tube which by 48 h.p.i, ended in a spherical, dark-walled appressorium [average diameter 64 pm; Figs 2, 3 (b)]. Results are similar on both stem and leaves. Appressoria of each race formed over the junctions of the anticlinal cells walls between adjacent epidermal cells [Figs 2, 3(a), (b)]. Appressorium maturation culminated with the migration of the conidial cytoplasm from the conidium to the appressorium via the germ tube [Figs 2, 3 (b)]. A septum developed between the germ tube and the conidium [Fig. 3 (b)]. When devoid of cytoplasm, conidia appeared as non-staining 'ghost cells' which subsequently collapsed [Figs 2, 3(b)]. Electron microscope analysis revealed that these 'ghost cells' were empty of identifiable cytoplasm. By 60 h.p.i appressoria formed penetration pegs and by 72 h.p.i, the epidermal ceils layers of both resistant and suceptible plants were colonized.
Structure of the appressorium and penetration peg formation Individual appressoria of both races of C. trifolii contain large numbers of free ribosomes, lipid bodies, mhochondria, and a single nucleus with a prominent nucleolus [Fig. 4 (a), (b)]. Retraction of the cytoplasm from the cell wall (Fig. 4) in appressoria forming penetration pegs allowed a close examination of the wall (Fig. 5). The threelayered appressorial wall has an overall thickness of 160 to 170 nm. The innermost layer is thin (!5 nm) and highly electron-dense while the second, thicker (70 nm) layer is electron-lucent. The third layer is electron-dense and thicker (80 nm) than the second layer. An electron-lucent, mucilaginous coat, which is continuous with the germ tube wall, is approximately 100 nm thick and contains numerous small, electron-dense particles. Where the appressorium contacts the host cuticle, the mucilaginous coating spreads over the host cuticle (Fig. 5). Close examination of the appressorium in Fig. 4(a) revealed a penetration pore approximately 200 nm in diameter and a small penetration peg [Fig. 6 (a)]. The peg does not contact the host cuticle at any point, the appressorium being attached to the
C. trifolii-alfalfa pathosystem. Part I
183 TABLE 1
Summary of the data from preliminary experiments to determine the timing of the interaction between 12. trifolii and M. sativa Host genotype Time (h.p.i.) 24 % conidia germinated 48 % germinated conidia forming appressorium 60. % appressoria forming penetration peg 72
96
120
S:S =
R:S b
R:R e
Rid-92 R2e-96
RI-94 RS-88
RI-93 R2-87
R1-85 R2-88
R1-90 R2-93
R1-91 R2-86
R1-51 R2-53
R1-53 R2-56
R1-50 R2-51
Initial penetration of the cutlcle, by both races, complete Penetration up to fourth cell layer by both races
Initial penetration of the cuticle, by both races, cgmplete RI restricted to single epidermal cell R2 penetration to 4th cell layer RI infected epidermal cell darkened R2 lesion development
Initial penetration of the cuticle, by both races, complete Restriction to single epidermal cell both races
Lesion development in both races
Infected epidermal ceils darkened in both races
aS:S plants are susceptible to races 1 and 2 ofC. trifolii (i.e. 5:5). bR:S plants are resistant to race 1 and susceptible to race 2 ofC. trlfolii (i.e. 1:5). e R : R plants are resistant to both races ofC. trifolii (i.e. 1:1). dR1 race 1 ofC. trifolii. eR2 race 2 of C. trifolii. host
cuticle
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r e c o n s t r u c t i o n o f t h e p o r e c o m p l e x [ F i g . 6 ( a ) - ( c ) ] r e v e a l e d it to b e a v e r y i n t r i c a t e s t r u c t u r e ( F i g . 7). T h e w a l l s o f t h e p o r e e x t e n d i n t o t h e a p p r e s s o r i a l c y t o p l a s m t o a p p r o x i m a t e l y o n e - q u a r t e r t h e d i a m e t e r o f t h e a p p r e s s o r i u m ( F i g . 7), f o r m i n g a c o n e . FIo. 1. Longitudinal sections showing chlorazol black E-stained appressoria (A) of C. trifolff distributed over the stem surface at 48 h.p.i. × 800. (a) Race 1-resistant/race 2-resistant ( R / R ) alfalfa plant v. race 2 ofC. trifolii. × 800. (b) Race l-susceptible/race 2-susceptible (S/S) alfalfa plant v. race I of G. trifolii, x 800. FIG. 2. Appressorium (A) of G. trifolli race 2 with attached conidial ghost cell (C). Appressorium has formed over the cell wall junction between two epidermal cells (arrowhead). Chlorazol black E-staining, 48 h.p.i, x 1100. FIo. 3. Toluidine blue 0-stained, uhramicrotome thick sections showing appressoria (A) of C. trifolii formed over the junctions between the anticlinal cell walls between adjacent epidermal cells (i, ii) at 48 h.p.i. Epidermal cell walls and cuticle are evenly stained and host cell cytoplasm exhibits no signs of any host response. × 1200. (a) R / R plant v. race 2. (b) S/S plant v. race 1. Appressorium is attached to conidial ghost cell (C) by a short germ tube (GT). A septum (S) is visible between the conidium and germ tube.
184
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Fno. 4. Transmission electron micrographs (TEMs) of the same sites shown in Figs 3 (a) and 3(b). Each appressorium (A) contains numerous lipid bodies (L), vacuoles (V) and a single nucleus (N). Cytoplasm o f t h e underlying epidermal cells (i, ii) has the same uhrastructure as in control tissue. Host cuticle (Cu) and epidermal cell wails show no alterations in their ultrastructure compared with control tissue• × 5850. (a) R / R plant v. race 2. A small penetration peg has formed at the base of the appressorium (arrowhead). (b) S/S plant v. race 1.
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FIG. 7. Three-dimensional diagrammatic representation of an appressorium ofG. trifolii based upon serial reconstruction from Figs. 5 and 6(a)-(c). Layers a-c and the mucilaginous coat (d) correspond to those in Fig. 5. The penetration peg (pp) forms at the base of the appressorium over the anticlinal wall junction between the epidermal cells. The elaborate cone-shaped pore complex ((2) extends into the appressorium approximately one quarter of the appressorial diameter. N o n e o f the appressorial wall layers a r e c o n t i g u o u s with the cone, a l t h o u g h the second layer projects ihto the appressorial c y t o p l a s m , f o r m i n g a t h i c k e n e d collar a r o u n d the p e n e t r a t i o n pore [Figs 6 (a), (b), 7]. T h e wall of the c o n e e x t e n d s d o w n t h r o u g h the pore to form the e l e c t r o n - l u c e n t wall o f the p e n e t r a t i o n peg. At the tip o f the p e n e t r a t i o n peg the cell wall is very thin [Fig. 6 (a)].
Timing and ultrastructure of the early host responses C o n t r o l p l a n t tissue is well preserved w h e n fixed in 3 % / 1 " 5 % g l u t a r a l d e h y d e / a c r o l e i n . T h e cuticle a n d e p i d e r m a l cell wall s t a i n e d u n i f o r m l y with t o l u i d i n e b l u e 0 (Fig. 8). Fresh free h a n d stem cross sections did n o t stain with chlorazol black E (Fig. 9), facilitating the analysis o f f u n g a l spores, g e r m tubes a n d appressoria o n the l e a f s u r f a c e [Figs l ( a ) , (b), 2]. U h r a s t r u c t u r a l analysis showed that, w i t h i n the e p i d e r m a l a n d cortical cells of n o n i n o c u l a t e d alfalfa stems, a thin layer of c y t o p l a s m s u r r o u n d s a large c e n t r a l v a c u o l e FIo. 5. High magnification of an area of the appressorium seen in Fig. 4 (a). The appressorial cell wall consists of three layers: an innermost, electron-dense layer (a), a second, electron-lucent layer (b), and a third, electron-dense layer (c). A mucilaginous coating (d) containing numerous small, electron-dense particles (arrowhead) spreads over the stem surface. × 67500. FIo. 6. TEMs from a group of serial sections taken in order to form a three-dimensional reconstruction of the penetration peg and cone complex seen in the appressorium of Fig. 4 (a). All magnifications × 48600. (a) Near-median section through the developing penetration peg (pp). An elaborate cone-shaped wall complex (C) extends into the appressorial cytoplasm. (b) Two seetioqs serial to Fig. 6(a) showing walls of the cone (C). (c) Four sections serial to Fig. 6(a) showing walls of the cone (C).
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Flo. 8. Toluidine blue 0-stained uhramicrotome thick section from a HzO-injected control plant. Epidermal cells (i) are devoid of any material in their vacuoles, and cell walls and cuticle are evenly stained, x 800. FIG. 9. Chlorazol black E-stained freehand section from H20-injected control plant. Epidermal (i) and cortical (ii) cells do not react with the stain. × 800. Fro. 10. (a) TEM from control plant. A thin layer of cytoplasm (arrow) surrounds a large, central vacuole (V). Epidermal cell wall (HCW) and cuticle (Cu) are uniform over the periclinal surface of the stem. × 3000. (b) TEM of bracketed area of Fig. 10 (a). Nucleus (N), chloroplasts (Ch), mitochondria (mt) and rough cndoplasmic reticulum are contained within the cortical cell's cytoplasm. × 3900. [Fig. 10 (a)]. T h e thin l a y e r o f c y t o p l a s m c o n t a i n s a single nucleus, chloroplasts w i t h starch grains, m i t o c h o n d r i a , a n d e n d o p l a s m i c r e t i c u l u m [Fig. 10 (b)]. H o s t responses b e g i n w h e n the f u n g a l p e n e t r a t i o n peg c o n t a c t s the host cuticle [Fig.
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11 (a)]. Serial section analysis of the cell depicted in Fig. 11 (a) and (b) confirmed that, in this case, the short penetration peg had not breeched tile cuticle. Neither enzymatic degradation nor inward bending of the cuticle and cell wall were evident around the penetration peg although the staining of these structures was altered around the point ofingress. In Fig. 11 (a) and (b), and in other similar cases involving both resistant and susceptible plants, small vacuoles a p p e a r in the host cytoplasm directly beneath tile point of pathogen ingress. Typically, papillae are just beginning to form and, at such an early stage, are homogeneous and electron-lucent [Fig. 11 (a)]. Whether race 1 or 2 of the pathogen is involved, compatible and incompatible reactions are indistinguishable until this stage of development. No host response has ever been observed prior to actual contact of the penetration peg with the host cuticle [Fig. 4 (a)]. Appressoria that do not form a penetration peg do not elicit a host response [Fig. 4 (b)]. DISCUSSION
Conidial and appressorial dimensions are in agreement with those previously reported [34]. However, previous studies have shown that C. trifolii completes conidial germination and appressorial formation by 16 and 24 h.p.i, respectively [7,28] which is faster than in the present study. Differences in tile timing of these events m a y be attributable to isolate, cultivar, inoculation a n d / o r incubation conditions. Neither the fungal race nor host genotype influenced the a m o u n t and timing of conidial germination or appressorial development. T h e formation of appressoria over the anticlinal wall junctions between epidermal cells is typical of Coltetotrichum species [16, 21]. T h e ultrastructure ofappressoria is also normal for such fungal cells,, although the cell wails are a p p a r e n t l y more complex than those previously described, consisting of three wall layers rather than two [3,16,27]. It is possible that the techniques used in earlier studies did not resolve the two innermost wall layers (i.e. Fig. 5, layers a and b). If the structural differences are real, the additional layer in C. trifolii m a y aid the survival of the fungus under adverse conditions [36]. T h e outermost wall layer and the mucilaginous coat a p p a r e n t l y correspond to those previously reported. Darkening of the wall is p r o b a b l y due to the deposition of melanin in the electron-dense layer (i.e. Fig. 5, layer c) and m a y be required for normal infection to occur [6,12,37]. T h e mucilaginous coat (i.e. Fig. 5, layer d) m a y also play several roles in the pathogenic process. With C. graminicola, the mucilage is composed of hemicellulose and serves to anchor the appressorium to the leaf surface [13]. Furthermore, with C. graminicola, this coat which is continuous over the germ tube and conidium, is known to contain proline-rich proteins that can bind phenolic exudates Fro. II. (a) TEM of a median section through a fungal penetration peg (pp) which has penetrated into, but not through, the host cuticle (Cu) of a susceptible plant. Bracketed area, shown at higher magnification in the insert ( x 23600), shows altered staining (arrowhead) of the cuticle and host cell wall (HCW) around the penetration peg. A small papilla (P) is developing between the host plasmalemma and cell wall and numerous small vacuoles (V) are evident in the epidermal cell's cytoplasm, x 16600. (b) TEM ofsame infection site seen in Fig. 11 (a) only one section serial. The penetration peg is no longer visible. Staining of the host cuticle and cell wall is uniform beneath the appressorium (A). The papilla (P) is just visible, while the small vacuoles (V) are still evident within the epidermal cell. x 10000.
192
M . J . R . Mould et al.
from the host, thus protecting the appressorium and conidium [19] from host-derived, toxic phenolic compounds. The development and dimensions of the penetration peg and associated cone-shaped pore complex are similar to those of C. lindemuthianum [16]. Elaborate cone structures also have been reported to be formed by other phytopattaogenie fungi such as C. lagenari,lm [37] and Gloeosporium plalani [31]. With C. trifolii the wall of the penetration peg is continuous with that of the cone. Since tile growing end of the penetration peg is essentially a hyphal tip, the tenuous nature of the cell wall at the apex is not unexpected [11]. It has been reported that disruption of the host cuticle precedes contact between C. trifolii and the alfalfa plant [28]. This was not found to be tile case in the present study and, nor was a host response initiated in resistant or susceptible plants until the penetration peg actually touched the cuticIe. The earliest plant responses include alterations in the staining properties of the host cuticle and epidermal cell wail, tile deposition of papillae and vacuolar fragmentation. The increase in the staining intensity of the host cuticle and cell wall in the immediate area of penetration is suggestive of a chemical, probably enzymatic, interaction between the host and patbogen [16, 21]. The absence of inward bending and mechanical damage to the cuticle suggests that little mechanical pressure is exerted by the peg at the infection point. According to the work of Dickman et al. [8] on C. gloeosporioides a cutinase enzyme present in the wall of the infection peg may cause highly localized degradation of the host cuticle, thereby allowing the infection peg to enter without causing mechanical damage to the host cuticle. No direct chemical evidence for cutinase involvement is available for the C. trifofii-alfalfa pathosystem. The development of papillae in the epidermal cells of the alfalfa plant is localized directly beneath the invading penetration pegs. The presence of numerous small vacuoles in the host cytoplasm adjacent to these wall appositions suggests that some components of the deposit m a y be conducted to the infection site by such vacuoles. In barley coleoptile cells responding to E. graminis hordei, small vacuoles in the cytoplasm appear to actively transport the osmiophillic precursor material to the infection site by vacuolar fragmentation [38]. The origin of the small vacuoles has not been determined. Unfortunately, no published observations of the very earliest host response between bean epidermal cells and C. lindemuthianum are available; however, no small vacuoles are observed in the host cytoplasm during papilla formation in corn leafepidermal cells responding to infection by C. graminicola [27]. T h e initial alfalfa response to C. lrifolii appears to be the same in resistant and sueeptible plants infected by either race of the pathogen. Cytological differences in responding plant cells only develop after the cuticle is actually breeched [18]. This work was supported by grants from tile Natural Sciences and Engineering Research Council of Canada. We would like to thank Anna Schajnoha for help in typing the manuscript. REFERENCES
1. BOLAND,G.J. & BROCIIU,L.D. (1989). Collelolrichumdestructirum on alfalfa in Ontario and cultivar responses to anthracnose. CanadianJournal of Plant Pathology11,303-307.
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30. SAUNDERS,J. ~V. & BINGIIAM,E. T. (1972). Production of alfalfa plants from callus tissue. CropScience 12, 804-808. 31. SE1FERS, D. & A.~MON, V. (1980). Mode of penetration of sycamore leaves by Gloeosporium platani. Ph.)'topathology 70, 1050-1055. 32. SPURR, A. R. (1969). A low-vlscosity epoxy resin embedding medium for electron microscopy, jTournal of UltrastructureResearch 26, 3143. 33. TIFFANY, L. H. & GIL.~mN,j . C. (1954). Species of Colletotrichum from legumes. Mycologia 46, 52-75. 34. WEt.TY, R. E. & MUELLER,J. P. (1979). Occurrence of a highly virulent isolate of Colletotrichnm trifolii on alfalfa in North Carolina. Plant Disease Reporter 63, 666-670. 35. WELTY, R.E. & RAWLtNGS,J . O . (1985). Effects of inoculum concentration and temperature on anthracnose severity in alfalfa. Ph.)'topathology75, 593-598. 36. WoLxow, P. M., SISLER, H. D. & VtGtL, E. L. (1983)- Effect of inhibitors of melanin biosynthesis on structure and function of appressoria of Colletotrichum lindemuthianum. Physiological Plant Pathology 22, 55-71. 37. XuH, X. L.,JXRLFORS, U. & Kt;d,J. (1988). Uhrastructural changes associated with induced systemic resistance of cucumber to disease: host response and development of Colletotrichum lagenarium in systemically protected Ieaves. Canadian ffournat of Botany 66, 1028-1038. 38. ZEYEN, R.J. & BrCSHNELL,W. R. (1979). Papilla response of barley epidermal cells caused by Eusiphe graminis: rate and method of deposition determined by microcinematography and transmission electron microscopy. Canadian ffournal of Botany 57, 898-913.