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Structure and significance of “blocked veins” in Verticillum-infected chrysanthemum
Physiological
Plant Pathology
(1983)
23,35-53
Structure and significance of “blocked Verticilllirm-infected chrysanthemum JANE ROBB~ Departments Canada
in
and L. V. BUSCH:
of Botany
and Gencticsf
(Accepted for publication
If basic
veins”
January
and Environmental
Biology:
University
of Gulph,
Guclph,
Ontario
NIG
2WI
1983)
fuchsin
is introduced into the vascular column of a chrysanthemum infected with most of the veins of the plant will turn red. However, certain veins associated with flaccid leaf lobes may remain unstained. This paper reports the cytological analysis of “blocked veins”. In such a dysfunctional vein all the vessels distal to the visual block are coated and terminally occluded by smooth vessel coating material; no basic fuchsin is present in or around these vessels. All uncoated vessels end at or proximal to the site of visual blockage; red dye only occurs in the secondary walls of and in parenchyma cells surrounding uncoated vessels. The results are summarized as a diagrammatic reconstruction of a “blocked vein” and, on the basis of this as well as other published structural and physiological data, a hypothesis is presented to explain the development of flaccidity in Verticilliuminfected chrysanthemums. The irreversible phase ofwilt is precipitated by the terminal occlusion of vein endings. Verficillium
dahlia
INTRODUCTION
Basic fuchsin has been used extensively as a vascular dye. When the stem of a plant is severed and the cut end is immersed in basic fuchsin normally the plant will take up the dye and eventually the entire vascular system will stain red. However, if the plant hosts a vascular pathogen the veins in certain parts of the plant, particularly in the leaves, may fail to stain [4]. The staining effect is somehow “blocked”. Studies of chrysanthemums infected with the fungus Verticillium dahliae have shown that the position of “blocks” is directly correlated with a decrease in relative water content and the development of the wilting syndrome (i.e. flaccidity) in the afi‘ected leaf lobes [41
61.
Currently, the use of basic fuchsin or a similar vascular dye [4, 16] is the only known way to determine which veins are non-functional in such systems. Yet, despite the obvious importance of vascular dysfunction to the development of the “wilting” syndrome the actual cause of the blockage of dye is unknown. Several hypotheses are possible. The stain may physically cease to move at the site of visual blockage because of the physical occlusion of the xylem elements: (1) at the site of the visual blockage, (2) proximal to the site of blockage [II] or (3) distal to the site of blockage. Alternatively, the stain may be present in the “blocked” veins but the red staining 004%4059/83/040035
+ 19 sO3.00/0
0
1983 Academic
Press Inc.
(London)
Limited
36
J. Robb and L. V. Busch
effect may somehow be prevented. At least three, or possibly all, of these possibilities may have a structural basis. We have, therefore, undertaken the cytological analysis of individual dysfunctional leaf veins of Verticillium-infected chrysanthemum plants treated with basic fuchsin in the hopes of finally determining the cause and significance of the stain blockage associated with vascular wilt disease. The interpretation ofany study employing basic fuchsin as a vascular dye must take into consideration a number of potential limitations inherent in the technique. Firstly, the position of the stain tells us nothing about water flow per se. Secondly, basic fuchsin is a strong reagent which may produce structural or chemical alterations that are unrelated to the actual disease. Little is currently known about such possible detrimental effects of the dye on either healthy or diseased plants. However, preliminary physiological studies of the same system indicate that basic fuchsin causes little damage to healthy chrysanthemum tissue within the timespan of the experiments (Robb & Street, unpublished data). We have also sought to minimize the possibility of misinterpretation due to any spurious effects of the dye by extensive use of controls in carrying out these experiments. MATERIAL Inoculation,
AND METHODS growth conditions and sampling Chrysanthemum cuttings (Chrysanthemum moriilium Ramat. cv. Brilliant Anne) were grown and inoculated with V. dahliae Kleb. as previously described [12]. Foliar
symptoms began to appear in chrysanthemum about 33 days after inoculation [I]. Infected plants were selected at an early stage of symptom expression when several leaves exhibited flaccidity of some of the lobes but no chlorosis was yet detectable. The selected plants, as well as several uninoculated controls, were severed near the soil line and the cut end of the stem was attached to a gravity-feed staining column as described by MacHardy et al. [6J. As the dye was carried through the vascular system of the plant the veins turned red in colour. Using this method, it only required 2-3 h to stain the entire vascular system of a plant approximately 30.4 cm high; the staining was considered to be complete when all leaf veins of the control plants were uniformly red and stain dripped from the severed end of one of the major lateral veins. Leaves containing blocked veins were removed from the plant and dropped immediately into a pool of glutaraldehyde (1 o/o in 0.07 M phosphate buffer, pH 6.8) at 4 “C. A light box was placed under the specimen in order to clearly visualize the blocked areas. A sample was then cut from each of four major lateral veins in which blocks occurred, each sample consisting of l-2 mm of red vein proximal to the block as well as the entire length of unstained vein distal to the block (i.e. 3-6 mm), up to and including the vein ending. Four vein samples of equivalent length and position were cut from an adjacent unblocked vein of the same leaf lobe and from veins of uninoculated plants. Figure 1 illustrates diagrammatically the sampling of stained chrysanthemum leaves. Prekaration for scanning electron microscopy (SEM) The samples were immediately transferred to a solution containing 2% glutaraldehyde and 1.5% acrolein in 0.07 M phosphate buffer, pH 6.8 and fixed at 4 “C for
Verticillium-infected
chrysanthemum
(0)
37
(b)
FIG. 1. Diagram stained stained tissue.
illustrating the position of blocks and sampling procedures in basic fuchsin leaves. Solid lines represent chrysanthemum leaves. (a) infected, (b) uninoculated veins; dotted lines represent unstained. veins. The stippled areas represent flaccid leaf
5-6 h. They were washed in the same buffer, and free-hand sectioned either longitudinally or as serial cross sections. All were subsequently treated by the ligandmediated osmium binding technique of Kelly et al. [5], dehydrated with acetone and critical-point dried with liquid COZ. They were then coated with 1 : 1 gold-palladium alloy and viewed with a gun potential of 10 kV with an ETEC Autoscan microscope. Preparation for transmissionelectron microscopy( TEM) and light microscopy(LM) The precut samples were fixed at 4°C in a solution containing 2% glutaraldehyde and 1.5% acrolein in 0.07 M phosphate buffer at pH 6.8. Half of the samples were post-fixed in 2% 0~04 and all were processed for light and electron microscopy as previously described [II, 121. Each vein was serial sectioned in sets, each set consisting of (1) one 1 pm thick monitor section (i.e. giving a grey interference colour) for LM, (2) three O-5 pm thick monitor sections (i.e. giving red-green interference colours) for LM, (3) 40 80-90 nm thin sections (i.e. giving a light gold interference colour) for TEM and (4) 15 0.5 pm thick sections (i.e. giving red-green interference colours) for LM histochemistry. The sets were numbered from the leaf margin (set 1) inwards, each set representing about 15 pm of vein. After staining with 0.5% aqueous uranyl acetate and lead citrate the thin sections were viewed using a Phillips 200A electron microscope or a JOEL JEM 100 CX electron microscope operating at 60 kV. The 1 pm thick monitor sections were viewed (LM) unstained to ascertain the presence and position of the basic fuchsin in each set (i.e. thick monitor section). The red-green monitor sections were stained with 0.5% Toluidine blue 0 in 1% sodium borate for the purpose of correlated LM-TEM analysis [II]. The remaining red-green sections were kept as a reservoir of material; they were used subsequently only when additional histochemical information was required to clarify any ambiguities arising from the correlated LM-TEM interpretation of vascular alterations [II, 241. Additional stains employed were: (1) Prussian blue, (2) Sudan black B and (3) Schiffs reagent following oxidation by HsOz (i.e. PAS reaction) as previously described [II]. The thick sections (LM) were photographed using a Nikon Labophot microscope.
38
J. Robb and L. V. Busch
Four blocked and four unblocked samples from infected leaves were serially sectioned to completion. Four control veins from uninoculated plants were cut in the same manner. In each case two of the veins were post-fixed in 0~04 and two were not. In those which were post-fixed the position of the block was marked with a notch beside the vein prior to post-fixation. RESULTS Observations
of blocked veins of infected plants In flaccid leaf lobes stain blockage occurred in the major lateral vein servicing the lobe as well as in some of the minor veins in the affected areas. At this early stage of symptom development, most of the blockages occurred within 3-5 mm of the vein ending. Figure 6(a) illustrates tissue which was fixed in glutaraldehyde-acrolein (i.e. no osmium), dehydrated and embedded in plastic. The vein in the left mould was from an infected symptomatic leaf; a stain blockage is clearly visible with the vascular tissue being red on the proximal side of the block (i.e. left) and unstained from the block to the vein tip (i.e. right). Tissue which had been post-fixed in osmium was black, obscuring the visual blocks.
Set 250
/
Stained
Visual block
3
Unstained
FIG. 2. Schematic diagram showing the position of the basic fuchsin stain and the structure of a blocked major lateral vein of a chrysanthemum plant infected with V. dahlias. The figure was reconstructed from the data obtained from the blocked vein illustrated in Figs 3-7 and 8(a). Note. once again. that throughout the study the sets of sections have been numbered from the leaf margin (set 1) inwards. H, Vessel lumen; 0, unstained vessel wall ; &I, pink vessel wall ; !& vessel coating material.
Figure 2 is a diagrammatic representation of the structure of a blocked leaf vein as revealed by the present study. In the unstained portions of veins distal to stain blockage, 92% of all vessels extending to the leaf margins were occluded (Fig. 3) at FIG. 3. (a) Set 4, LM, red-green monitor section stained with Toluidine blue 0 showing terminal plugging of all vessel endings (arrows). en. x 450. (b) Set 1, TEM, showing first two vessel cross sections encountered (A, B). Note the homogeneous lead-positive terminal plugs. ca. x 4700. (c) Set 2, TEM, showing occlusion of all vessels. Vessels A, B, C and D labelled for orientation. C(I. x 3000.
FIG. 4. Set 3, TEM, coated
vessel D labeled
showing two coated vessels for orientation. co x 3000.
(Cl).
Occluded
vessels A, B and
C, and
Verticillium-infected
chrysanthemum
FIG. 5. Set 7, TEM, showing that all vessels are coated. The proximal surfaces of terminal are occluded by plugs are visible in two vessels, E and F; none of the other vessel lumina electron-dense lipid-rich material. Vessels G-J contain spherical and tubular luminary inclusions. Vessels A, B and C are labelled for orientation. C(I. x 3000.
42
J. Robb and L. V. Busch
the vessel endings. These terminal plugs stained a deep blue (i.e. without osmium) or purple (i.e. with osmium) with Toluidine blue 0 [Fig. 3(a)] and, using TEM, appeared to be homogeneous in composition and lead positive [Figs 3(b), (c) ; 4 ; 51. We have used the phrase “terminal plug” to refer to any occlusion which exhibited the aforementioned histochemical reactions and which sealed the end of an individual vessel whether that vessel continued to the ending of the entire leaf or not (i.e. Fig. 2). Terminal plugs varied in thickness from 20-60 pm and hence a single plug frequently appeared in four or five consecutive sets of sections (Figs 3, 4). Distal to the point of blockage the walls of all xylem vessels were lined with smooth coating material (Fig. 5) ; the structure and staining properties of this type of coating (Cl) have been previously described in chrysanthemum [II, 12, II]. The coatings appeared to be continuous with the terminal plugs; and the two were similar in structure and staining properties (Fig. 4). Figures 6(b)-(g), 7 and 8 illustrate correlated LM-TEM analysis through the site of visual blockage of the vein illustrated in Fig. 6(a). Figure 6(b), (c) shows, respectively, the 0.5 and 1 pm thick monitor sections (LM) from set 230 of this vein; Fig. 7(a) shows a correlated thin section visualized with the TEM. Set 230 is representative of all sets distal to the visual stain block. The 1 pm thick monitor section was colourless [Fig. 6(c)] and the corresponding Toluidine blue-stained, red-green section [Fig. 6(c)] and the corresponding thin section [Fig. 7(a)] showed that all of the vesselswere coated. The first tissue which was stained with basic fuchsin appeared in set 248 in which the 1 pm thick monitor section showed a single new vessel with pink secondary cell walls and a pink terminal occlusion. By set 250 the plug had disappeared but in the 1 pm thick monitor section the secondary walls remained pink [Fig. 6(e), arrowhead] ; correlated analysis [Figs 6(d) ; 7 (b) , arrowheads] indicated that this vessel was also the only uncoated vessel in the vein cross section. Four other new vessels were present in set 250; all had pink walls and all were occluded by terminal plugs [Figs 6(d), (e) ; 7(b), asterisks]. When plugs and secondary vessel walls were stained pink with basic fuchsin [Fig. 6(d)], the same structures stained purple with Toluidine blue 0 [Fig. 6(e) J. Wh en no basic fuchsin was present in the walls and plugs, both structures were blue with the same stain. The plugs disappeared from all of the new vessels within two or three sets and all were uncoated. Set 265 contained five additional uncoated vessels, all of which were new and all of which exhibited FIG. 6. (a) Whole mount of plastic embedded material showing infected blocked vein on the left [Figs 3-5; 6(b)-(g); 7; 8(a), (b)]; arrow indicates the visual point of blockage. Also showing uninoculated control on the right. C(I. x 9. (b)-(g) S erial sections through visual stain block of the same infected vein (a) fixed in glutaraldehyde-acrolein without 0~0~. The vessels are extensively colonized by fungus (F). Arrowsindicateorientation. (b) Set 232, LM, red-green monitor section stained with Toluidine blue 0; all vessels are lined with deep blue coating material. cd. x 700. (c) Set 32, LM, I pm thick monitor section showing no basic fuchsin in the vessels. C(I. x 700. (d) Set 250, LM, red-green monitor section stained with Toluidine blue 0 showing four terminal plugs (*) and one uncoated vessel (arrowhead). ca. x 700. (e) Set 50, LM, thick monitor section showing pink coloration of terminal plugs (*) and secondary walls of four vessels and pink secondary wall of uncoated vessel (arrowhead). The pink colour indicates the presence of basic fuchsin. C(I x 700. (r) Set 265, LM, red-green monitor section stained with Toluidine blue 0 showing uncoated vessels (arrowheads). ~(1. x 700. (g) Set 265, LM, thick monitor section showing pink coloration in many vessel walls (arrowheads). cn. x 700.
J. Robb and L. V. Busch
44
pink secondary vessel walls [Figs 6(f), (g); 8(a)]. In subsequent sets all newly appearing vessels were uncoated and all had secondary walls which stained pink. Some of the new vessels were terminally occluded; others were not. Lining material decreased and disappeared from many of the coated vessels and, in such cases, 1 pm thick monitor sections showed that the secondary walls and surrounding parenchyma also became pink. Similar results were obtained from the second sample prepared without osmium; in all cases the walls of coated vessels were colourless while those of uncoated vessels were stained pink. When red-green sections were subsequently stained with the PAS reaction or basic fuchsin all vessel walls stained pink throughout the entire length of the samples in both control situations as well as in the infected blocked veins, indicating that all the vessel walls have the capacity to stain with the dye. Figure 8(b) is an enlargement of part of the vein illustrated in Fig. 8(a). Portions of four vessels are shown, the two on the right being coated and colourless, and the two on the left being uncoated and pink. In the “uncoated” vessels electron-dense material does appear in patches along the walls but the material is discontinuous and does not involve the pit membranes. Other uncoated, pink-staining vessels had the same appearance. In the present study fungus occurred only in the coated vessels [Figs 7; 8(a)]. Some of the fungal walls were coated and others not; interestingly uncoated fungal hyphae always appeared to be necrotic and in various stages of degeneration while coated hyphae were ultrastructurally intact. Other than the terminal plugs no other absolute occlusion (i.e. plugs, tyloses) of vessel lumina occurred at any point in the colonized veins whether or not stain was visually blocked. Partial occlusion of lumina by vessel coating materials and fungus did, however, occur. Additionally, in the sample prepared without osmium the lumina of coated vessels were frequently filled with spherical or tubular inclusions which appeared at intervals throughout the vessels [Figs 7(b) ; 81. When exposed to basic fuchsin this material stained positively whether the stain was applied internally or over the section. The inclusions were also osmium negative and their presence was obscured when post-fixed by massesof osmiophilic fibrillar material which were interspersed among them. Similar inclusions were clearly visible in blocked veins using SEM techniques [Fig. 9(b), (c)]. In longitudinal sections through the block the occurrence and concentration of these vascular inclusions increased in the direction of the vein endings [Fig.g(b), (c)l; tha t is, the concentration was consistently higher towards the unstained part of the blocked vein [Fig. 9(c)]. None of this material was seen in the inoculated control veins [Fig. 9(d)] and none has ever been observed in previous studies of any part of the vascular system other than the dysfunctional vein endings. Observations of the controls
The experiment included two controls: (1) an internal one which consisted of observaFIG. 7. (a) Set 232, TEM, thin section (PM). co. x 2800. (b) Set 250, TEM, coated vessel (arrowhead). ca. x 2800.
showing coated vessels (Cl) and infused pit membranes thin section showing terminal plugs (*) and one un-
J. Robb and L. V. Busch
46
tions of unblocked veins from infected plants and (2) observations of veins from uninoculated chrysanthemum plants. Both controls were stained uniformly red with the basic fuchsin but, when processed and embedded the internal controls were stained a pale pink while the uninoculated veins became completely colourless [Fig. 6(a)]. Less than 5% of the vessel endings were occluded by terminal plugs in the infected controls (Fig. lo), and no plugs were observed in healthy leaves. In both cases all vessels in the terminal 3 mm of vein were uncoated [Figs 9(d) ; 1 l] ; however, coating did appear in some of the vesselsof the unblocked, infected veins in the more internal parts of the leaf. All the thick monitor sections appeared colourless in both controls. Aside from the fungus and coating material in infected plants [Fig. 11(b)] no other plugging or blocking agents were observed. DISCUSSION
In Verticillium-infected chrysanthemum plants the irreversible wilt, which ultimately causes death, apparently results from vascular dysfunction [6J. The cause is the occlusion of the vessel endings by a substance which is continuous with and which structurally and histochemically resembles lipid-rich coating [II]. As such, the site of visual stain blockage is largely incidental. The possibility arises that the use of basic fuchsin in some way generates the formation of the terminal plugs. In the light of the internal control this seems improbable. Moreover, no stain is visible in the walls, the terminal plugs or the inclusions in the lumina of terminally occluded vessels for 810 mm from the tips indicating that the basic fuchsin is not nor ever was present in this part of the vein. Any presumed effect of basic fuchsin would, therefore, have to act at a very great distance. The visual staining effect stops where the uncoated vessels end (Fig. 2). Perhaps the simplest explanation is that uncoated vessels allow the free passage of basic fuchsin but terminally occluded, coated vesselsdo not. Inherent in such an explanation is the assumption that coated and terminally sealed vesselscannot transport the stain either axially or laterally and so, in essence, the fluid is contained in “pipes which have no outlet” [.?, II, 131. Another possibility although more complex is that the presence of the stain in the “uncoated” vessels is artefactual. Basic fuchsin is forced through the vascular system under pressure; therefore, it is conceivable that the staining solution may dissolve, alter, or remove some structure or substance which would ordinarily resist the passage of fluid. In many of the uncoated vessels viewed with TEM, electron-opaque material does occur on the secondary walls of the vessel but the material is discontinuous and could be construed as resulting from the erosion of a previously complete barrier. Stain could not move, even under pressure, in the terminally sealed vessels because the fluid would have no outlet [2, II, 231 but it could be forced artificially through any unsealed vessel. Some of the stained vessels also have terminal occlusions which stain red. To follow this interpretation one would FIG. 8. (a) Set 265, TEM, thin section showing many uncoated vessel walls (arrowheads). x 2800. (b) Set 265, TEM, higher magnification of bracketed area of Plate 15 showing coated vessels (Cl) on the right with an infused full border pit (PM]) between and two uncoated vessels on the left (arrowheads) with unaltered full border pit (PM2) between. Note also the presence of uncoated (Fl) and coated (F2) hyphae and luminary inclusions (I) in the coated vessels. cn x 14000. ca.
48
J. Robb and L. V. Busch
have to assume either that the plugs were newly formed after the stain had reached the vessel ending or that they were formed from the debris produced by the basic fuchsin-generated erosion of some barrier (e.g. lipid-rich coating material). Experiments are currently in progress to determine whether basic fuchsin is capable of removing resistance barriers in Verticillium-infected plants. The success of this technique is dependent upon the fact that the red coloration is retained by the vessel walls of infected plants after processing and embedding in plastic. Little or no dye is retained by the uninoculated controls. Freehand sections of freshly stained tissue show that the vessel walls of both healthy and infected plants do stain red; also basic fuchsin applied over the thick plastic sections stains the secondary walls in both cases. This suggests that the dye penetrates the walls of infected and healthy plants equally but that the binding capacity of the walls differs. The evidence provided here and elsewhere [I, 3, 6, 7, II, 12, 141 suggests a possible sequence of events which results in the irreversible wilting (i.e. leaf flaccidity) of Verticillium-infected chrysanthemums. The fungus first proliferates and spreads through the largest of the xylem vesselsof the major lateral leaf veins; these are located in the centre of the vascular column in chrysanthemum and extend to the leaf margins. The fungus produces cell wall degrading enzymes, hormones and other compounds which may be harmful to the plant [9, 201 and which initiate wound responses in the xylem parenchyma cells [8, 131. 0 ne of the elicited responses is the formation of vessel coating material [3, 13, 151. Colonization procedes outward to the vessel endings. The terminal openings of chrysanthemum vessels are quite small (i.e. diameter 0.25 p) and eventually are sealed by an accumulation of coating material across the terminal pore. Once all the vessels at the end of a major lateral vein are coated and terminally sealed water cannot move axially or laterally and a section of the leafwill be permanently deprived ofwater. As the colonization spreads into and up the smaller peripheral vessels and minor veins of the vascular column these too will be coated and closed off progressively until whole leaf lobes are deprived of water and the tissue will eventually die. The irreversible stage of wilt is preceeded by a reversible stage in which the leaves become flaccid during the day but recover at night [4]. A number of factors which have been shown to increase resistance to waterflow in other hosts [4] may FIG. 9. (a) TEM. High magnification of tubular and spherical luminary inclusions (I). ca. x 27000. (b), (c) SEM. Longitudinal sections through a stain-blocked vein of an infected plant, showing fungal colonization (F) and increasing density of luminary inclusions (I) toward vein ending. Arrow indicates direction offlow. (b) Proximal to visual block. cc. x 750. (c) at site of visual block. ca. x 700. (d) SEM. Colonized (F) unblocked control. cn. x 800. FIG. 10. Serial sections through ending of a colonized unblocked control vein, fixed in glutaraldehyde-acrolein without 0~0~. Arrows indicate same orientation. (a) Set 4, LM, redgreen monitor section stained with Toluidine blue 0 showing a single terminal plug (*). ~(1. x 450. (b) Set 2, TEM, showing first three vessel cross sections encountered (A, B, C). Vessel C is approximately 1 pm in diameter; the section is very close to the ending. All are open. ca. x 4600. (c) Set 4, TEM, showing all vessel endings open and uncoated. ca. x 4000.
FIG. 11. (a) Serial section through ending of a colonized unblocked control vein, fixed in s h owing first fungal hyphae encountered. glutaraldehyde-acrolin without 0~04. Set 6, TEM, ca. x 3100. (b) Set 6, TEM, high magnification of colonized vessel in (a) showing coated hypha (Cl) and uncoated vessel. CB. x 11 800.
FIG. 9.
FIG.
10.
FIG. 11.
J. Robb and L. V. Busch
52
contribute to the developing of this initial water stress in chrysanthemum: progressive terminal occlusion, tyloses, fungal proliferation, occlusion or partial occlusion of vessels by various gums, gels and coating material and perhaps even toxic effects on the mesophyll and epidermal cells [4]. However, in Vertz’cillium-wilt of chrysanthemum it is doubtful that the stress incurred by factors other than terminal occlusion ever becomes limiting. It is the terminal sealing off of entire veins which apparently precipitates the irreversible stage of wilt. In our opinion the above hypothesis best suits the available data on the development of the leaf flaccidity response in VerticiElium-infected chrysanthemums. However, it must be pointed out that because of the systemic nature of the infection and the extended length of time between the initial penetration of the fungus into the root and the appearance of symptoms (i.e. 33 days) a precise chronological order is difficult to establish in most vascular diseases. For example, the occlusion of vessel endings or end plate pores may precede rather than follow the coating of vessel walls. The temporal ordering of the cell biological events which culminate in the vascular disease syndrome requires further study and is fundamental to our understanding of the primary causes of symptom development and of the concepts of susceptibility and resistance. CONCLUSIONS
When basic fuchsin is introduced into the vascular system of a Verticillium-infected chrysanthemum plant the passage of the stain is blocked in some veins of flaccid leaf lobes. Ultrastructure analysis of “blocked” major veins and suitable controls indicates the following: ( 1) vascular occlusion does not occur at the site of blockage; (2) terminal occlusion of all vessel endings does occur in “blocked” veins; (3) all terminally occluded vesselsare coated vessels (i.e. ClC3 coating material, [14] ; (4) the terminal plugs appear to be an accumulation of vessel coating material; (5) terminal occlusion of all the vessels of a major lateral vein initiates the irreversible stage of leaf flaccidity; (6) when leaves have been prepared for electron microscopy the dye is completely removed from uninoculated controls but continues to stain the unblocked veins of infected leaves. In infected leaves the red stain occurs only in the uncoated vessels of infected plants and in adjacent parenchyma cells; (7) the visual stain block occurs where the last uncoated vessels end. This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada. We wish to thank Herbert Gruning, Barbara Lee, Alexandra Smith and Elizabeth Smith for technical assistance and Dr P. F. S. Street for helpful discussion in the preparation of the manuscript. REFERENCES I. ALEXANDER,
S. J. & HALL, R. (1974). Verticillium wilt of chrysanthemum: anatomical observations roots, stems and leaves. Canadian Journal of Botany 52, 783-789. 2. CORDEN, M. E. & CHAMBERS, H. L. (1966). Vascular dysfunction in FGsarium wilt of tomato. American Journal of Botany 53, 284-287. 3. DOUGLAS, S. M. & MACHARDY, W. E. (1981). The relationship between vascular alterations and symptom development in Verticillium wilt ofchrysanthemum. Physiological Plant Pathology 19, 31-39. 4. HALL, R. & MACHARDY, W. E. (1981). Water Relations. In Fungaf Wilt Diseases of Pfanls, Ed. by M. E. Mace, A. A. Bell and C. H. Beckman, pp. 193-253, Academic Press, London.
on colonizationof
Verticillium-infected
chrysanthemum
R. O., DEKKAR, apphcation tn coating
5. KELLY,
R. A. F. & BLUEMINK, J. G. (1973). Ligand-mediated biological specimens for scanning electron microscopy.
53 osmium Journal
binding:
its
of 1Wrastructure
Research 45, 254-258.
6. MACHARDY, W. E., BUSCH, L. V. & HALL, R. (1976). Verricillium wilt of chrysanthemum: quantitative relationship between increased stomata1 resistance and local vascular dysfunction preceeding wilt. Canadian Journal of Botany 54, 1023-1034. 7. MACHARDY, W. E., HALL, R. & BUSCH, L. V. (1974). Verticillium wilt of chrysanthemum: relative water content and protein, RNA, and chlorophyll levels in leaves in relation to visible wilt symptoms. Canadian Journal of Botany 52, 49-54. 8. MOREAU, M. & CATESSON, A. M. (1981). Behaviour of the cells of the xylem parenchyma after a vascular stress. 34th International Verticillium Symposium, Bari, Italy. p. 45 (Abstr.). 9. PEGG, G. F. (1981). Biochemistry and physiology of pathogenesis. In Fungal Wilt Diseases of Plants, Ed. by M. E. Mace, A. A. Bell and C. H. Beckman, pp. 193-253, Academic Press, London. 10. PUHALLA, J. E. & BELL, A. A. (1981). Genetics and biochemistry of wilt pathogens. In Fungal Wilt Diseases of Plants, Ed. by M. E. Mace, A. A. Bell and C. H. Beckman, pp. 146192, Academic Press, London. 11. ROBB, J., BRISSON, J. D., BUSCH, L. V. & Lu, B. C. (1979). Ultrastructure of wilt syndrome caused by Verticillium dahliae. VII. Correlated light and transmission electron microscope identification of vessel coatings and tyloses. Canadian Journal of Botany 57,822-834. 12. ROBB, J., BUSCH, L. V. & Lu, B. C. (1975). Ultrastructure of wilt syndrome caused by Verticillium dahliae. I. In chrysanthemum leaves. Canadian Journal of Botany 53,901-913. 13. ROBB, J., BUSCH, L. & RAIJSER, W. E. (1980). Zinc toxicity and xylem vessel wall alterations in white beans. Annals of Botany 46,43-50. 14. ROBB, J., SMITH, A., BRISSON, J. D. & BUSCH, L. V. (1979). Ultrastructure of wilt syndrome caused by Verticillium dahliae. VI. Interpretive problems in the study of vessel coatings and tyloses. Canadian Journal of Botany 57, 795-821. 15. ROBB, J., SMITH, A. & BUSCH, L. (1982). Wilts caused by Vnticillium species. A cytological survey of vascular alterations in leaves. Canadian Journal of Botany 60,825-837. 16. SCHEFFER, R. P. & WALKER, J. C. (1953). The physiology of Fusarium wilt of tomato. Phytopathology 43, 116125. 17. TALBOYS, P. W. & BUSCH, L. V. (1970). Pectic enzymes produced by Verticillium species. Transactions of the British Mycological Society 55, 367-38 1.
Plant Pathology
(1983)
23,35-53
Structure and significance of “blocked Verticilllirm-infected chrysanthemum JANE ROBB~ Departments Canada
in
and L. V. BUSCH:
of Botany
and Gencticsf
(Accepted for publication
If basic
veins”
January
and Environmental
Biology:
University
of Gulph,
Guclph,
Ontario
NIG
2WI
1983)
fuchsin
is introduced into the vascular column of a chrysanthemum infected with most of the veins of the plant will turn red. However, certain veins associated with flaccid leaf lobes may remain unstained. This paper reports the cytological analysis of “blocked veins”. In such a dysfunctional vein all the vessels distal to the visual block are coated and terminally occluded by smooth vessel coating material; no basic fuchsin is present in or around these vessels. All uncoated vessels end at or proximal to the site of visual blockage; red dye only occurs in the secondary walls of and in parenchyma cells surrounding uncoated vessels. The results are summarized as a diagrammatic reconstruction of a “blocked vein” and, on the basis of this as well as other published structural and physiological data, a hypothesis is presented to explain the development of flaccidity in Verticilliuminfected chrysanthemums. The irreversible phase ofwilt is precipitated by the terminal occlusion of vein endings. Verficillium
dahlia
INTRODUCTION
Basic fuchsin has been used extensively as a vascular dye. When the stem of a plant is severed and the cut end is immersed in basic fuchsin normally the plant will take up the dye and eventually the entire vascular system will stain red. However, if the plant hosts a vascular pathogen the veins in certain parts of the plant, particularly in the leaves, may fail to stain [4]. The staining effect is somehow “blocked”. Studies of chrysanthemums infected with the fungus Verticillium dahliae have shown that the position of “blocks” is directly correlated with a decrease in relative water content and the development of the wilting syndrome (i.e. flaccidity) in the afi‘ected leaf lobes [41
61.
Currently, the use of basic fuchsin or a similar vascular dye [4, 16] is the only known way to determine which veins are non-functional in such systems. Yet, despite the obvious importance of vascular dysfunction to the development of the “wilting” syndrome the actual cause of the blockage of dye is unknown. Several hypotheses are possible. The stain may physically cease to move at the site of visual blockage because of the physical occlusion of the xylem elements: (1) at the site of the visual blockage, (2) proximal to the site of blockage [II] or (3) distal to the site of blockage. Alternatively, the stain may be present in the “blocked” veins but the red staining 004%4059/83/040035
+ 19 sO3.00/0
0
1983 Academic
Press Inc.
(London)
Limited
36
J. Robb and L. V. Busch
effect may somehow be prevented. At least three, or possibly all, of these possibilities may have a structural basis. We have, therefore, undertaken the cytological analysis of individual dysfunctional leaf veins of Verticillium-infected chrysanthemum plants treated with basic fuchsin in the hopes of finally determining the cause and significance of the stain blockage associated with vascular wilt disease. The interpretation ofany study employing basic fuchsin as a vascular dye must take into consideration a number of potential limitations inherent in the technique. Firstly, the position of the stain tells us nothing about water flow per se. Secondly, basic fuchsin is a strong reagent which may produce structural or chemical alterations that are unrelated to the actual disease. Little is currently known about such possible detrimental effects of the dye on either healthy or diseased plants. However, preliminary physiological studies of the same system indicate that basic fuchsin causes little damage to healthy chrysanthemum tissue within the timespan of the experiments (Robb & Street, unpublished data). We have also sought to minimize the possibility of misinterpretation due to any spurious effects of the dye by extensive use of controls in carrying out these experiments. MATERIAL Inoculation,
AND METHODS growth conditions and sampling Chrysanthemum cuttings (Chrysanthemum moriilium Ramat. cv. Brilliant Anne) were grown and inoculated with V. dahliae Kleb. as previously described [12]. Foliar
symptoms began to appear in chrysanthemum about 33 days after inoculation [I]. Infected plants were selected at an early stage of symptom expression when several leaves exhibited flaccidity of some of the lobes but no chlorosis was yet detectable. The selected plants, as well as several uninoculated controls, were severed near the soil line and the cut end of the stem was attached to a gravity-feed staining column as described by MacHardy et al. [6J. As the dye was carried through the vascular system of the plant the veins turned red in colour. Using this method, it only required 2-3 h to stain the entire vascular system of a plant approximately 30.4 cm high; the staining was considered to be complete when all leaf veins of the control plants were uniformly red and stain dripped from the severed end of one of the major lateral veins. Leaves containing blocked veins were removed from the plant and dropped immediately into a pool of glutaraldehyde (1 o/o in 0.07 M phosphate buffer, pH 6.8) at 4 “C. A light box was placed under the specimen in order to clearly visualize the blocked areas. A sample was then cut from each of four major lateral veins in which blocks occurred, each sample consisting of l-2 mm of red vein proximal to the block as well as the entire length of unstained vein distal to the block (i.e. 3-6 mm), up to and including the vein ending. Four vein samples of equivalent length and position were cut from an adjacent unblocked vein of the same leaf lobe and from veins of uninoculated plants. Figure 1 illustrates diagrammatically the sampling of stained chrysanthemum leaves. Prekaration for scanning electron microscopy (SEM) The samples were immediately transferred to a solution containing 2% glutaraldehyde and 1.5% acrolein in 0.07 M phosphate buffer, pH 6.8 and fixed at 4 “C for
Verticillium-infected
chrysanthemum
(0)
37
(b)
FIG. 1. Diagram stained stained tissue.
illustrating the position of blocks and sampling procedures in basic fuchsin leaves. Solid lines represent chrysanthemum leaves. (a) infected, (b) uninoculated veins; dotted lines represent unstained. veins. The stippled areas represent flaccid leaf
5-6 h. They were washed in the same buffer, and free-hand sectioned either longitudinally or as serial cross sections. All were subsequently treated by the ligandmediated osmium binding technique of Kelly et al. [5], dehydrated with acetone and critical-point dried with liquid COZ. They were then coated with 1 : 1 gold-palladium alloy and viewed with a gun potential of 10 kV with an ETEC Autoscan microscope. Preparation for transmissionelectron microscopy( TEM) and light microscopy(LM) The precut samples were fixed at 4°C in a solution containing 2% glutaraldehyde and 1.5% acrolein in 0.07 M phosphate buffer at pH 6.8. Half of the samples were post-fixed in 2% 0~04 and all were processed for light and electron microscopy as previously described [II, 121. Each vein was serial sectioned in sets, each set consisting of (1) one 1 pm thick monitor section (i.e. giving a grey interference colour) for LM, (2) three O-5 pm thick monitor sections (i.e. giving red-green interference colours) for LM, (3) 40 80-90 nm thin sections (i.e. giving a light gold interference colour) for TEM and (4) 15 0.5 pm thick sections (i.e. giving red-green interference colours) for LM histochemistry. The sets were numbered from the leaf margin (set 1) inwards, each set representing about 15 pm of vein. After staining with 0.5% aqueous uranyl acetate and lead citrate the thin sections were viewed using a Phillips 200A electron microscope or a JOEL JEM 100 CX electron microscope operating at 60 kV. The 1 pm thick monitor sections were viewed (LM) unstained to ascertain the presence and position of the basic fuchsin in each set (i.e. thick monitor section). The red-green monitor sections were stained with 0.5% Toluidine blue 0 in 1% sodium borate for the purpose of correlated LM-TEM analysis [II]. The remaining red-green sections were kept as a reservoir of material; they were used subsequently only when additional histochemical information was required to clarify any ambiguities arising from the correlated LM-TEM interpretation of vascular alterations [II, 241. Additional stains employed were: (1) Prussian blue, (2) Sudan black B and (3) Schiffs reagent following oxidation by HsOz (i.e. PAS reaction) as previously described [II]. The thick sections (LM) were photographed using a Nikon Labophot microscope.
38
J. Robb and L. V. Busch
Four blocked and four unblocked samples from infected leaves were serially sectioned to completion. Four control veins from uninoculated plants were cut in the same manner. In each case two of the veins were post-fixed in 0~04 and two were not. In those which were post-fixed the position of the block was marked with a notch beside the vein prior to post-fixation. RESULTS Observations
of blocked veins of infected plants In flaccid leaf lobes stain blockage occurred in the major lateral vein servicing the lobe as well as in some of the minor veins in the affected areas. At this early stage of symptom development, most of the blockages occurred within 3-5 mm of the vein ending. Figure 6(a) illustrates tissue which was fixed in glutaraldehyde-acrolein (i.e. no osmium), dehydrated and embedded in plastic. The vein in the left mould was from an infected symptomatic leaf; a stain blockage is clearly visible with the vascular tissue being red on the proximal side of the block (i.e. left) and unstained from the block to the vein tip (i.e. right). Tissue which had been post-fixed in osmium was black, obscuring the visual blocks.
Set 250
/
Stained
Visual block
3
Unstained
FIG. 2. Schematic diagram showing the position of the basic fuchsin stain and the structure of a blocked major lateral vein of a chrysanthemum plant infected with V. dahlias. The figure was reconstructed from the data obtained from the blocked vein illustrated in Figs 3-7 and 8(a). Note. once again. that throughout the study the sets of sections have been numbered from the leaf margin (set 1) inwards. H, Vessel lumen; 0, unstained vessel wall ; &I, pink vessel wall ; !& vessel coating material.
Figure 2 is a diagrammatic representation of the structure of a blocked leaf vein as revealed by the present study. In the unstained portions of veins distal to stain blockage, 92% of all vessels extending to the leaf margins were occluded (Fig. 3) at FIG. 3. (a) Set 4, LM, red-green monitor section stained with Toluidine blue 0 showing terminal plugging of all vessel endings (arrows). en. x 450. (b) Set 1, TEM, showing first two vessel cross sections encountered (A, B). Note the homogeneous lead-positive terminal plugs. ca. x 4700. (c) Set 2, TEM, showing occlusion of all vessels. Vessels A, B, C and D labelled for orientation. C(I. x 3000.
FIG. 4. Set 3, TEM, coated
vessel D labeled
showing two coated vessels for orientation. co x 3000.
(Cl).
Occluded
vessels A, B and
C, and
Verticillium-infected
chrysanthemum
FIG. 5. Set 7, TEM, showing that all vessels are coated. The proximal surfaces of terminal are occluded by plugs are visible in two vessels, E and F; none of the other vessel lumina electron-dense lipid-rich material. Vessels G-J contain spherical and tubular luminary inclusions. Vessels A, B and C are labelled for orientation. C(I. x 3000.
42
J. Robb and L. V. Busch
the vessel endings. These terminal plugs stained a deep blue (i.e. without osmium) or purple (i.e. with osmium) with Toluidine blue 0 [Fig. 3(a)] and, using TEM, appeared to be homogeneous in composition and lead positive [Figs 3(b), (c) ; 4 ; 51. We have used the phrase “terminal plug” to refer to any occlusion which exhibited the aforementioned histochemical reactions and which sealed the end of an individual vessel whether that vessel continued to the ending of the entire leaf or not (i.e. Fig. 2). Terminal plugs varied in thickness from 20-60 pm and hence a single plug frequently appeared in four or five consecutive sets of sections (Figs 3, 4). Distal to the point of blockage the walls of all xylem vessels were lined with smooth coating material (Fig. 5) ; the structure and staining properties of this type of coating (Cl) have been previously described in chrysanthemum [II, 12, II]. The coatings appeared to be continuous with the terminal plugs; and the two were similar in structure and staining properties (Fig. 4). Figures 6(b)-(g), 7 and 8 illustrate correlated LM-TEM analysis through the site of visual blockage of the vein illustrated in Fig. 6(a). Figure 6(b), (c) shows, respectively, the 0.5 and 1 pm thick monitor sections (LM) from set 230 of this vein; Fig. 7(a) shows a correlated thin section visualized with the TEM. Set 230 is representative of all sets distal to the visual stain block. The 1 pm thick monitor section was colourless [Fig. 6(c)] and the corresponding Toluidine blue-stained, red-green section [Fig. 6(c)] and the corresponding thin section [Fig. 7(a)] showed that all of the vesselswere coated. The first tissue which was stained with basic fuchsin appeared in set 248 in which the 1 pm thick monitor section showed a single new vessel with pink secondary cell walls and a pink terminal occlusion. By set 250 the plug had disappeared but in the 1 pm thick monitor section the secondary walls remained pink [Fig. 6(e), arrowhead] ; correlated analysis [Figs 6(d) ; 7 (b) , arrowheads] indicated that this vessel was also the only uncoated vessel in the vein cross section. Four other new vessels were present in set 250; all had pink walls and all were occluded by terminal plugs [Figs 6(d), (e) ; 7(b), asterisks]. When plugs and secondary vessel walls were stained pink with basic fuchsin [Fig. 6(d)], the same structures stained purple with Toluidine blue 0 [Fig. 6(e) J. Wh en no basic fuchsin was present in the walls and plugs, both structures were blue with the same stain. The plugs disappeared from all of the new vessels within two or three sets and all were uncoated. Set 265 contained five additional uncoated vessels, all of which were new and all of which exhibited FIG. 6. (a) Whole mount of plastic embedded material showing infected blocked vein on the left [Figs 3-5; 6(b)-(g); 7; 8(a), (b)]; arrow indicates the visual point of blockage. Also showing uninoculated control on the right. C(I. x 9. (b)-(g) S erial sections through visual stain block of the same infected vein (a) fixed in glutaraldehyde-acrolein without 0~0~. The vessels are extensively colonized by fungus (F). Arrowsindicateorientation. (b) Set 232, LM, red-green monitor section stained with Toluidine blue 0; all vessels are lined with deep blue coating material. cd. x 700. (c) Set 32, LM, I pm thick monitor section showing no basic fuchsin in the vessels. C(I. x 700. (d) Set 250, LM, red-green monitor section stained with Toluidine blue 0 showing four terminal plugs (*) and one uncoated vessel (arrowhead). ca. x 700. (e) Set 50, LM, thick monitor section showing pink coloration of terminal plugs (*) and secondary walls of four vessels and pink secondary wall of uncoated vessel (arrowhead). The pink colour indicates the presence of basic fuchsin. C(I x 700. (r) Set 265, LM, red-green monitor section stained with Toluidine blue 0 showing uncoated vessels (arrowheads). ~(1. x 700. (g) Set 265, LM, thick monitor section showing pink coloration in many vessel walls (arrowheads). cn. x 700.
J. Robb and L. V. Busch
44
pink secondary vessel walls [Figs 6(f), (g); 8(a)]. In subsequent sets all newly appearing vessels were uncoated and all had secondary walls which stained pink. Some of the new vessels were terminally occluded; others were not. Lining material decreased and disappeared from many of the coated vessels and, in such cases, 1 pm thick monitor sections showed that the secondary walls and surrounding parenchyma also became pink. Similar results were obtained from the second sample prepared without osmium; in all cases the walls of coated vessels were colourless while those of uncoated vessels were stained pink. When red-green sections were subsequently stained with the PAS reaction or basic fuchsin all vessel walls stained pink throughout the entire length of the samples in both control situations as well as in the infected blocked veins, indicating that all the vessel walls have the capacity to stain with the dye. Figure 8(b) is an enlargement of part of the vein illustrated in Fig. 8(a). Portions of four vessels are shown, the two on the right being coated and colourless, and the two on the left being uncoated and pink. In the “uncoated” vessels electron-dense material does appear in patches along the walls but the material is discontinuous and does not involve the pit membranes. Other uncoated, pink-staining vessels had the same appearance. In the present study fungus occurred only in the coated vessels [Figs 7; 8(a)]. Some of the fungal walls were coated and others not; interestingly uncoated fungal hyphae always appeared to be necrotic and in various stages of degeneration while coated hyphae were ultrastructurally intact. Other than the terminal plugs no other absolute occlusion (i.e. plugs, tyloses) of vessel lumina occurred at any point in the colonized veins whether or not stain was visually blocked. Partial occlusion of lumina by vessel coating materials and fungus did, however, occur. Additionally, in the sample prepared without osmium the lumina of coated vessels were frequently filled with spherical or tubular inclusions which appeared at intervals throughout the vessels [Figs 7(b) ; 81. When exposed to basic fuchsin this material stained positively whether the stain was applied internally or over the section. The inclusions were also osmium negative and their presence was obscured when post-fixed by massesof osmiophilic fibrillar material which were interspersed among them. Similar inclusions were clearly visible in blocked veins using SEM techniques [Fig. 9(b), (c)]. In longitudinal sections through the block the occurrence and concentration of these vascular inclusions increased in the direction of the vein endings [Fig.g(b), (c)l; tha t is, the concentration was consistently higher towards the unstained part of the blocked vein [Fig. 9(c)]. None of this material was seen in the inoculated control veins [Fig. 9(d)] and none has ever been observed in previous studies of any part of the vascular system other than the dysfunctional vein endings. Observations of the controls
The experiment included two controls: (1) an internal one which consisted of observaFIG. 7. (a) Set 232, TEM, thin section (PM). co. x 2800. (b) Set 250, TEM, coated vessel (arrowhead). ca. x 2800.
showing coated vessels (Cl) and infused pit membranes thin section showing terminal plugs (*) and one un-
J. Robb and L. V. Busch
46
tions of unblocked veins from infected plants and (2) observations of veins from uninoculated chrysanthemum plants. Both controls were stained uniformly red with the basic fuchsin but, when processed and embedded the internal controls were stained a pale pink while the uninoculated veins became completely colourless [Fig. 6(a)]. Less than 5% of the vessel endings were occluded by terminal plugs in the infected controls (Fig. lo), and no plugs were observed in healthy leaves. In both cases all vessels in the terminal 3 mm of vein were uncoated [Figs 9(d) ; 1 l] ; however, coating did appear in some of the vesselsof the unblocked, infected veins in the more internal parts of the leaf. All the thick monitor sections appeared colourless in both controls. Aside from the fungus and coating material in infected plants [Fig. 11(b)] no other plugging or blocking agents were observed. DISCUSSION
In Verticillium-infected chrysanthemum plants the irreversible wilt, which ultimately causes death, apparently results from vascular dysfunction [6J. The cause is the occlusion of the vessel endings by a substance which is continuous with and which structurally and histochemically resembles lipid-rich coating [II]. As such, the site of visual stain blockage is largely incidental. The possibility arises that the use of basic fuchsin in some way generates the formation of the terminal plugs. In the light of the internal control this seems improbable. Moreover, no stain is visible in the walls, the terminal plugs or the inclusions in the lumina of terminally occluded vessels for 810 mm from the tips indicating that the basic fuchsin is not nor ever was present in this part of the vein. Any presumed effect of basic fuchsin would, therefore, have to act at a very great distance. The visual staining effect stops where the uncoated vessels end (Fig. 2). Perhaps the simplest explanation is that uncoated vessels allow the free passage of basic fuchsin but terminally occluded, coated vesselsdo not. Inherent in such an explanation is the assumption that coated and terminally sealed vesselscannot transport the stain either axially or laterally and so, in essence, the fluid is contained in “pipes which have no outlet” [.?, II, 131. Another possibility although more complex is that the presence of the stain in the “uncoated” vessels is artefactual. Basic fuchsin is forced through the vascular system under pressure; therefore, it is conceivable that the staining solution may dissolve, alter, or remove some structure or substance which would ordinarily resist the passage of fluid. In many of the uncoated vessels viewed with TEM, electron-opaque material does occur on the secondary walls of the vessel but the material is discontinuous and could be construed as resulting from the erosion of a previously complete barrier. Stain could not move, even under pressure, in the terminally sealed vessels because the fluid would have no outlet [2, II, 231 but it could be forced artificially through any unsealed vessel. Some of the stained vessels also have terminal occlusions which stain red. To follow this interpretation one would FIG. 8. (a) Set 265, TEM, thin section showing many uncoated vessel walls (arrowheads). x 2800. (b) Set 265, TEM, higher magnification of bracketed area of Plate 15 showing coated vessels (Cl) on the right with an infused full border pit (PM]) between and two uncoated vessels on the left (arrowheads) with unaltered full border pit (PM2) between. Note also the presence of uncoated (Fl) and coated (F2) hyphae and luminary inclusions (I) in the coated vessels. cn x 14000. ca.
48
J. Robb and L. V. Busch
have to assume either that the plugs were newly formed after the stain had reached the vessel ending or that they were formed from the debris produced by the basic fuchsin-generated erosion of some barrier (e.g. lipid-rich coating material). Experiments are currently in progress to determine whether basic fuchsin is capable of removing resistance barriers in Verticillium-infected plants. The success of this technique is dependent upon the fact that the red coloration is retained by the vessel walls of infected plants after processing and embedding in plastic. Little or no dye is retained by the uninoculated controls. Freehand sections of freshly stained tissue show that the vessel walls of both healthy and infected plants do stain red; also basic fuchsin applied over the thick plastic sections stains the secondary walls in both cases. This suggests that the dye penetrates the walls of infected and healthy plants equally but that the binding capacity of the walls differs. The evidence provided here and elsewhere [I, 3, 6, 7, II, 12, 141 suggests a possible sequence of events which results in the irreversible wilting (i.e. leaf flaccidity) of Verticillium-infected chrysanthemums. The fungus first proliferates and spreads through the largest of the xylem vesselsof the major lateral leaf veins; these are located in the centre of the vascular column in chrysanthemum and extend to the leaf margins. The fungus produces cell wall degrading enzymes, hormones and other compounds which may be harmful to the plant [9, 201 and which initiate wound responses in the xylem parenchyma cells [8, 131. 0 ne of the elicited responses is the formation of vessel coating material [3, 13, 151. Colonization procedes outward to the vessel endings. The terminal openings of chrysanthemum vessels are quite small (i.e. diameter 0.25 p) and eventually are sealed by an accumulation of coating material across the terminal pore. Once all the vessels at the end of a major lateral vein are coated and terminally sealed water cannot move axially or laterally and a section of the leafwill be permanently deprived ofwater. As the colonization spreads into and up the smaller peripheral vessels and minor veins of the vascular column these too will be coated and closed off progressively until whole leaf lobes are deprived of water and the tissue will eventually die. The irreversible stage of wilt is preceeded by a reversible stage in which the leaves become flaccid during the day but recover at night [4]. A number of factors which have been shown to increase resistance to waterflow in other hosts [4] may FIG. 9. (a) TEM. High magnification of tubular and spherical luminary inclusions (I). ca. x 27000. (b), (c) SEM. Longitudinal sections through a stain-blocked vein of an infected plant, showing fungal colonization (F) and increasing density of luminary inclusions (I) toward vein ending. Arrow indicates direction offlow. (b) Proximal to visual block. cc. x 750. (c) at site of visual block. ca. x 700. (d) SEM. Colonized (F) unblocked control. cn. x 800. FIG. 10. Serial sections through ending of a colonized unblocked control vein, fixed in glutaraldehyde-acrolein without 0~0~. Arrows indicate same orientation. (a) Set 4, LM, redgreen monitor section stained with Toluidine blue 0 showing a single terminal plug (*). ~(1. x 450. (b) Set 2, TEM, showing first three vessel cross sections encountered (A, B, C). Vessel C is approximately 1 pm in diameter; the section is very close to the ending. All are open. ca. x 4600. (c) Set 4, TEM, showing all vessel endings open and uncoated. ca. x 4000.
FIG. 11. (a) Serial section through ending of a colonized unblocked control vein, fixed in s h owing first fungal hyphae encountered. glutaraldehyde-acrolin without 0~04. Set 6, TEM, ca. x 3100. (b) Set 6, TEM, high magnification of colonized vessel in (a) showing coated hypha (Cl) and uncoated vessel. CB. x 11 800.
FIG. 9.
FIG.
10.
FIG. 11.
J. Robb and L. V. Busch
52
contribute to the developing of this initial water stress in chrysanthemum: progressive terminal occlusion, tyloses, fungal proliferation, occlusion or partial occlusion of vessels by various gums, gels and coating material and perhaps even toxic effects on the mesophyll and epidermal cells [4]. However, in Vertz’cillium-wilt of chrysanthemum it is doubtful that the stress incurred by factors other than terminal occlusion ever becomes limiting. It is the terminal sealing off of entire veins which apparently precipitates the irreversible stage of wilt. In our opinion the above hypothesis best suits the available data on the development of the leaf flaccidity response in VerticiElium-infected chrysanthemums. However, it must be pointed out that because of the systemic nature of the infection and the extended length of time between the initial penetration of the fungus into the root and the appearance of symptoms (i.e. 33 days) a precise chronological order is difficult to establish in most vascular diseases. For example, the occlusion of vessel endings or end plate pores may precede rather than follow the coating of vessel walls. The temporal ordering of the cell biological events which culminate in the vascular disease syndrome requires further study and is fundamental to our understanding of the primary causes of symptom development and of the concepts of susceptibility and resistance. CONCLUSIONS
When basic fuchsin is introduced into the vascular system of a Verticillium-infected chrysanthemum plant the passage of the stain is blocked in some veins of flaccid leaf lobes. Ultrastructure analysis of “blocked” major veins and suitable controls indicates the following: ( 1) vascular occlusion does not occur at the site of blockage; (2) terminal occlusion of all vessel endings does occur in “blocked” veins; (3) all terminally occluded vesselsare coated vessels (i.e. ClC3 coating material, [14] ; (4) the terminal plugs appear to be an accumulation of vessel coating material; (5) terminal occlusion of all the vessels of a major lateral vein initiates the irreversible stage of leaf flaccidity; (6) when leaves have been prepared for electron microscopy the dye is completely removed from uninoculated controls but continues to stain the unblocked veins of infected leaves. In infected leaves the red stain occurs only in the uncoated vessels of infected plants and in adjacent parenchyma cells; (7) the visual stain block occurs where the last uncoated vessels end. This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada. We wish to thank Herbert Gruning, Barbara Lee, Alexandra Smith and Elizabeth Smith for technical assistance and Dr P. F. S. Street for helpful discussion in the preparation of the manuscript. REFERENCES I. ALEXANDER,
S. J. & HALL, R. (1974). Verticillium wilt of chrysanthemum: anatomical observations roots, stems and leaves. Canadian Journal of Botany 52, 783-789. 2. CORDEN, M. E. & CHAMBERS, H. L. (1966). Vascular dysfunction in FGsarium wilt of tomato. American Journal of Botany 53, 284-287. 3. DOUGLAS, S. M. & MACHARDY, W. E. (1981). The relationship between vascular alterations and symptom development in Verticillium wilt ofchrysanthemum. Physiological Plant Pathology 19, 31-39. 4. HALL, R. & MACHARDY, W. E. (1981). Water Relations. In Fungaf Wilt Diseases of Pfanls, Ed. by M. E. Mace, A. A. Bell and C. H. Beckman, pp. 193-253, Academic Press, London.
on colonizationof
Verticillium-infected
chrysanthemum
R. O., DEKKAR, apphcation tn coating
5. KELLY,
R. A. F. & BLUEMINK, J. G. (1973). Ligand-mediated biological specimens for scanning electron microscopy.
53 osmium Journal
binding:
its
of 1Wrastructure
Research 45, 254-258.
6. MACHARDY, W. E., BUSCH, L. V. & HALL, R. (1976). Verricillium wilt of chrysanthemum: quantitative relationship between increased stomata1 resistance and local vascular dysfunction preceeding wilt. Canadian Journal of Botany 54, 1023-1034. 7. MACHARDY, W. E., HALL, R. & BUSCH, L. V. (1974). Verticillium wilt of chrysanthemum: relative water content and protein, RNA, and chlorophyll levels in leaves in relation to visible wilt symptoms. Canadian Journal of Botany 52, 49-54. 8. MOREAU, M. & CATESSON, A. M. (1981). Behaviour of the cells of the xylem parenchyma after a vascular stress. 34th International Verticillium Symposium, Bari, Italy. p. 45 (Abstr.). 9. PEGG, G. F. (1981). Biochemistry and physiology of pathogenesis. In Fungal Wilt Diseases of Plants, Ed. by M. E. Mace, A. A. Bell and C. H. Beckman, pp. 193-253, Academic Press, London. 10. PUHALLA, J. E. & BELL, A. A. (1981). Genetics and biochemistry of wilt pathogens. In Fungal Wilt Diseases of Plants, Ed. by M. E. Mace, A. A. Bell and C. H. Beckman, pp. 146192, Academic Press, London. 11. ROBB, J., BRISSON, J. D., BUSCH, L. V. & Lu, B. C. (1979). Ultrastructure of wilt syndrome caused by Verticillium dahliae. VII. Correlated light and transmission electron microscope identification of vessel coatings and tyloses. Canadian Journal of Botany 57,822-834. 12. ROBB, J., BUSCH, L. V. & Lu, B. C. (1975). Ultrastructure of wilt syndrome caused by Verticillium dahliae. I. In chrysanthemum leaves. Canadian Journal of Botany 53,901-913. 13. ROBB, J., BUSCH, L. & RAIJSER, W. E. (1980). Zinc toxicity and xylem vessel wall alterations in white beans. Annals of Botany 46,43-50. 14. ROBB, J., SMITH, A., BRISSON, J. D. & BUSCH, L. V. (1979). Ultrastructure of wilt syndrome caused by Verticillium dahliae. VI. Interpretive problems in the study of vessel coatings and tyloses. Canadian Journal of Botany 57, 795-821. 15. ROBB, J., SMITH, A. & BUSCH, L. (1982). Wilts caused by Vnticillium species. A cytological survey of vascular alterations in leaves. Canadian Journal of Botany 60,825-837. 16. SCHEFFER, R. P. & WALKER, J. C. (1953). The physiology of Fusarium wilt of tomato. Phytopathology 43, 116125. 17. TALBOYS, P. W. & BUSCH, L. V. (1970). Pectic enzymes produced by Verticillium species. Transactions of the British Mycological Society 55, 367-38 1.