Ultrastructure of wilt syndrome caused by Verticillium dahliae. IV. Evidence for a- and P-conidial forms

J . D. BRISSON"~ Depur!met~! of Bolany ernd Gerlelics, Ut~iversify of Grrelph, Glrelpll, Onf . , Ccrr~ada NIG 2WI

A N D

JANE ROBB A N D LLOYD BUSCH~ Depcrr!r?ienf of Envirorrnle?~!al Biology, Universify of G~relph, Grrelpll, On!., Coneldo N l G 2WI

Received July 19, 1977

BRISSON, J. D., J. ROBB, and L. B u s c ~ . 1978. Ultrastructure of wilt syndrome caused by Ver~icillirrr?~ dohliae. IV. Evidence for a - and P-conidial forms. Can. J . Bot. 56: 1458-1465.

Scanning electron microscope observations of leaf petioles of chrysanthemums infected with V . dalrliue suggest that two spore forms are present: the normal conidiospore ( a conidium) and a sickle-shaped secondary spore or P spore (beta spore). Conidia of V. dahliue appear to be ovoid in shape. possibly septate, and in the host plant, covered by a coating material. Phialides are rarely seen in the host, although the conidia are numerous. Conidia can apparently give rise, simultane- ously, to two secondary structures: germ tubes and P spores. The P spores are sickle-shaped structures of constant diameter and when mature are free floating in the vessels.

BRISSON, J. D.. J . ROBB et L. B u s c ~ . 1978. Ultrastructure of wilt syndrome caused by Verlicil- lirrr?~ dahliue. IV. Evidence for a - and P-conidial forms. Can. J. Bot. 56: 1458-1465.

Des observations effectuees au microscope electronique B balayage sur des g t io les de feuilles provenant de chrysanthemes infectes par le V. clul~licre suggerent la presence de deux formes de spores: une conidiospore normale (a-conidie) ainsi qu'une conidie secondaire en forme defaucille ou P-spore (bkta-spore). Les conidies no~.males de V. dnhliue sont d'une forme ovolde, septees et enveloppees, dans la plante hbte, d'un materiel de recouvrement. Les phialides sont rarement observees dans la h6te bien que les conidies soient nombreuses. Lesconidies peuvent apparem- ment donner naissance simultanement a deux structures differentes: des tubes germinatifs et des P-spores. Les P-spores sont des structures en forme de faucille d'un diametre constant et qui, a maturite, flottent librement dans les elements de vaisseaux.

Introduction Verticillium dahlicre is a causative agent of vascu-

lar wilt disease ina large variety of host plants (Pegg 1974). Normally, the fungus penetrates the vascu- lar column in the host root and quickly spreads along the xylem vessels in the direction of water flow. The speed of colonization is frequently faster than would be expected on the basis of hyphal tip growth alone. Light microscope (LM) studies suggested that the conidium is an important agent of fungal dispersal in chrysanthemum (Alexander and Hall 1974). This hypothesis is supported by two facts: first, the rate of growth, and second, the discontinuous nature of fungal colonization.

Actual conidia, which are about 2 pm in diame- ter, are difficult to see in the vessels with the LM (Alexander and Hall 1974; Garber and Houston 1966; Nelson 1950). The scanning electron micro- scope (SEM) has been used to show conidial pro-

'The senior author was a holder of an Ontario-Quebec ex- change fellowship.

'Present address: Division of Biological Sciences, National Research Council of Canada, Ottawa, Ont., Canada KIA OR6.

'Reprint requests should be sent to Dr. L. Busch.

duction by hyphae of other Verticilliutn species in different hosts (Cooper and Wood 1974; Locci et al. 1970). In the present study, we have used the SEM to study some aspects of conidial morphology and ontogeny in chrysanthemum plants infected with V . dahliae. For comparative purposes, we have also made SEM observations of conidia produced by the fungus in culture.

Materials and Methods lnoclrlrr!iotl crr~d Plnrlr Grotvlh Condi~ior~s

Chrysanthemum plants (C/~,yscrn!hernrr,n rnorifolirrr?l Ramat cv. Brilliant Anne) were grown and inoculated with Verlicillirrm clolllicre Kleb. a s previously reported (Robb el 01. 1975). Foliar symptoms begin to appear in chrysanthemum about 33 days after inoculation.

F~rngnl C~rltrrres Fung;ll colonies were started from a previous inoculum and

plated on a dextrose medium (Ainsworth 1968). The colonies were allowed to grow from I to 2 weeks.

Fixcr!iorc crrld Elcc!ror~ Microscopy For the SEM samples. inoculated and uninoculated leaf

petioles were fixed for 2 h at room temperature in a solution containing 2% glutaraldehyde and 1% osmium tetroxide in 0.07M phosphate buffer, pH 6.8. They were subsequently treated by the ligand-mediated osmium-binding technique of Kelley er crl. (1973). dehydrated with acetone. and critical-point

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I N ET A L . 1459

dried from acetone with liquid CO,. Samples ofgrowing f u n g ~ ~ s colonies were removed on pieces of agar, fixed in OsO, vapour, and dried over Drierlite.

All samples were coated with a 1 : l gold-palladium alloy and viewed with a gun potential of 10 kV with an ETEC Autoscan. Difficulties were encountered in eliminating charging effects when viewing p spores since most of those observed were deep i n the xylem vessels or under the curve of the vessel walls.

. . . . . Stereopairs were made to avoid controversial interpretation: the . . . . photos were taken at a7" t i l t each way from the central axis and . . . .

the microgfitphs were mounted with the lower t i l t angle on the left.

The cells illustrated in Fig. 9 were prepared for transmission electron microscopy (TEM) as previously described (Robberril. 1975).

Results By the time that a leaf of achrysanthemum plant

infected with V . dalzliue develops advanced foliar symptoms (i.e., leaf flaccidity and chlorosis), spore-producing hyphae and numerous conidia are observed in the petioles (Fig. 1). Phialides are rarely seen in the hyphae growing in the host plant (Fig. 3, arrow; Fig. 4). Figure 2 illustrates a phialide in cultui-e. This particular phialide has a well de- fined collar (Fig. 2); in our material, the collar ap- pears to be prominent only when conidia are form- ing. In chrysanthemum, the production of conidia appears to occur at random; that is, hyphae in adja- cent vessels do not necessarily produce spores at the same level and conidial production does not seem to be associated with end-vessel obstruction, as previously observed in cotton (Beckman et ul . 1974).

Most of the conidia observed, either in culture (Fig. 5) or within host xylem vessels (Figs. 1,6-8), are ovoid in shape. When 20 conidia were mea- sured, they averaged l .9 pm in diameter (range 1.3-3.1) and 4.4pm in length (range 2.2-7.7). In

. . culture, most of the conidia have a median constric- tion (Fig. 5). In the plant, more of the spores lack this prominent constriction (Figs. 6 ,7) than have i t (Fig. 8).

In the host plant, many of the conidia are as- sociated with germ tubes which tend to be straight and of variable diameter (Figs. 1 , 8, 9, 13). In a number of cases, such gel-m tubes have been ob- served to constrict and then swell again in a consis-

. . . . . . . . . . . . tent pattern (Figs. 10, 14). Note that in Fig. 10, the . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . fungus changes direction after the constriction. . . . . . . . . . . . . . . . . . . . . . .

Conidial germ tubes can also invade adjacent vessels through pits in the vessel walls (Fig. 9). Figure 9, which is a TEM micrograph, also illus- trates the fact that when observed in thin sections many conidia are coated by an electron-dense sheath of material. The conidia generally appear to be free in the vessels, but sometimes they are at-

tached to the vessel walls or to each other by threadlike structures (Figs. 1, 6 ,7 , an-ows).

Apart from normal germ tubes, some conidia apparently give rise to curved or sickle-shaped structures (Figs. I , s; 10, s; 12; 13, s) which aver- aged 1.1 pm in diameter (range 1 .O- 1.2) and 6 pm in length (range 5-7). Figures 1 . 10, 12, and 13 are stereopairs provided to illustrate the three- dimensional contours of the sickle-shaped struc- tures. A single conidium can apparently produce both structures simultaneously, the germ tubes basipetall y and the second structure acropetally (Figs. 1 , 10). The sickle-shaped structiires may be seen attached to a conidium (Figs. 1, 10, 13) or free in the vessels (Figs. 11, 12). The diameter of the attached secondary structures appears to be less (Figs. 10, 13) than that of the free form (Figs. 12, 13).

Figures 1 1 and 12 show that sickel-shaped struc- tures sometimes occur in the vessels in a position removed from the hyphal colony in the direction of water flow. This suggests that they are probably free floating when mature. Such structures have only been seen in the pathogenic situation; re- peated attempts to find them on fungal culti~res of V. dahlicce have failed.

The number of sickle-shaped structures is very low compared with the enormous numbers of con- idia and germ tubes. To date, we have counted only six p conidia in which the entire contour of the structure could be seen distinctly enough to make stereopairs.

Discussion The cell type considered to characterize Verticil-

iilrn~ spore ontogeny is the phialide (Barron 1968). Such phialides produce spores in chains bound to- gether by a mucous sheath. To the best of our knowledge, the only PI-evious illustration of a Ver- ticillium phialide in a parasitic situation was re- ported by Locci et al. (1970, Plate 11, Fig. 5) for Hetnileia vastatrix, a coffee leaf rust parasitized by V. lzemileiae. The low number of phialides and the high number of conidia observed in chrysan- themum vessels suggest that there is a secondary mode of conidium formation, possibly budding (Fig. 1 ,b) as observed in shake cultures of V. albo- atrum by Vessey and Pegg (1973).

The median constriction observed on many of the spores in culture may be an external manifesta- tion of septation within; however, this point re- quires confirmation by sectioning conidia. Pre- sumably the sheath of material coating the conidia in the host plant obscures the median constriction so frequently observed in culture. The presence of

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CAN. J. BOT. VOL. 56. 1978

FIG. 1. Stereopairs of a general view of Verticilli~rtn dahlirre growing inside a late metaxylem vessel at the fore of a major leaf vein. Most conidia have germ tubes some of which pass into the adjacent metaxylem vessel (beneath) through large bordered pit pairs. Some conidia appeal- to be budding ( h ) and threads link conidia to host cell walls or to each other (arrows). Two sickle-shape conidia (s) are present. x 4465.

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BRlSSON ET AL. 1

FIG. 2. Verficillilrm dcrhlirre growing on agar; production of a conidium from a phialide @) which has a distinct collar (ar~.ow). x 9930. FIG. 3 u . Hyphae occupying a metaxylem vessel with one of them showing a phialide end. x 2590. FIG. 3h. Enlarged view of the outlined portion of Fig. 3u showing a phialophore collar scar (arrow). x 3780. FIG. 4. Hypha bearing a phialophore within ;I host vessel. x 3500. FIG. 5 . SepVate conidia (arrows) growing over the agar. X 9455. FIG. 6. Twoconidia present in a vessel are covered with a mucous sheath. Threads (arrows) join the conidial wall to the vessel wall. x 10900.

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1462 C A N . J . BOT. VOL. 56. 1978

FIG. 7. Two conidia present in n vessel :\re covered with n mucous sheath. Threads (arrow) join the conidial wall to the vessel wall. x I 1 h65. FIG. 8. Septi~te c o n i d i ~ ~ m (arrow) borne on a hypha inside a vessel member. The adjacent hyphal stl-and has thre21ds to host cell wall. x 5065. F I G . 9. A TEM picture showinga germ tube passing through the primary wi~ll ofan early metaxylem vessel. An electron-dense deposit is present around the germinating conidium and germ tube. The nature of the primary wall ischanged. x 38 625. FIG. 10. Stereopairs of the portion outlined in Fig. 1 but viewed at adifferent angle to show the sickle-shape conidi~~ni(s) lying over the pit pairs and the iterative germination mode with 21 constricted coll:u. region (arrow). (Figure 14 is a schematic representation of this area.) x 82 15.

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FIGS. 11-13. General view of a late protoxylem vessel oriented such that the basipetal direction is downwards (Fig. 11). X

775. The portions outlined are enlarged in Figs. 12 ( X 4690) and 13 ( x 2200). Figure 12 illustrates two free sickle-shaped conidia (arrows). Figure 13 shows a germ tube (6') and two sickle-shaped conidia (s). One of the sickle-shaped structures is attached to an a-conidium; the other is free. Note the differences in diameter of the germ tube and the free and attached sickle-shaped structures. The stereoscopic effect clearly shows the free spore to be lying over the pit rather than to be passing through it.

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1464 C A N . J . BOT. VOL. 56. 1978

FIG. 14. Production of a hyphal strand by transformation of a short secondary spore prod~lced by a germ tube - conidiophore complex. c, rest of collar; gt, germ tube: hs, hyphal strand; s, short conidium.

the constriction is not associated with the produc- tion of agerm tube. Swinburne (1976) has shown by SEM that in Colletotrich~ltn r n ~ ~ s a e a similar con- striction appears only when the conidium forms a second germ tube and when the first one is trans- formed into an appressorium.

Since germ tubes appear to be straight in the vast majority of cases, the abrupt change in direction which may accompany a consistent pattern of con- striction followed by swelling suggested the hypothesis that germ tubes can themselves develop into conidiophores which produce short secondary spores which in turn give rise to hyphal strands (Fig. 14). This hypothesis requires confirmation.

The only sexual state known for a Verticilliurn species (i.e., V. olivaceut-urn) belongs to the genus Nectt-ia (Hastie 1976). The presence of dimorphic spores is common in the asexual state of fungi be- longing to this genus (e.g., Cylindrocarpon and F~lsari~lnz; Barron 1968; Booth 1971). Normally, the two spore forms are called macroconidia and microconidia. These forms are produced by differ- ent phialide structures; both are capable of germi- nation and both have a pathogenicity potential. Other genera are known to produce dimorphic con- idia. In some of these genel'a, on1 y one of the spore forms meets the above criteria of ontogeny, germi- nation and pathogenicity; therefore, a different terminology has been adopted. The spore form which does meet the above criteria is called the a spore (alpha spore); the spore form which does not is sometimes called the P spore (beta spore) (Ainsworth 1963). The characteristic features of B conidia are that they are secondary structures of constant diameter, that they can be produced by conidia but are narrower in diameter than the germ tubes, and that they are usually sickle shaped and sterile.

In the pathogenic state of V. dahliae, we have observed free, sickle-shaped structures similar in appearance to P spores and we have also observed similar sickle-shaped structures attached to normal

conidia. This suggests that the attached form gives rise to the free form and that V. dcrhliae produces two spore types which we will subsequently refer to as the a conidium and the p conidium. We cannot entirely rule out the possibility that the free sickle- shaped structures are contaminants since the number observed was very low. However, no con- taminants were found when sections of the same petioles were incubated in culture. Also the P con- idia do not appear to be coated or attached to the vessel walls and may tend to be removed during processing.

The diameter of a P conidium, when compared with that of agerm tube or of an a conidium, seems to vary during its initiation in two ways. Firstly, although the diameter of the germ tube of the a conidium changes over its length, notably at the constriction, the diameter of the p conidium seems to remain constant from one end to the other. Sec- ondly, the diameter of a P conidium still attached to an a conidium is less than that of a free p conidium. This could be explained if P conidia remain at- tached when immature or if P conidia were capable of subsequent enlargement once separated from the a conidium. In V. dahliae, the term 'secondary spore' is applied to a conidium whose initiation is iterative or dual. The term 'iterative germination' is applied to a germination in which the germ tube itself becomes the conidiophore giving rise to the secondary spore (Mangemot and Reisinger 1976). The term 'dual germination' is applied to the situa- tion in which a conidium gives rise to two germ tubes, but the second can only be initiated after the first has differentiated into a conidiophore giving rise to the secondary spore (Buckley et al. 1969) or an appressorium (Swinburne 1976). The secondary sickle-shaped structures, which we have observed, appear to arise directly from the conidium; the germ tube and sickle-shaped structure also seem to extend simultaneously. Therefore, the term 'sec- ondary spore' does not seem to be appropriate to the sickle-shaped structure. In our opinion, if the sickle-shaped, free-floating structures are, in fact, an alternative spore form produced by V. dahliae, then that spore form should be referred to as a P spore and the normal conidial form should be re- ferred to as an a spore.

Acknowledgments Grants from the National Research Council of

Canada to L. V. Busch, R. L. Peterson, and B. C. Lu are gratefully acknowledged. The authors thank Betsy Smith for her technical assistance and Dr. George L. Barron for helpful review of the manu- script.

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BKISSON ET AL. 1465

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