Physiological and Molecular Plant Pathology (1989) 35, 357-366

In vitro formation of haustoria of the cowpea rustfungus, Uromyces vignae, in the absence of a livingplant cellI . Light microscopy

MICHÈLE C. HEATHBotany Department, University of Toronto, 25 Willcocks Street, Toronto, Ontario M5S 3B2, Canada

(Accepted for publication June 1989)

Haustoria of the cowpea rust fungus were induced to form on oil-containing collodion membranes,in the absence of any living plant material, by the application of an inactivated commercialenzyme preparation . No neckband was detected along haustorial necks and mithramycin stainingshowed that nuclei did not complete their migration into haustoria from the haustorial mothercells (HMCs) . Haustorial walls fluoresced only slightly with calcofluor and at an intensity muchless than walls of the infection structures or haustorial mother cells . Walls of germ tubes,appressoria, infection pegs, substomatal vesicles, infection hyphae, HMCs and haustoria allfluoresced uniformly but weakly when treated with (FITC)-Concanavalin A, indicating thepresence of mannose, glucose or acetyl glucosamine residues of the fungal surface ; fluorescence wasvirtually eliminated by prior treatment of the fungus with unlabelled lectin or by incubating thelectin with its hapten . With FITC-WGA, walls of germ tubes, appressoria, the thickened regionof the HMC wall surrounding the infection peg and the haustorial neck wall all fluoresced morebrightly than other parts of the fungus . Although fluorescence could be eliminated by mixing theFITC-WGA with chitin hydrolysate, the ability of unlabelled WGA to reduce subsequentbinding of FITC-WGA to appressoria and HMCs depended on the concentration of the labelledlectin and in no instance was haustorial wall fluorescence significantly reduced . These and otherdata suggest that FITC-WGA binding to the haustorium may not indicate the presence of chitin .

INTRODUCTION

Many biotrophic fungal plant parasites are characterized by their formation ofdeterminate intracellular structures known as haustoria [1] . Although the degree ofgrowth exhibited by these fungi in the absence of a living host plant variesconsiderably, until recently there had been no reports of indisputable haustoriadeveloping in the absence of a plant cell . Cytological and cytochemical studies ofhaustoria have been limited, therefore, to studies of these structures in planta [e .g . 2-5],with the accompanying problem of distinguishing fungal from plant material at thehost-parasite interface . In this report, evidence for the in vitro formation of haustoriaof the cowpea rust fungus, U. vignae Barcl ., is presented . Therefore, it is now possible toexamine intact and undamaged haustoria in the absence of the host . The present paperalso describes some morphological aspects of in vitro haustorium formation and reports

Abbreviations used in text : Con A, Concanavalin A ; FITC, fluorescein isothiocyanate ; HMC, haustorialmother cell ; WGA, wheat germ agglutinin .

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on the presence of lectin and calcofluor binding sites on and in the haustorial wall asrevealed by light microscopy .

MATERIALS AND METHODS

Induction of haustoriaWashed urediospores [9] of U . vignae were inoculated, using an air stream, onto thesurface of oil-containing collodion membranes prepared in glass Petri dishes [11] andthe membranes were then sprayed lightly with sterile, double distilled water . Thedishes were sealed with parafilm, wrapped in aluminium foil, and incubated at 20-22 °C for 6-7 h . The membranes were then sprayed with a solution of 5 mg ml -1 driselase(Sigma) that had been incubated in a boiling water bath for 30 min (to inactivate wall-degrading enzymes) and centrifuged at 500 g for 10 min to remove any precipitate .All solutions were sprayed onto the membranes in a sterile hood using a 3 ml sterileplastic syringe with a 30 guage needle. In some cases, the driselase was ejected througha sterile filter (0 . 2 Itm pore size) ; however, relatively few bacteria were observed on themembranes when the fungus was harvested at 27 h even when a sterile filter was notused. After driselase treatment, the dishes were sealed and wrapped as before andincubated further until 27, 30 or 48 h after inoculation .

Mithramycin staining of nucleiAt 30 or 48 h after inoculation, drops of 5 % glutaraldehyde in 0 .07 M potassiumphosphate buffer, pH 6 .8, were added to the membrane surface and left for 10 min .The underlying membrane pieces were then excised with a spatula and mounted onglass sides in 200 µg/ml-1 mithramycin (Pfizer Inc . New York, U .S.A .) in 300 mmMgCl, [8] . The fungus was examined with a Reichert Jung Polyvar microscope usinginterference contrast optics . The yellow fluorescence of nuclei was detected byepifluorescence microscopy using blue light irradiation (exciter filter BP 450-495,dichroic mirror DS 510, barrier filter LP 520) .

Calcofluor bindingPieces of membrane were mounted on glass slides in 1 % calcoflour white ST(American Cyanamid Co.) . The fluorescence of haustoria and infection structures wasdetermined by epifluorescence microscopy using ultraviolet irradiation (exciter filterBP 330-380, dichroic mirror DS 420, barrier filter LP 418) .

Lectin bindingFor wheat germ agglutinin (WGA) binding, FITC-WGA (Sigma) was prepared in0 .01 M potassium or sodium phosphate buffer, pH 6 . 8, containing 0 . 15 M NaCl. Unlessmentioned as otherwise, membrane pieces were havested at 27 h after inoculation andfloated fungus side down on a 1, 0 . 5 or 0 .2 mg ml-1 FITC-WGA solution for 5 min .After rinsing in two changes of the phosphate buffered saline, the membrane pieceswere mounted in the same solution. The specificity of the lectin binding was examinedby floating membrane pieces either on a similar solution of unlabelled WGA (Sigma)prior to FITC-WGA treatment, or by mixing the FITC-WGA solution with an equalvolume of chitin hydrolysate [7] suspension 10 min prior to adding the membrane .

In vitro formed U. vignae haustoria

359For concanavalin A (Con A) treatment, FITC-Con A (Sigma) (1 mg ml -1 or

0 .5 mg ml-1 ) was prepared in 0.01 M sodium phosphate buffer, pH 6.8, containing0 . 01 M NaCl and 0.001 M each MnCl2 , MgCl,, and CaCl2 . Membrane pieces werefloated on the FITC-Con A solution, rinsed, and mounted as described for theFITC-WGA. The specificity of the binding was examined by floating the membranefor 5 min on a 1 mg ml -' solution of unlabelled Con A (Sigma) before FITC-Con Atreatment and by adding the FITC-Con A to an equal volume of 40 mg ml -1 a methyl-D-mannoside (Sigma) in the same buffered salt solution 10 min before adding themembrane .

In all cases, at least ten haustoria were examined per treatment . Fungal fluorescencewas observed under blue light epifluoresence irradiation .

RESULTS

Haustorium morphologyOn oil-containing collodion membranes, urediospore-derived germs tubes of U. vignaedifferentiate to sequentially form an appressorium, an infection peg, a substomatalvesicle, an infection hypha and a haustorial mother cell (HMC) [11, 12] (Fig . 1) .Fortuitously, it was found that the supernatant from a boiled, enzymatically inactive,preparation of driselase (a crude preparation of fungal-derived enzymes that degradeplant cell walls) induced up to 30% of HMCs to form a haustorium-like structure ifapplied at about 6 h after inoculation . Like young haustoria formed in susceptibleplants [10], haustoria on membranes consisted of a neck and a terminal, almostspherical body (Figs 1,3,7,9,10) . In most cases, these haustoria appeared identical insize, shape, and site of origin on the HMC to haustoria formed in the plant [9],except that some haustorial necks widened as they joined the HMC (Figs 4,5) . Carefulfocussing of the microscope revealed that in such cases, the penetration peg thattraversed the HMC wall was no wider than when the neck was of uniform width (Fig .5) . Even by 48 h after inoculation, no sign was seen of any structure interpretable asa neckband [15] (Fig. 11), although these can be detected using the same optics onhaustoria observed in cleared leaves (Heath, unpublished work) . As in infected leaves[10], the HMC became increasingly vacuolate as the haustorium developed and a largevacuole was commonly observed in the haustorial body by 48 h after inoculation (Fig .11) . By this time, each fungal individual with a haustorium also had a secondary hyphaattached either to the substomatal vesicle or, more commonly, to the region of theinfection hypha adjacent to the HMC septum (as in Figs 1,9) . These varied in lengthfrom 5 to 90 gm, as did secondary hyphae in individuals that lacked haustoria. By48 h after inoculation, most HMCs lacking haustoria appeared dead as describedpreviously [12] .

Mithramycin stainingWhen treated with mithramycin at 30 h after inoculation, over 90% of HMCscontained four small, brightly fluorescent nuclei ; only two nuclei were observed in theremainder. Of HMCs with haustoria, 96 % contained four nuclei (Figs 1,2) . No nucleiwere seen in the haustorial neck or body . At 48 h after inoculation, two of the 20haustoria observed had a single nucleus in the haustorial neck . The secondary hypha

360 M . C . Heath

FIGS 1 and 2 . A germinated spore (SP) that has formed an appressorium (A), substomatalvesicle (S), infection hypha (IH), haustorial mother cell (HMC), haustorium (arrow) andsecondary hypha (2 °H) ; treated with mithramycin and viewed under differential interferencecontrast (DIC) microscopy (Fig . 1) or LJV irradiation (Fig . 2) . Note that three of the four brightlyfluorescent nuclei can be seen in the HMC and two larger, less fluorescent nuclei (arrow, Fig . 21are in the infection hypha . No nuclei were observed in the haustorium . Two nuclei are detectablein an ungerminated spore (upper right) . x 454 .

FIGS 3-6 . Haustorium (H) and haustorial mother cell (HMC) mounted in calcofluor andobserved under DIC microscopy at different focal levels (Figs 3-5), and under t; V irradiation(Fig . 6) at the same focal level as in Fig . 5, Note the swelling of the haustorial neck (arrow, Fig .4) where it joins the HMC, and the much smaller diameter of the penetration peg (arrow, Fig .5) which traverses the HMC wall . The haustorium and the thickened region of the HMC wallsurrounding the penetration peg do not detectably fluoresce (Fig . 6) although a slight fluorescenceof the haustorial wall was detected visually . x 2221

In vitro formed U. vignae haustoria

361usually contained four nuclei while the infection hypha sometimes appeared vacuolateand anucleate . However other similarly vacuolate infection hyphae contained twolarge nuclei (Fig . 2) ; often these showed only faint diffuse fluorescence as if they werein the process of being degraded . Similarly faint fluorescence was observed in the nucleiof moribund HMCs that lacked haustoria ; some of these HMCs lacked detectable nucleialtogether.

Calcofluor bindingAs observed previously [12], calcofluor fluorescence of appressorial and haustorialmother cell walls was greater than that of the walls of germ tubes, substomatal vesicles,or infection hyphae. Haustoria originated from the circular non-fluorescent area of theHMC previously concluded to be the thickening of the HMC wall through which thehaustorial penetration peg normally develops [12] (Fig . 6) . Those haustoria thatconsisted of a neck without a terminal body generally did not fluoresce . Haustoria withbodies fluoresced very slightly (undetectable in Fig . 6) and at a much lower intensitythan infection hyphae .

Lectin bindingAll infection structures and haustoria fluoresced after FITC-Con A treatment ; the wallof appressoria and HMCs fluoresced with approximately the same intensity as those ofgerm tubes, substomatal vesicles, infection hyphae and haustoria (Figs 7,8) . Fluorescencewas similar irrespective of whether I or 0.5 mg ml -1 solutions were used and regardlessof the length of incubation (5 or 40 min) . Treating the fungus with unlabelled Con Aprior to FITC-Con A treatment totally prevented any fluorescence of the fungal walls,including those of the haustoria . Adding a methyl-D-mannoside to the FITC-Con Asolution before adding the fungus similarly eliminated any wall fluorescence .

With FITC-WGA, fungal structures fluoresced more strongly than with the sameconcentration (w/v) of FITC-Con A, with germ tubes and appressoria fluorescingmore strongly than other infection structures (Figs 9,10) . Walls of HMCs oftenexhibited a wide band of slightly brighter fluorescence than seen in the rest of the HMC(Figs 10,12) which sometimes correlated with an underlying region of nonvacuolatecytoplasm (Figs 11,12) . Many haustorial mother cells that lacked haustoria exhibiteda brightly fluorescent ring in the region of the wall contacting the collodion membrane(Figs 13,14) . This ring was similar in diameter and position to the non-fluorescent wallthickening revealed after calcofluor staining [12] (Fig . 6) . The central non-fluorescentregion in the lectin-treated material was similar in diameter to that of the penetrationpeg .

Haustorial walls generally fluoresced brightly in FITC-WGA regardless ofconcentration, with the neck often exhibiting brighter fluorescence than the body (Figs11,12) . Adding chitin hydrolysate to the labelled lectin before adding the fungus totallyeliminated all fungal fluorescence . In contrast, variable results were obtained followingprior treatment of the fungus with unlabelled WGA. One to 25 mg ml-1 of theunlabelled lectin applied for up to 30 min had little or no effect on the subsequentfluorescence produced by incubation in 1 mg ml -1 FITC-WGA. A reduction in thefluorescence following treatment with a 10 mg ml -1 unlabelled WGA solution for 5 minwas observed only when the labelled lectin solution was diluted with phosphate

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FIGS 7-14 . For legend see opposite .

M. C. Heath

In vitro formed U. vignae haustoria

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buffered saline to about 0 .2 mg ml- ' . Such a reduction in fluorescence intensity wasdetectable only for germ tubes and appressoria ; the fluorescence of infection hyphaeand haustoria seemed little changed .

DISCUSSION

This investigation has confirmed that the haustoria-like structures of U. vignae formedon collodion membranes are indeed true haustoria, since they develop from thethickened region of the HMC wall as do the haustoria formed in the plant . These latterhaustoria develop neckbands just prior to the migration of nuclei and cytoplasm fromthe HMC into the haustorium [10] . However, no neckbands were detected in thehaustoria formed in vitro and although nuclear migration began in a few haustoria, itwas not completed, even by 48 h after inoculation . Given that the first-formedhaustorium in the plant is usually fully formed by 24-36 h after inoculation it appearsthat some additional signals or nutrients are necessary to support further haustorialdevelopment in vitro .

Con A normally interacts specifically with a-o-mannopyranosyl and, to a lesserextent, a-D-glucopyranosyl and a-D-N acetylglucosamine residues [14] . Such specificityapparently was exhibited by the FITC-Con A with respect to binding to fungalinfection structures and haustoria since all fluorescence was virtually eliminated aftermixing the labelled Con A with a methyl-n-mannoside or after pretreatment of thefungus with unlabelled Con A . Since the Con A protein is probably too largemolecular mass over 1000001 to penetrate far into the fungal walls, the observations

of uniform, low fluorescence, of the walls of all fungal structures, including haustoria,suggest that they all hear mannose, glucose, or acetyl glucosamine residues on theirouter surfaces . This observation is of interest given that the outer layer of the haustorialwall is continuous with an inner wall layer of the HMC, and although the outer wallsoft he HMC, infection hypha, substomatal vesicle, and infection peg are all continuous,they develop from the inner wall of the appressorium [15] . Such observations imply achange in wall structure as the fungus enters the leaf during the formation of theinfection peg, and as it forms a haustorium [15] . Notwithstanding such changes,however, with respect to Con A binding sites, the outer surface of germ tubes, infectionstructures and haustoria seem remarkably uniform . A low level of fairly uniform

Fins 7 and 8 . Haustorium i H haus(orial mother cell !HcMC) and infection hypha (LH) treatedwith FITCCon A and viewed under DIC microscopy Fig 7 and blue light irradiation (Fig . 8j,x 2221Fins 9 and 10 . Fungal structures (legend as for Fig . I , treated with FITC-WGA and viewed

under DIC microscopy (Fig . 9'9 and blue light irradiation (Fig . 10,! . Note the stronger fluorescenceof the walls of the germ tube 'GT, appressorium and parts of the haustorial mother cell andsecondary hypha as compared to the walls of the haustorium !arrow : . x 507

Fins 11 and 12 . Vacuolate haustorium iH and haustorial mother cell (HMC) treated withFITC-AVGA and viewed under DIC microscopy (Fig . I 1 ; or blue light irradiation (Fig . 12) . Notethe strong fluorescence of the haustorial neck wall and the regions of the HMC wall overlying thenote% acuolate regions of the cytoplasm . x 2519

Fins 13 and 14. Haustorial mother cell treated with FITCAVGA and viewed under DICmicroscopy Fig . 13, or blue light irradiation fFig . 14i at the fungus-collodion membraneinterface . Note the bright fluorescent ring corresponding to the thickened region of the HMC wallsurrounding the penetration peg (arrow ; . x 2221

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M. C. Heathfluorescence also has been reported for infection structures, lacking HMCs orhaustoria, of the bean rust fungus, U. appendiculatus after treatment with FITC-Con A[17] . Chong et al . [5] have shown in an ultrastructural study of infected plants thatgold-labelled Con A binds to the outer wall layer of the young haustorium and theHMC of the wheat stem rust fungus, Puccinia graminis Esp . tritici. Different species of rustfungi, therefore, appear rather similar in terms of Con A binding sites on the surfaceof infection structures and haustoria .

Previous ultrastructural studies of P. coronata Esp . avenae [3] and P. graminis Esp . tritici[5] in host plants have shown that gold-labelled WGA binds to the HMC wall,including the thickened region surrounding the penetration peg ; however it does notbind to the wall of the young haustorium . These observations were interpreted toindicate that the walls of the young haustorium lack chitin, a point that may besignificant in terms of the recognition (or lack of it) of the haustorium by the host cell .That haustorial walls of U. vignae similarly lack chitin is suggested by the low incidenceof binding sites for calcofluor, a fluorochrome often used to detect cellulose and chitinmicrofibrils as well as other glucans [13] . However, it must be pointed out that thethickened region of the HMC wall surrounding the haustorial penetration peg of U.vignae also does not fluoresce with calcofluor [12] despite the binding of gold-labelledWGA to thin sections of this region in Puccinia spp. in planta [3,5] and the indicationfrom a colorometric assay that this thickening in U. vignae HMCs contain chitin [12] .It is possible, therefore, that the low level of calcofluor binding to the haustorial wallmay be due to the exclusion of the brightener or the masking of binding sites as mayoccur in the thickened region of the HMC wall [12] .

The presence or absence of chitin in the haustorial wall was not further elucidatedby the application of FITC-WGA. This lectin generally is assumed to interactspecifically with polymerized acetyl glucosamine residues such as are found in chitin[14] . Previous studies have shown that on collodion membranes such as used in thepresent investigation, appressoria and germ tubes of U. vignae [17] and U. appendiculatus[16] fluoresce brightly after treatment with FITC-WGA, with less fluorescenceexhibited by substomatal vesicles and infection hyphae. The present study hasconfirmed this observation and shown that walls of HMCs and haustoria also bindFITC-WGA. However, at the same concentration (w/v) as used for FITC-Con A, thefluorescence of no part of the fungus could be inhibited by prior treatment withunlabelled lectin, even at concentrations that should have saturated all availablebinding sites . This observation suggests that at the concentrations of FITC-WGA used,the lectin binds to the fungus nonspecifically or may rapidly displace the unlabelledlectin from the fungus . At low concentrations of the FITC-WGA, fluorescence of thewalls of the appressoria and germ tubes were reduced, but not eliminated, by priortreatment with unlabelled lectin . The labelled lectin, therefore, may have been bindingto some specific sites associated with the walls of these infection structures, but none ofthe data prove that the FITC-WGA was binding solely to chitin . Binding patterns ofFITC-WGA that do not seem to reflect the presence of acetyl glucosamine residueshave been reported in plant tissue [6] and it has been suggested that charge, as well ashapten affinity, may influence binding [18] . The possibility, therefore, that WGA maybind to wall components other than chitin, and the fact that the fluorescence of thehaustorium was not detectably reduced by the unlabelled lectin treatment even though

In vitro formed U. vignae haustoria 365some infection structures did show reduced fluorescence under the same conditions,suggest that the haustorium fluorescence may not indicate the presence of chitin .Moreover, this study supports the claim [6] that the binding of FITC-lectins,particularly WGA, may be more complicated and less specific than usually assumed .Indeed, there appears to be no easily applied control to prove whether or not theobserved FITC-WGA binding can be attributed to the presence of chitin ; even priorremoval of chitin with purified chitinase may lead to the loss of other bindingmolecules. It is possible, given the lack of binding to haustoria noted in ultrastructuralstudies [3, 5], that the same problems of specificity may not apply to the gold-labelled,protein-complexed WGA used in such studies . However, the hapten-binding controlsused in light and electron microscope investigations of lectin binding to rust fungi[3-5,16,17] test only whether the hapten can compete with the fungus for lectinbinding sites ; they do not prove that the same hapten is involved in binding the lectinto the fungus . The present investigation suggests that neither calcofluor or FITC-WGAare totally reliable for the detection of chitin in fungal walls .

Using gold-labelled WGA applied to thin sections of haustoria of P . graminis f.sp .trilici formed in the plant, Chong et al . [4, 5] reported that only the inner layers of theintercellular hyphae and the HMCs become labelled . They also showed that theinterpolated additional wall layer (the annulus) surrounding the penetration pegbound both gold-labelled WGA and Con A [5] . If the distribution of binding sites issimilar in U. vignae, then the present observations suggest that the WGA protein,because of its relatively low molecular mass (36000), may penetrate to sites insidefungal walls rather than bind solely to their surfaces . In particular, penetration into thewall by FITC-WGA but not FITC-Con A would explain why only the formerrevealed the presence of the annulus . Why the annulus should bind FITC-WGA butnot calcofluor is still a question that needs further investigation .

This paper represents the first report of indisputable haustoria being formed in theabsence of a living plant cell . Thus, it seems that the latter is not mandatory for at leastthe early stages of haustorium formation . Moreover, the present work supports theearlier conclusion [12] that the lack of haustorium formation on collodion membranesis due to the premature senescence of the HMC that may be prevented by plant-derived factors. What these factors may be, and whether these are the samehaustorium-inducing factors present in the driselase solution used here, are underinvestigation .

I thank K . Chilikoff for excellent technical assistance, and K . Sault for printing themicrographs. This work was funded by the Natural Sciences and Engineering ResearchCouncil of Canada .

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MONSIGNY, M ., ROCHE, A., SENE, C ., MAGET-DANA, R . & DELMOTTE, F . (1980) . Sugar-lectininteractions : how does wheat-germ agglutinin bind sialoglycoconjugates? European Journal ofBiochemistry 104, 147-153 .