Staining Paraffin Embedded Sections of Scald of Barley before Paraffin Removal

K. Xi1 and P. A. Burnett2 'Agriculture and Agri-Food Canada, Lacombe Research Center, 6000 C 6; E Trail,

Lacombe, Alberta, Canada T4L 7 Wl and ILethbridge Research Center, Highway 3 East, P.O. Box 3000, Main, Lethbridge, Alberta, Canada T7J 487

ABSTRACT. Staining of paraffin embedded sec- tions with periodic acid-Schiff reagent and fast green before paraffin removal resulted in differ- entiation of barley seed and leaf tissue from fungal structures of Rhynchosporium secalk. Crystal violet, toluidine blue 0 and aniline blue also successfully stained fungal structures of R. secalis in barley leaf tissqes. Staining of embed- ded sections before paraffin removal allows sim- ple processing of a series of sections, saves time and reduces solvent consumption.

Key words: leaf infection, paraffin embedment, PAS reagent, Rhynchosporium secalis, scald of barley, seed infection

araffin embedding of infected plant material P allows easypreparationofaseries ofsections, making it possible to monitor the development of hyphae and fungal related structures. This methodgenerallyrequires removal ofthe paraffin beforethesectionedmaterialcanbe stainedwith dyes in aqueous or alcoholic solutions (Sass 1958). As a result, sectioned materials are sub- jected to hydration and dehydration after paraf- fin removal. This process is time-consuming, requires a large quantities of solvents and in- creases the risk of tissue damage. Sakai (1973) simplified the paraffin method by differentially staining plant materials using toluidine blue 0 withoutpriorremovalofparaffh. Maetal. (1993) also differentially stained plant materials using safranin and fast green before paraffin removal. In a histopathological study on blackleg of rape- seed, Xi and Morrall (1993) differentiated Lep-

Correspondence to: K. Xi, Agriculture and Agri-Food Canada, Lacombe Research Centre, 6000 C & E Trail, Lacombe, Alberta T4L 1 W1 Canada

BlOTECHNlC & HISTOCHEMISTRY Q by Williams & Wilkins 1052-0295/97/173-177 Volume 72, Number 4

tosphaeriamaculans (Desm.) Ces. 8rdeNot. from rapeseed (Brassica nap& L.) tissue by staining with toluidine blue 0 before removing paraffin.

Several researchers have conducted histolog- ical studies on the scald ofbarley (Hordeum uul- gare L.) caused by Rhynchosporium secalis (Oudem.) J. J. Davis. Ayesu-Offei and Clare (1 970) investigated the infection process in bar- leyleaves usingparaffin embedding. No detailed procedures were given for staining, making it unclear whether their embedded sections were stained before or after paraffin removal. After dewaxing, Doken (1988) stained infected barley leaf sections with periodic acid-Schiff (PAS) reagent and counterstained with methylene blue. Lehnackers and Knogge (1990) located fun- gal structures of R. secalisusinganimmunolog- ical procedure. Whole mount preparations were used to investigate seed infectionwithR. secaZis (Skoropad 1959, Kayand Owen 1973).

Successful staining of blackleg infected spring rapeseed before paraffin removal (Xi and Morralll993) has indicated that this method is potentially useful in histopathology, including studies on barley leaf infection with R. secalis. Furthermore, we wanted to determineifthis sim- plified procedure could be applied to microtomed seed sections to reveal the precise distribution of R. secalisinfection in barley seed tissue. The PAS stain has been the method of choice for demon- strating fungi in animal tissue (Kligman and Mescon 1950) because periodic acid causes chemical changes in fungi rich in polysaccha- rides. Periodic acid hydrolysis produces aldehy- des that combine with the colorless Schiff reagent, allowing a color reaction to develop (Sharvilll952). To develop a rapid protocol that could be efficiently used to assess samples, we tested the PAS reagent fordifferential stainingof sections of paraffin embedded barley leaves and

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174 Biotechnic & Histochemistry

seeds infected with R. secalis without removing paraffin. Several dyes were also used to uni- formly stain sections of paraffin embedded bar- ley leaves infected with R. secalis without removing paraffin.

MATERIALS AND METHODS Leaves ofbarley cv. Harringtonwith scald le-

sions were sampled from greenhouse grown plants 20 days after inoculation with an isolate ofR. secalis, that had been isolated from cv. Har- rington at the Agriculture &Agri-Food Canada (AAFC), Lacombe Research Center. Spikes of cv. Argyle grown in the greenhouse at growth stage 5 1-55 (Zadoks et al. 1974) were inoculatedwith the same isolate, and infected grains were harvested45 days after inoculation. The inocu- lation procedure was similar to that described by Xue et al. (1995). Harrington leaves with scald lesions and Argyle grains with lesions re- sembling 2-eyed symptoms (Skoropad 1959) were also sampled from experimental plots that were naturally infected by R. secalis at AAFC, Lacombe Research Center. The leaves were cut into pieces approximately 0.5 cm long and fixed in 3% glutaraldehydein 0.025 M sodium phos- phatebuffer(SPB), pH6.8, a t 4 C overnightorin a mixture oflactopheno1:methanol:chloroform (5:3:2, v/v) (Ayres and Owen 1971) for 24 hr at room temperature. After a thorough washing in SPB, the leaf samples were dehydrated bypas- sage through a graded ethanol series from 5 to 80%. The grains were cut into quarters and soakedin distilled water at 80 C for 2 hr, fixed in 70% ethanol for 48 hr (Agrawal et al. 1987) and dehydrated in 80% ethanol. The leafand grain materials were further dehydrated in a n ethanol/n-butanol series (Johansen 1940) and paraffin chips were added gradually to replace butanol. The tissues were embedded in Para- plast8 X-tra.

Transverse and longitudinal 10-15 pm sec- tions of leaf and grain tissues were cut per- pendicular to the leaf blade on a Tissue-Tek I1 microtome. Paraffin ribbons were affixed to slides with Haupt's adhesive (Jensen 1962) and dried on a hot plate at 40-45 C. A modification [Ayesu-Offei and Clare 1970) based on the PAS reagentwas used for differential staining ofbar- ley tissues infected by R. secalis where the sec- tions were immersed in 1% periodic acid for 5 min and washed in distilled water for 5 min. The leafsections were immersed in Schiff's reagent for 2.5-4.5 hr, depending on the amount of stainable tissues present in a section. Grain

sections were immersed for 2-3 min. The sam- ples werethen immersed in 10% aqueous potas- sium metabisulfite solution for 5 min and washed in distilled water for 5 min. Leafsections were stained in 0.5% fast green in 95% ethanol for3-7.5 hrdependingonthematerialandgrain sections were stained for 4.5 hr. Several dyes were used to stain individual sections from R. secalis infected barley tissue: 0.05% toluidine blueOinSPBfor 15-20min. 0.1Vocrystalviolet for 15 min, or 1% aniline blue for 3.5 hr. After washing in distilled water for 5 min, the sections were air dried, dewaxed in three changes of xy- lene and mounted in PemountB for light mi- croscopic examination. Photomicrographs were prepared with Kodak T-Max or Fuji color films using a Zeiss Axioskop and Olympus micro- scope with attached camera. The testwith each dye was repeated at least twice.

RESULTS AND DISCUSSION For leaf sections, differential staining with

PAS and fast green stained hyphae and spores of R. secalisred to purple, and the mesophyllgreen (Figs. 1-2). Occasionally, the cell wall stained red (Fig. 2) but this does not lead to any confu- sion in identifjmg the pathogen. For grain sec- tions, the PAS reagent and fast green stained hyphaeofR. secalisred to purple, thehusklayer green, and the pericarp layer red to purple (Fig. 3). Contrast is sufficient to differentiate the husk tissue and hyphae because hyphae were mostly observed between the husk and the peri- carp layer (Skoropad 1959, Kay and Owen 1973). The morphology and location of fungal hyphae were similar to those in a sectionwith ar- tificial inoculation (Fig. 4), confirming the pres- ence of fungal hyphae in the grain section sampled from the field. The optimum contrast in sectioned material was achieved by overstain- ingin decolorized basic fuchsin, then counter- stainingin fast green, withthelength ofstaining time being determined by monitoring the color density. Although plant tissues, hyphae and spores were uniformly stained with crystalvio- let (Figs. 5-6), toluidineblue 0 (Fig. 7) or aniline blue (Fig. 8), there was little difficulty in identi- fying subcuticular hyphae and spores because these structures are usually on or near the sur- face ofhost tissues. Among the dyes, crystalvi- olet and toluidine blue 0 appear to be better than aniline blue for the short period of staining required to obtain optimum color density. When fungal structures were in the host leaf tissues, such as in themesophyll due to heavy infection,

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Staining Paraffin Embedded Sections 175

Fig. 1 . Longitudinal section perpendicular to the blade of barley leaf infected by R. secalis stained with PAS and counter- stained with fast green. A stoma (St) in the upper epidermis of the section, and hyphae (h) in the rnesophyll cells (rn) collapsed as a result of heavy infection. x 230. Fig. 2. Transverse section of barley leaf infected by R. secalis stained with PAS and counterstained with fast green. A strorna (St) was in the upper epidermis and spores (5) and hyphae (h) were in the vicinity. X 690. Fig. 3. Section of barley grain with natural infection by R. secalis stained with PAS and counterstained with fast green. Hy- phae (arrows) were in the husk layer (HL) and colonized the pericarp tissue (p). X 430. Fig. 4. Section of barley grain artificially inoculated with R. secalis stained with PAS and counterstained with fast green. Hy- phae (arrows) were in the husk layer (HL). x 430.

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176 B iotechn i c & H istochern istry

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Fig. 5. Spores and subcuticular hyphae (arrows) of R. secalis in the upper epidermis of a transverse section of barley leaf stained with crystal violet. x 230. Fig. 6. Subcuticular hyphae (arrow) of R. secalis in a longitudinal section perpendicular to the blade of barley leaf stained with crystal violet. x 230. Fig. 7. Sporulation (arrow) of R. secalis in the upper epidermis of barley leaf stained with toluidine blue 0. X 230. Fig. 8. Subcuticular hyphae (arrow) of R. secalis in a longitudinal section perpendicular to the blade of barley leaf stained with aniline blue. X 460.

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Staining Paraffin Embedded Sections 177

PAS and fast green were superior for differenti- ating hyphae and spores from host tissues.

In addition to saving time and reducing solvent consumption, Maet al. (1 993) indicated that the risk of tissue damage was reduced or eliminated by stainingsections before paraffin removal. Ex- perience in our laboratory has shown that PAS stainingafterremovalofparaffin, followedbyre- peated hydration and dehydration, sometimes caused dewaxed tissue to detach from the slide because Haupt’s adhesive is dissolved by aque- ous solutions. The method with differential or uniform staining described in the current study allows alarge number ofsections to be processed, and easier detection and location of R. secalisin barley. The same method with toluidine blue 0 resulted in distinct, contrasting colors between rapeseed tissue and L. maculans making it pos- sible to detect latent infection and to examine hy- phal development (Xi and Morrall 1993). Toluidine blue 0, however, cannot differentiate tissues using the conventional method because the dye is highly soluble alcohol and metachro- masia is lost (Sakai 1973). I t is noteworthy that scald of barley and blackleg of rapeseed repre- sent different modes of fungal infection. The former is characterized by the growth of subcu- ticular hy-phae in the leaf(Ayesu-Offei and Clare 1970) and the latter by systemic latent infection from the petiole to the basal stem (Hammond et al. 1985). Successful staining of sections from the two fungal diseases suggests that staining before paraffin removal may be useful for study- ing other host-pathogen systems.

ACKNOWLEDGMENT We gratefully acknowledge the financial sup- port from the National Science and Engineering Research Council of Canada through Visiting Fellowships in Canadian Government Labora- tories and Alberta Barley Commission.

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