CUTICLE, CELL WALL ULTRASTRUCTURE ANDDISEASE RESISTANCE IN MAIDENHAIR EERN
BY K. J. ARCHER* AND A. L. J. COLE
Department of Plant and Microbial Sciences, University of Canterbury,Christchurch, New Zealand
{Accepted 10 January 1986)
SUMMARY
Light and scanning electron microscopy showed the pinnules of maidenhair fern, Adiantumraddianum var. raddianum (Hoshizaki), possess hydrophobic, crystalline deposits overlying awell developed cuticle on both abaxial and adaxial surfaces. In response to attempted infectionby Botrytis cinerea (Pers. ex Fr.), browning, cell wall thickening and papilla formation occurred.
Transmission electron microscopy revealed infection pegs are surrounded by electron-densepapillae forming partial barriers to further colonization.
There is a paucity of recorded diseases on ferns (Moore, 1959; Dingley, 1969;Pirone, 1970; Westcott, 1971) and ahhough this may reflect a lack of research itmay also indicate that ferns exhibit a marked resistance to pathogenic attack.
Cultivated ferns grown under glass have received more attention from patho-logists than their wild counterparts because of their greater economic importancebut, in general, even these appear remarkably free from disease problems(Hoshizaki, 1979). Swain (1980) reported ferns to be immune from both pathogenicand herbivorous attack. Botrytis cinerea (Pers. ex Fr.), Phythium sp., and Fusariumsp. have caused problems on young cultivated ferns in local nurseries but do littledamage to older plants. This report concerns an investigation of disease resistancein ferns using the horticulturally important maidenhair fern, Adiantum raddianumvar. raddianum (Hoshizaki) and a common phytopathogen, Botrytis cinerea, whichhas been recorded (Dingley, 1969) as causing a grey mould of Adiantum in NewZealand.
Many mechanisms have been proposed to explain the resistance of higher plantsto pathogens. In ferns this is not the case although it has been suggested that anumber of secondary compounds which exhibit antimicrobial activity could beimplicated (Wills, 1956; Challenger et al., 1957; Nilsson, 1959; WoUenweber,1978; WoUenweber & Dietz, 1981).
The obvious hydrophobicity of Adiantum pinnules and possible importance asa disease escape mechanism led to this study of pinnule surface topography andpinnule structure. Attempted infection by B. cinerea was followed using light andtransmission electron microscopy.
* Present address; School of Forestry, University of Canterbury, Christchurch, New Zealand.
Plant materialYoung sporophytes oi A. raddianum were potted in a soil mixture containing
equal parts of sterile peat, leaf compost and sand. The plants were grown underglass away from direct sunlight. Vigorous growth was maintained by monthlyapplications of liquid NPK (Phostrogen, Shell Chemicals Ltd.) and occasionaladditions of dolomite and urea.
InoculationPlant material was inoculated with spore suspensions of B. cinerea. Sterile,
double glass distilled water containing 0'05 % v/v 'Tween 80' surfactant waspoured onto a sporulating culture. The resulting suspension was filtered throughseveral layers of the fine muslin and the filtrate centrifuged at 1000 g' for 5 min. Thesupernatant was then removed and the spores resuspended in fresh, sterile, doubleglass distilled water to give a final concentration of 20000 spores per ml. Pinnuleswere inoculated by placing \0 /A droplets of spore suspension onto the pinnulesurface.
Where spores were suspended in a nutrient solution this was prepared accordinglyto Hargreaves et al. (1976) and contained sucrose, cas-amino acids, KHjPO^ andMgSO,.
Detached leaf cultureMost experiments were conducted using detached Adiantum pinnules floating
on kinetin solution (20 mg 1~̂ ) in 5 cm petri dishes. Twenty such dishes wereplaced on wet newspaper in plastic trays. The trays were illuminated at 21 °C underconstant photon flux of 5 /lE m~^ s~^ from fluorescent tubes.
Infection studiesInoculated pinnules were removed from incubation after 24 h. The opacity
of the leaves made microscopic observation of spores difficult — a leaf clearingtechnique adapted from that described by Ryan & Clare (1974) was used.Inoculated pinnules were placed on a sheet of Whatman No. 1 chromatographypaper and a decolourizing solution, Carnoy's fluid (Dey, 1919), was allowed todescend the paper carrying chlorophyll pigments with it. Fungal inocula were notdislodged with this technique, and cleared whole pinnules with attached sporeswere stained with lactophenol cotton-blue and examined with conventional brightfield microscopy.
Optical microscopyPinnules were prepared for optical microscopy by fixation in formalin-acetic
acid followed by dehydration in an ethanol-tertiary butyl-alcohol series and finallyembedded in 'paraplast' wax. Sections (3 to 10 fim) were cut on a Jung rockingmicrotome and stained by the periodic acid-SchiiT technique (Preece, 1959) whenfungal material stained bright pink and host tissue green-blue. Suberized materialwas visualized with Sudan III stain and 'lignified' tissues using a thionin orange'G'/erythrosin/light-green stain (Margolena, 1932).
Resistance of Adiantum to Botrytis 343
Scanning electron microscopySolvents used in standard SEM dehydration procedures have been shown to
afTect the presence and composition of leaf surface waxes (Baker & HoUoway, 1971;Reed, 1982), therefore, small portions of fresh pinnules were attached to aluminiumstubs with double sided cellotape, coated with gold in a Polaron sputter coater andexamined immediately in either a Cambridge Stereoscan 600 at 15 kV or aJEOL35 SEM at 10 kV.
Transmission electron microscopyPinnules were removed from healthy plants and immersed in 0-025 M phosphate
buffer pH 7-2, cut into small segments and immediately fixed in 5 % v/v gluta-raldehyde in 0-025 M phosphate buffer for 4 h at 4 °C. After two washes in freshbuffer the specimens were dehydrated in an acetone series and then infiltrated withan acetone - Spurr's (1969) resin mixture on a rotary mixer for 5 d under vacuum.Following infiltration, the specimens were placed in fresh Spurr's resin and leftfor 4 d to ensure the complete removal of acetone and they were then embeddedin a further change of fresh Spurr's resin. Ultra-thin sections were cut on an LKBultratome and mounted on formvar coated copper grids. Sections were stained withuranyl acetate in 50% ethanol for 20 min then momentarily stained with leadcitrate (Sato, 1967) and examined with an Hitachi HS7-S microscope at 50 kV.
RESULTS
Pinnule structureWhen young or mature pinnules were immersed in water they were found to
be strongly hydrophobic. The presence of extensive crystalline aggregations wereevident using S.E.M. These deposits were evenly spread on both pinnule surfacesbut were most prolific on mature adaxial surfaces [Fig. l(a) to (d)].
In transverse section, under light microscopy, the pinnule showed a simplestructure. The upper and lower epidermal surfaces were usually in direct contactbut in some cases one, or at most two, layers of mesophyll were present. Whenstained with Sudan I l i a distinct orange layer could be discerned on the outermostsurface indicating the presence of lipidic material [Fig. l(e)].
Transmission electron microscopy of the adaxial surface of mature pinnulesrevealed a thin, translucent epicuticular layer overlying a complex cuticularmembrane. Using the scheme of Wattendorf & HoUoway (1980) it was possibleto distinguish a distinct outer cuticle proper and an inner cuticular layer with alamellate reticulate substructure [Fig. l(f)]. The intense staining reaction withosmium suggests a high lipid content (Hayat, 1970). The abaxial epidermis inmature pinnules showed a similar structure to that of the adaxial surface [Fig. 2(a)].In contrast, the outer cell layers on both the adaxial and abaxial surfaces of youngpinnules were much thinner and lacked the well defined osmiophilic zone[Fig. 2(b)].
Infection of pinnulesWithin 4 h of inoculation of the upper surfaces of pinnules, 99 % of B. cinerea
conidia germinated and produced a short (8 /im) germ tube which usually swelledat the tip to form an appressorium prior to penetration. However, penetration alsooccurred directly from germ tubes without appressorial formation. When nutrientswere added to the infection droplet prior to germination an increased number of
344 K. J. ARCHER AND A. L. J. COLE
Fig. 1 (a) to (d). Scanning electron micrographs of pinnule surfaces showing wax deposits, (a)Mature pinnule, adaxial surface (b) Mature pinnule, ahaxial surface (c) Young pinnule, adaxialsurface (d) Young pinnule, abaxial surface, (e) Light micrograph showing a cross section of a maturepinnule, stained with Sudan IH. Note the accumulation of stain (arrowed) in the outer wall layer,(f) Transmission electron micrograph of the outer wall layer from the adaxial epidermis of a maturepinnule. Ahhreviations: w, epicuticular wax layer; cp, cuticle proper; cl, cuticular layer; cw, cell
wall; Material Hxed in glutaraldehyde/OsOj and stained with uranyl acetate/lead citrate.
Resistance of Adiantum to Botrytis 345
(a)
Fig. 2. Transmission electron micrographs of ultra-thin sections of pinnules of Adiantumraddianum. (a) Outer epidermal wall layer of a mature pinnule, (b) Outer epidermal wall layerof a young pinnule, (c) Early stages of cell wall thickening induced in the abaxia! epidermalcell wall of a young pinnule, (d) Attempted penetration of the abaxial epidermis of a youngpinnule by Botrytis cinerea. (e) The interface between B. cinerea and the Adiantum pinnuleseen in (d). (0 The interaction of B. cinerea ana the abaxial epidermis of a young pinnule close
to a cell wall junction.
Abbreviations: A, appressorium; cp, cuticle proper; cw, cell wall; cl, cuticular layer; few,fungal cell wall; g, golgi body; gm, granular material; ip, infection peg; m, mitochondrion;ocw, original cell wall; pi, plasmalemma; p, papilla; tew, thickened cell wall. All specimens
fixed in glutaraldehyde/OsOj and stained with uranyl acetate/lead citrate.
346 K- J- ARCHER AND A. L. J. COLE
appressoria formed. They usually formed above the anticlinal walls betweenadjacent epidermal cells. A greater number of attempted penetrations wereobserved on young pinnules, where the cuticle was much thinner, than on maturepinnules [c.f. Fig. 2(a), (b)].
At the point of penetration the appressorium appeared to be surrounded bymucilage which may have functioned to attach it to the cuticle. A circularpenetration hole was formed under the appressorium by the penetration peg, thesmooth outline of which is indicative of an enzymatic process having occurred.
Considerable discolouration and thickening of the wall was evident within 4 h ofits being challenged by an appressorium or germ tube [Fig. 2(c)]. The thickenedregion under the appressorium was more osmiophilic than the original cell walland appeared to consist of an electron translucent layer, similar in appearance tothe original cell wall, and an electron dense layer which was almost twice as thickas the original cell wall. Cytoplasmic organelles aggregated beneath the thickenedregion. The abundance of mitochondria, vesicles and endoplasmic reticulumsuggest it was a site of intense metabolic activity.
The degree of browning and papilla size increased up to 24 h after infection.Figures 2(d), (e) and (f) show an appressorium in close contact with the lowerepidermal wall of a young pinnule, with the epidermal wall forming a dome shapedpapilla protruding into the cell lumen but remaining external to the plasmalemma.The shape of the induced papilla was variable but it commonly took the form of atapered cylinder completely surrounding the infection peg. Papillae usuallymeasured 4 to 5 /iva at the base and protruded 2 to 3 /im into the cell lumen. Wallthickenings and papillae gave positive reactions to Sudan III and Margolena'sstain indicating the possible presence of suberized and lignified material.
The fungus appeared unable to break the papillar barrier except where a hostcell was subjected to five or six simultaneous penetrations. In this case the cell waskilled and a progressive rot developed. Detached pinnules, killed by immersionin boiling water for 24 s or by exposure to chloroform vapour for 24 h, showedno evidence of papilla formation.
D I S C U S S I O N
The fronds of A. raddianum appear to have several structural features which maycontribute to resistance to infection by pathogens such as B. cinerea.
Both abaxial and adaxial pinnule surfaces possess hydrophobic, crystalline, waxydeposits overlying a well developed distinct cuticle. The presence of cuticularwaxes in ferns has seldom been reported and a discrete cuticular membrane,structurally similar to that found in higher plants, has been established incomparatively few lower plants. Wada & Staehelin (1981) reported a multilayeredlipid-like coat on the outer layers of Adiantum capillus veneris (L.) gametophytes.In New Zealand, Hall & Burke (1974) showed that Blechnum capense (L. Schecht.)had little or no wax deposits, and in Cyathea dealbata (Forst. f. Swartz) wax wasconfined to the abaxial pinnule surfaces only. Thick cuticle and waxy layers havebeen shown to be structures important for disease 'escape' in a number of plants.Disease 'escape' is not truly a resistance mechanism because the fungus does notchallenge the host, i.e. resistance is not put to the test (Wood, 1967). Possessionof an intrinsic 'escape' mechanism such as wax deposition is important, however,because it is to the plant's advantage if a potentially parasitic fungus can beprevented from staying on the plant long enough to penetrate the tissues.
Resistatice of Adiantum to Botrytis 347
The hydrophobic nature of the fronds would also present an unfavourableenvironment to Botrytis spores since free water, or high humidity, is required forgermination and infection (Blakeman, 1980). Hydrophobicity of plant surfaces hasbeen previously implicated in disease escape mechanisms; Jennings (1962) relatedthe resistance of raspberry canes to Botrytis to their waxy coatings and Heather(1967) suggested that the resistance of Eucalyptus bicostata to Phaeoseptoriaeucalypti was related to the thickness of the waxy cuticle and to the resulting poordeposition of spores.
A well developed cuticle is present on both pinnule surfaces of A. raddianum.There is little record of the presence of cuticles in ferns. Priestly & RadclifTe (1924)claimed that ferns were largely ' indifferent" with respect to cuticle formation andin those ferns that possessed a cuticle it was generally confined to the region aboveanticlinal walls between adjacent epidermal cells.
The difference in thickness between young and mature cuticles in Adiantum isreflected in the reduced number penetrations in mature pinnules where the cuticleis thick. In terms of purely mechanical penetration, the thickness and hardnessof the combined cuticle and cell wall would govern the ease of penetration (Marks,Berbee & Riker, 1965; Jhooty & KcKeen, 1965). However it is likely thatpenetration is mediated to a large extent by enzymes. A number of studies(Verhoeff & Warren, 1972; Verhoeff", Warnaar & Schijven, 1979; Verhoeff, 1980)have shown that B. cinerea produces cutin degrading enzymes in vivo and in vitro.As a response to challenge by B. cinerea, browning, cell wall thickening and papillaformation occurs. These are relatively common responses of plants to pathogens(Kuc, 1972; Matta, 1982). Many correlations exist between the production ofpapillae and resistance to fungal penetration (Aist, 1976). In Adiantum the papillaeare formed rapidly by living tissue and once formed the infection hypha iseffectively contained except where several penetrations of a cell occur.
There is still confusion concerning the chemical composition of papillae butcallose, lignin and suberin appear common constituents (Ride, 1983). Althoughthe role of papillae in resistance to pathogens is still open to conjecture, and othermechanisms may also be operating, the present work indicates papilla formationis an important response by Adiantum to pathogenic attack.
REFERENCES
AIST, J. R. (1976). Papillae and related wound plugs of plants. Annual Review of Phytopathology, 14, 145-163.BAKER, E. A. & HOI.LOWAY, P. J. (1971). Scanning electron microscopy of-waxes on plant surfaces. Micron,
2, 364-380.BLAKEMAN, J. P. (1980). Behaviour of conidia on aerial plant surfaces. In: The Biology of Botrytis (Ed. by
J. R. Coley-Smith, K. Verhoeff & W. R. Jarvis), pp. 115-151. Academic Press, London.CHALLENGER, F., BYWOOD, R., THOMAS, P. & HAYWARD, B. J. (1957). Sulfoxide compounds in ferns.
Archives of Biochemistry and Biophysics, 69, 514—528.DEY, P. K. S. (1919). Studies in the physiology of parasitism V: infection hy Cntletotrichum lindetnuthianum.
Annals of Botany, 33, 305-312.DiNGLEY, J. M. (1969). Records of Plant Diseases in New Zealand. New Zealand Department of Scientific
and Industrial Research Bulletin 192.HALL, D . M. & BURKE, W. (1974). Wettahility of leaves of a selection of New Zealand plants. New Zealand
Journal of Botany, 12, 283-298.HARGREAVES, J. A., MANSFIELD, J. W. & COXON, D . T . (1976). Conversion of wyerone to wyerol hy Botrytis
cinerea and B.fabae in-vitro. Phytochemistry, 15, 651-653.HAYAT, M . A. (1970). Principles and Techniques of Electron Microscopy, vol. 1, Van Nostrand, Reinhold
Company, New York.
12 ANP 103
348 K. J. ARCHER AND A. L. J. COLE
HEATHER, W . A. (1967). Leaf characteristics of Eucalyptus bicostata Maiden et al. seedlings affecting thedeposition and germination of spores of Phaeoseptoria eucalypti (Hansf.) Walker. Australian Journalof Biological Science, 20, 11 55-11 60.
HOSHIZAKI, B . J. (1979). Fern Growers Manual. Alfred A. Knopf, New York.JENNINGS, D . L . (1962). Some evidence on the influence of the morphology of raspherry canes upon their
liability to be attacked by certain fungi. Horticultural Research, 1, 100-111.JHOOTY, J . S. & MCKEEN, W . E . (1965). Studies on powdery mildew of strawberry caused hy Sphaerotlieca
macularis. Phytopathology, 55, 281-285.Klic, J. (1972). Phytoalexins. Annual Review of Phytopathology, 10, 207-232.MARGOLENA, L . A. (1932). Erythrosin for Stoughton's thionin orange ' G ' . Stain Technology, 1, 25-27.MARKS, G . C , BERBEE, J. G. & RIKEB, A. J. (1965). Direct penetration of leaves of Populus tremuloides by
Colletotrichum gloeosporioides. Phytopathology, 55, 408-412.MATTA, A . (1982). Mechanisms in non-host resistance. In; Active Defence Mechanisms in Plants. (Ed. hy
R. K. S. Wood), pp. 130-132, Academic Press, London.MOORE, W . C . (1959). British Parasitic Fungi, Camhridge University Press.NILSSON, M . (1959). The structure of ceroptene. Acta Chemica Scandinavica, 13, 750-757.PlRONE, P. P. (1970). Diseases and Pests of Ornamental Plants, 4th Edn. Ronald Press Co, New York.PREECE, T . E . (1959). A staining method for the study of apple scah infections. Plant Pathology, 8, 127-129.PRIESTLEY, J. H. & RADCLIFFE, F . M . (1924). A study of the endodermis in the Filicinae. New Phytologist,
23, 161-193.REED, D . W . (1982). Wax alteration and extraction during electron microscopy preparation of leaf cuticles.
In: The Plant Cuticle (Ed. by D. E. Cutler, K. L. Alvin & C. E. Price), pp. 181-195. Linnaean SocietySymposium series No. 10. Academic Press, London.
RIDE, J. P. (1983). Cell walls and other structural harriers in defence. In: Biochemical Plant Pathology. (Ed.hy J. A. Callow), pp. 215-236. John Wiley, London.
RYAN, C . C . & CLARE, B . G . (1974). Coating of leaf surfaces with agarose to retain fungal inoculum 'in-situ'for staining. Stain Technology, 49, 15-18.
SATO, T . (1967). A modified method for lead staining of thin sections. Journal of Electron Microscopy, 16,133.
SPURR, A . R . (1969). A low-viscosity epoxy resin emhedding medium for electron microscopy. ^o»(-»a/ ofUltrastructural Research, 26, 31-43.
SWAIN, T . (1980). The importance of flavonoids and related compounds in fern taxonomy and ecology: anoverview of the symposium. Bulletin of the Torrey Botanical Club, 107, 113-115.
VERHOEFF, K . (1980). The infection process and host pathogen interactions. In: The Biology of Botrytis.(Ed. hy J. R. Coley-Smith, K. Verhoeff & W. R. Jarvis), pp. 153-180. Academic Press, London.
VFRHOEFF, K . & WARREN, J. M. (1972). In vitro and in vivo production of cell wall degrading enzymes hyBotrytis cinerea from tomato. Netherlands Journat of Plant Pathology, 78, 179-185.
VFRHOEFF, K . , WARNAAR, E . & SCHIJVEN, E . J. P. (1979). Infection of young tomato plants hy Botrytiscinerea. Acta Botanica Neerlandica, 28, 242.
WADA, M . & STAEHELIN, L . A. (1981). Ereeze-fracture ohservations on the phistna-memhrane, the cell walland the cuticle of growing protonema of Adiantum capillus-veneris. Planta, 151, 467-468.
WATTENDORF, J. & HOLLOWAY, P. J. (1980). Studies on the ultrastructure and histochemistry of plantcuticles: the cuticular memhrane of Agave arnericana L. /H sttti. Annals of Botany, 46, 13—28.
WESTCOTT, C . (1971). Plant Diseases Handbook (3rd Ed.) Van Nostrand Reinhold Company, New York.WILLS, E . D . (1956). Enzyme inhibition hy allicin the active principle of garlic. Biochemical Journat, 63,
514-520.WoLLENWEBEii, E. (1978). The distrihution and chemical constituents of the farinose exudates in gymno-
grammoid ferns. American Fern Journal, 68, 13-28.WOLLENWEBER, E . & DIFTZ, V. H. (1981). Scale insects feeding on farinoses of Pityrogramma species.
American Fern Journal, 71, 10—12.WOOD, R. K . S . (1967). Physiological Plant Pathology. Blackwell Scientific Puhlications, Oxford.
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