Plant and Soil 167: 121-126, 1994. @ 1994 KluwerAcademicPublishers. Printedin the Netherlands.

Impact of the exodermis on infection of roots by Fusarium culmorum

Susan A. Kamula, Carol A. Peterson and Colin I. Mayfield Department of Biology, University of Waterloo, Waterloo, ON, Canada N2L 3G1

Key words: Casparian bands, exodermis, Fusarium culmorum, hypodermis, passage cells, short cells, suberin lamellae

Abstract

Patterns of infection with Fusarium culmorum (W G Smith) Saccardo were observed in seedling roots of barley (Hordeum vulgare L.), wheat (Triticum aestivum L.), maize (Zea mays L.) and asparagus (Asparagus officinalis L). Apical regions of the main roots were not infected. Since penetration into the root occurred several days after inoculation and the roots were growing during the experiment, these regions had apparently not been in existence long enough to be infected. In older regions of barley, wheat and asparagus, hyphae entered through the tips of lateral roots. In barley and wheat, which had not developed any suberin lamellae in their subepidermal layer, infection occurred randomly over the remainder of the root. In maize, the fungus penetrated the epidermis at many sites but did not breach the exodermis in which all cells possessed both Casparian bands and suberin lamellae. Maize roots, therefore, sustained only minimal infections. In asparagus, the fungus grew through the short (passage) cells but never the long cells of the exodermis. In doing so, it penetrated cells possessing Casparian bands but lacking suberin lamellae. The results support the hypothesis that suberin lamellae provide effective barriers to the growth of F. culmorum hyphae.

Introduction guson and Clarkson, 1975; Peterson, et al., 1982). The exodermis of young roots of asparagus, on the oth- Deposition of suberin in plant cell walls is known to er hand, is dimorphic and consists of long and short deter fungal infection (Kolattukudy, 1984). Thus, the cells (Kroemer, 1903). At maturity, the long cells and suberin in the Casparian bands and suberin lamellae of possibly some of the short cells have both Casparian the exodermis, a specialized layer in the roots of many bands and suberin lamellae but the remainder of the angiosperm species (Perumalla et al., 1990; Peterson short cells have only Casparian bands (Hussey, 1982; and Perumalla, 1990), may play a role in preventing Kamula, 1991). Inclusion of a species with a dimorphic or delaying fungal invasion. Since the exodermis is exodermis made it possible to test the effects of Cas- the outermost layer of the cortex, it is well positioned parian bands and suberin lamellae on fungal growth to protect a large area of the organ. To determine the separately. potential importance of the exodermis as an antifun- gal layer, we compared the pattern of infection of four species by pathogen Fusarium culmorum (W G Smith) Materials and methods Saccardo. According to Perumalla et al. (1990), barley (Hordeum vulgare L.) and wheat (Triticum aestivum Caryopses of barley (Hordeum vulgare L.) and wheat L.) have nonexodermal roots. Maize (Zea mays L.) (Triticum aestivum L.) were purchased from Carolina and asparagus (Asparagus officinalis L.) have exoder- Biological Supply, Burlington, NC, USA ; caryopses mal roots (Peterson et al., 1982; Peterson and Peru- of maize (Zea mays L. cv. Seneca Chief) and seeds of malla, 1990). The exodermis of maize is similar, com- asparagus (Asparagus officinalis L. var. Mary Wash- prised of cells of uniform length and structure (Kroe- ington) were obtained from Ontario Seed Company, mer, 1903). At maturity, all cells of the exodermis Ltd., Waterloo, ON, Canada. All propagules were sur- have both Casparian bands and suberin lamellae (Fer- face sterilized in 5.2% sodium hypochiorite (commer- 122

cial Javex bleach) for five min and soaked overnight in mounted on microscope slides with props at the ends cold, running tap water. Plants were grown in vermi- of the coverslips to prevent squashing the specimens. culite, initially watered to saturation and subsequently To determine the location of suberin lamella develop- every 2-4 d with tap water, in a Conviron PGW-36 ment, cross sections were taken near the apices of exo- growth chamber (Controlled Environment, Winnipeg, dermal species and stained with Sudan red 7B which MN, Canada) at 25 °C during 16 h light (intensity 215 partitions into lipids, imparting a red colour to them /rE s -1 m -2) and 23 °C during 8 h darkness, relative (Brundrett et al., 1991). The experiment was repeated humidity 75-85%. Maize, barley and wheat grown for three times. 5 d and asparagus grown for 21 d were used in the All experimental specimens were observed with a experiments. Zeiss Axiophot microscope. Colour photographs were A culture of Fusarium culmorum (W.G. Smith) taken on Kodak Ektachrome 200 ISO, 35 mm slide Saccardo was provided by Dr Robert Hall, Department film, black and white on Ilford Pan F 50 ISO, 35 mm of Environmental Biology, University of Guelph, ON, negative film. Black and white prints were made from Canada, who had isolated it from the crown of a winter black and white negative film or from internegatives wheat plant in Elora, ON, Canada. The fungus was made from colour slides using Ilford Pan F 50 ISO, 35 grown on potato-dextrose agar (Difco Laboratories, mm negative film. Detroit, MI, USA) at room temperature in a dimly lit area for two months. A spore suspension was prepared by flooding the mycelium with sterile, distilled water Results and adjusting the volume to give 2 x 10s spores mL- 1. To begin an experiment, seedlings were gently No signs of contamination or infection were appar- uprooted and their root systems washed and laid hor- ent on control roots. The distinctive macroconidia of izontally in large, moist, sterile chambers. The pri- Fusarium culmorum were clinging to the surfaces of mary roots of each species were inoculated with E all treated roots and the majority of the conidia had ger- culmorum by pipetting 2 mL of the spore suspen- minated within 3 d. By 7 days, copious hyphae were sion over them. This resulted in an inoculum ranging present on the surfaces of the roots and were beginning between 14 and 38 spores mm -I root length. Roots to invade them. Penetration sites were at midlength and which served as controls were placed in similar sur- more proximal regions of the roots, never in the api- roundings and treated with water. The chambers were cal regions. Several distinct patterns of infection were covered with aluminum foil so that the roots were in discerned. darkness and the shoots emerged from the side. The In barley and wheat, infection occurred randomly containers were incubated at room temperature and in the midlength to basal regions of the roots (average the contents were wetted with sterile distilled water total root length about 170 mm). Hyphae grew through as necessary to maintain a moist environment around epidermal cells ( Fig. 1) and subsequently many runner, the roots. To determine the time course of infection, branched and coiled hyphae proliferated throughout three seedlings of each species were inoculated as the cortex (Fig. 2). Infection also occurred in some described above and individual roots were harvest- young lateral roots, often through their root caps (Fig. ed 0.5, 3, 7, 9, 14 and 21 d later. This experiment 3). Occasionally, black staining of the host tissue near was repeated three times. A further comparison of the an infection site was observed (Fig. 1) indicating that infection patterns in the four plant species was made suberin and/or lignin had been deposited as a wound by inoculating three seedlings of each and harvesting response. them after 21 d. Entire roots were analyzed from both In maize roots, the infection process was arrested at experiments. They were cut into segments 10-30 mm the exodermis. This layer became mature 40 mm from long which were cleared and stained with chlorazol the main root tip and very close to the tips of the later- black E (CBE) according to the method of Brundrett et als. Hyphae frequently penetrated the epidermis in mid al. (1984) with the following modifications. An auto- and basal regions of the main roots, as well as in the clave was used (20 rain at 121" C) for both cleating and young laterals. However, growth did not proceed into staining steps, the concentration of CBE was 0.03%, the cortex (Fig. 4). From 9 to 21 days after inocula- and the roots were mounted in 70% glycerin. With tion, masses of hyphae had formed in some epidermal this procedure, fungal walls, lignified plant walls and cells but the fungus had still not penetrated further into Casparian bands stained black. Root segments were the root (Fig. 5). Furthermore, hyphae did not grow 123 through the tips of the lateral roots. The pathogen, invasion in zones with an immature exodermis would therefore, produced only a minimal infection of the be anticipated. root, i.e. in the epidermis. A comparison of the infection patterns of various The exodermis of asparagus roots also influ- species indicates that the exodermis plays a role in enced the infection process. In these roots, Caspar- the invasion process and that the type of exodermis ian bands and suberin lamellae had formed 10 mm is critical for the outcome. Specifically, the failure of from the main root apex. The dimorphic nature of the fungus to penetrate the uniform exodermal cells the exodermis was apparent in clearings of control of maize and the long cells of asparagus indicates roots (Fig. 6). Fungal invasion of the root proceed- that suberin lamellae were the structures responsible ed through the epidermis overlying the short cells for prevention of its ingress. The Casparian band had and then through the short cells themselves, form- no such effect, since it was present in the short cells ing hyphal coils (Fig. 7). As an example of the of asparagus through which the hyphae grew. These extent of short cell penetration, in samples taken 14 results are reasonable because the Casparian band is days post inoculation, hyphae were present in 41% located in the anticlinal (radial and transverse) walls of of the 115 short cells tallied. In contrast, penetra- the cells whereas the lamellae also cover the tangential tion of a long cell was never observed anywhere in walls through which the fungus penetrates. Our obser- the roots. The fungus also gained entry by grow- vations agree with those of Gallaud (1905) who noted ing through the lateral roots, either through the short that Fusarium grew through the short cells but not the cells of the exodermis or directly through the root long cells in the exodermis of Cyripedium barbatum. tips. Noelle (1910) also described penetration of short cells (as well as unsuberized, immature hypodermal cells) by fungal hyphae as a general phenomenon in conifers. Discussion Shishkoff (1986) found that hyphal penetration by Pythium coloratum on Allium cepa roots occurred with A consistent feature of the invasion of all four plant much greater frequency through unsuberized short and species with Fusarium culmorum was that it did not long cells, although she did find a few cases where the occur in young regions of the roots. This result is not fungus had grown through the suberized wall of a long typical of Fusarium infections, which often proceed cell. The question of whether or not the exodermis con- through the root cap (Pennypacker, 1981; Smith and stitutes a resistant layer was also approached by Perry Peterson, 1985). In the present investigation, this result and Evert (1983) and Brammall and Higgins (1988) was probably a consequence of the relatively long time with Solanum tuberosum and Lycopersicon esculen- required by the fungus to invade the root. Since the rum, respectively. It is not clear from the literature roots were elongating by tip growth throughout the whether the exodermis of these plants is dimorphic or experiment, the youngest regions would not have been uniform (Shishkoff, 1987). In the infected zones, most in existence long enough to be infected. In species of the exodermal walls in S. tuberosum and some of the with an exodermis (maize and asparagus), the layer exodermal walls in L. esculentum lacked suberin lamel- was mature before the fungus succeeded in gaining lae and none of the electron micrographs illustrate entry into the roots. The maturation of the exodermis hyphal growth into an exodermal cell with a lamella. in rapidly growing maize roots occurs about 120 mm Therefore, it is possible that the invasion of the exo- from the root tip but is within 5 mm of the tip in roots dermis which led to the formation of lignitubers in the which have ceased growing (Perumalla and Peterson, case of S. tuberosum (Perry and Evert, 1983), or fur- 1986). The intermediate maturation distance of 40 mm ther invasion of the cortex in L. esculentum (Brammall obtained in the present study was a consequence of and Higgins, 1988), occurred through cells lacking a reduced growth rate caused by forcing the roots to suberin lamellae. Although the evidence is somewhat grow horizontally. In the case of maize and aspara- scattered at the present time, much of it indicates that gus, the apical, uninfected zone correlated with the suberin lamellae are effective in preventing growth of region of immature exodermis, in which a large area of pathogenic fungi through exodermal cells. plasmalemma is exposed to the soil solution (Kamula, Whether or not a root system is successfully 1991). However, if the fungus were capable of a more attacked by a pathogenic fungus depends not only on rapid infection than the strain used in the present study, the exodermis but also on the existence of other sites the length of the uninfected zone would be reduced and of invasion. For example, Fusarium spp. frequently 124

Fig. l-5. Whole roots which had been infected with Fusarium culmorum, fixed in FAA, cleared with KOH and stained with chlorazol black E. Scale bars = 25 pm. Fig. 1. Median longitudinal, optical section of barley root with E culrnorum hyphaein epidermal cell (arrow). The cell layer under the epidermis underwent a wound reaction (arrowheads). Fig. 2. Paradermal, optical section through the cortex of a heavily infected wheat root. Runner hyphae (arrowheads) occupy the intercellular spaces and branched hyphae (arrow) have grown into cortical cells. Fig. 3. Median longitudinal, optical section of a young, lateral root of barley. Hyphae of E culmorum had grown through the root cap (arrow) and continued into the root. Fig. 4. Median longitudinal, optical section of a maize lateral root with E cdmorum hyphae in its epidermis (e). The pathogen did not grow into the exodermis (ex), central cortex (c) or xylem(x). Fig. 5. Paradermal, optical section of a maize main root with extensive hyphal coiling in some epidermal cells (arrowheads). 125

Fig. 6 and 7. Whole asparagus roots which had been cleared and stained with chlorazol black E. Paradermal, opticalsections focus.& on the exodermis. Scale bars = 25 pm. Fig. 6. Uninfected root. Some short cells of the dimorphic exodermis are indicated by asterisks. Fig. 7. Infected root with a hyphal coil (arrow) within a short cell. enter plants through wounds and the apices of later- In all these cases, the fungi appear to sense the pres- al roots (Pennypacker, 1981). In the present study, ence of the short cells underlying the epidermis. The the fungus was unsuccessful in invading the maize mechanism of this interaction remains a mystery. It root through these other potential sites (for unknown may have evolved to attract mycorrhizal fungi to an reasons) and the interior of the root remained free of appropriate entry site, and was later used to advantage infection. However, in the case of asparagus, invasion by pathogenic fungi. occurred through both young lateral roots and short cells so that the root was thoroughly colonized. Thus, the presence of suberin lamellae in the exodermis can Acknowledgements only provide protection to a limited area of the root and successful pathogens can use other sites of entry. This research was supported by grants to C A P and It is interesting to note that when F: culmorum C I M from the Natural Sciences and Engineering attacked the roots of asparagus, it entered the epider- Research Council of Canada. We thank MS Daryl ma1 cells overlying the short cells of the exodermis. Enstone for reviewing the manuscript and preparing Shishkoff (1986) found that zoospores of Pythium col- the plates. orutum tended to encyst on the epidermis over the short cells of Allium cepa and that after germination, a large fraction of their hyphae grew through the short References cells. There are several reports that endomycorrhizal fungi also enter the roots through the short cells (see Brammall R A and Higgins V J 1988 A histological comparison of Shishkoff, 1986 lit. cit.; Peterson, 1992 and lit. cit.). fungal colonization in tomato seedlings susceptible or resistant to Fusurium crown and root rot disease. Can. J. Bot. 66,915-925. 126

Brundrett M C, Kendrick B and Peterson C 1991 Efficient lipid Perry J W and Evert R F 1983 Histopathology of Verticillium dahliae staining in plant material with Sudan red 7B or Fluorol yellow within mature roots of Russet Burbank potatoes. Can. J. Bot. 61, 088 in polyethylene glycol-glycerol. Biotechnic Histochem. 66, 3405-3421. 111-116. Perumalla C J and Peterson C A 1986 Deposition of Casparian bands Brundrett M C, Pich6 Y and Peterson R L 1984 A new method for and suberin lamellae in the exodermis and endodermis of young observing the morphology of vesicular-arbuscular mycorrhizae. corn and onion roots. Can. J. Bot. 64, 1873-1878. Can. J. Bot. 62, 2128-2134. Perumalla C J, Peterson C A and Enstone D E 1990 A survey of Ferguson I B and Clarkson D T 1975 Ion transport and endodermal angiosperm species to detect hypodermal Casparian bands. I. suberization in the roots of corn. New Phytol. 75, 69-79. Roots with a uniseriate hypodermis and epidermis. Bot. J. Linn. Galland I 1905 Etudes sur les mycorrhizes endophytes. Rev. Gtn Soc. 103,93-112. Bot. 17, 313-325. Peterson C A, Ernanuel M E and Wilson C 1982 Identification of a Hussey R B 1982 Interactions between V-A mycorrhizal fungi and Casparian band in the hypodermis of onion and corn roots. Can. Asparagus o~cinalis L. roots. M Sc Thesis, University of Guelph, J. Bot. 60, 1529-1535. Guelph, ON, Canada. Peterson C A and Perumalla C J 1990 A survey of angiosperm Kamula S A 1991 The significance of suberin lamella development species to detect hypodermal Casparian bands. II. Roots with in the dimorphic exodermis. MSc Thesis, University of Waterloo, a multiseriate hypodermis or epidermis. Bot. J. Linn. Soc. 103, Waterloo, ON, Canada. 113-125. Kolatmkudy P E 1984 Fungal penetration of defensive barriers in Peterson R L 1992 Adaptations of root structure in relation to biotic plants. In Structure, Function and Biosynthesis of Plant Cell and abiotic factors. Can. J. Bot. 70, 661-675. Walls. Eds. W M Dugger and S Bartnicki-Garcia. pp 302-343. ShishkoffN 1986 The dimorphic hypodermis of plant roots: its dis- American Society of Plant Physiologists, Rockland, MD. tribution in the angiosperms, staining properties, and interaction Kroemer K 1903 Wurzelhaut, Hypodermis und Endodermis der with root-invading fungi. PhD Thesis, Comell University, Ithaca, Angiospermenwurzel.Biblioth. Bot. 12, 1-159. NY. Noelle F W 1910 Studien zur vergleichenden Anatomic und Mor- ShishkoffN 1987 Distribution of the dimorphie hypodermis of roots phologie der Koniferen wurzeln mit Rticksicht auf die System- in angiosperm families. Ann. Bot. 60, 1-15. atik. Bot. Zeitung 68, 169-266. Smith A K and Peterson R L 1985 Histochemical features of wall Pennypacker B W 1981 Anatomical changes involved in the patho- appositions in Asparagus root rneristems infected by Fusarium. genesis of plants by Fusarium. In Fusarium: Diseases, Biology, Can. J. Plant Pathol. 7, 28-36. and Taxonomy. Eds. P E Nelson, T A Toussoun and R J Cook. pp 400--408. Penn. State Univ. Press, University Park and London. Section editor: H Lambers