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Hyphal growth in wood of Osmanthus armatus.

ABSTRACT

Pit membranes are structural components connecting one tracheary element to another. They are constructed so as to permit water movement but inhibit passage of air bubbles. One mechanism that has evolved to carry out these functions involves a pit membrane with a central, thickened torus surrounded by a permeable margo. The torus blocks the aperture leading to the neighboring cell and prevents transfer of air embolisms. Osmanthus armatus is a species whose wood contains such pit membranes, and a fungus-infected stem of this species was investigated to see whether the thickened tori could also inhibit movement of hyphae from one tracheary element to the next. The wood was infected by at least three different fungi and contained hyphae of different diameters. Hyphae can grow through the torus and through simple pit pairs of parenchyma cells. Hyphae also can grow from one vessel member to another through perforated end walls as well as directly through secondary wall material by using an appressorium. It appears as if the torus-bearing pit membranes' only function is associated with the inhibition of passage of air embolisms and not with inhibiting fungal movement.

INTRODUCTION

Bordered pit pairs control movement of water from one tracheary element to the next in the wood of vascular plants (Zimmermann, 1983; Pittermann et al., 2005). The key component of the pit pairs is the porous pit membrane, which allows passage of water molecules but not air embolisms. One mechanism by which these functions are accomplished is found in conifers, Ginkgo and Ephedra (Dute et al. 2014; Dute 2015) and involves a central impermeable thickening (torus) of the pit membrane surrounded by a permeable fibrillar ring, the margo. Water molecules can pass through the margo. In the presence of air embolisms, the pit membrane is displaced or aspirated so that the torus blocks entry into the adjoining cell (Zimmermann, 1983). Such a mechanism insures that air bubbles are confined and are not spread throughout the wood. Most bordered pit pairs in woods of angiosperms lack pit membranes differentiated into torus and margo. Rather, angiosperm pit membranes are homogeneous and have a screen with much smaller openings than those in the margos of conifers, Ginkgo etc. (Dute et al., 1992; Dute, 1994; Pittmerman et al., 2005; Dute et al., 2008). Such pit membranes are able to prevent passage of air embolisms, but at the cost of reduced water flux (Pittermann et al., 2005).

Recently, a number of species in various genera of the angiosperms have been observed to have bordered pit pairs possessing an impermeable torus as well as a surrounding margo with openings the size of the pit membrane of typical angiosperms rather than the larger openings of the margos of gymnosperms. Wheeler (1983) and Dute and Rushing (1987) attempted to explain those "hybrid" pit membranes by hypothesizing that the thickened torus prevented rupture of the membrane during aspiration.

Recently, a specimen of Osmanthus armatus used in a previous study (Dute and Elder, 2011) showed signs of stress as indicated by desiccating leaves which subsequently abscised. Preliminary investigation correlated these symptoms with the presence of hyphae in the wood. It was decided to study the pathway of hyphal growth through the wood cells to see whether, perhaps, the thickened tori of the pit membrane not only prevented passage of air bubbles from cell to cell, but also inhibited the progress of hyphal growth.

MATERIALS AND METHODS

A specimen of Osmanthus armatus (spiny olive) used in a previous study (Dute and Elder, 2011) was observed over a period of weeks to lose its leaves. The leaves exhibited water stress, then died and dropped from the stem.

1. The stem of the infected plant was cut into 1 cm segments, wrapped in moist paper toweling, and brought into the laboratory. Some segments were boiled for half an hour and cut into sections 20-40 [micro]m thick that were placed in formalin acetic acid-alcohol (FAA, Johanson 1940), kept for ten minutes under vacuum and remained in the preservative overnight at atmospheric pressure.

2. Thinner sections of 15-20 [micro]m were stained in toluidine blue O (TBO), mounted as a wet mount on a slide, viewed, and photographed with a light microscope.

3. Other wood segments were not boiled but sectioned directly with the sliding microtome (20-40[micro]m) and put into FAA overnight with a brief vacuum treatment as in #1.

Preserved sections of treatments one and three were dehydrated in an ethanol series and either air dried overnight between two glass slides for scanning electron microscopy (SEM), or infiltrated with JB-2 resin (Dute et. al, 2012). These latter specimens were later polymerized in the same resin. Sections of 2-6 [micro]m in thickness were cut from the resin blocks using a Sorvall MT-2b ultramicrotome, mounted on microscope slides, stained with TBO (Ruzin. 1999) and viewed with a light microscope.

Specimens for SEM were mounted on aluminum stubs and sputter-coated with gold. Next they were viewed with a Zeiss EVO 50 at 20 kV accelerating voltage.

Stem tissue from the infected plant was diced into 1 mm pieces and surface sterilized for ten seconds in 90% ETOH followed by a 1 minute wash in 6 % NaOCl solution. The surface sterilized cross sections were then aseptically placed in water agar media (VWR International), ten diced tissue segments per petri dish. Cultures were kept at room temperature (22.2 C-24.4 C) for 5 to 14 days. Fungal cultures were identified, and then subcultured for storage at -20 C. Fungal colonies were identified by placing small 1 mm sections of mycelium on glass sides, staining with cotton blue solution (VWR International), observed with either a Nikon Ellipse at 300 to 600X or were viewed and photographed using a Nikon Biophot microscope with a Nikon D-70 digital camera. Sudan Black B solution (0.07% w/vol in 70% ETOH) was used for identification of lipids in living hyphae according to the procedure found in Ruzin (1999).

Healthy wood tissue of O. armatus that previously had been macerated by the method of Wheeler (1983) was used for the observation of individual wood cells. Macerated tissue was separated into individual cells using teasing needles, stained with TBO and viewed using a light microscope (LM).

RESULTS

Fungus

Fungi recovered from the infected stem show branched, septate hyphal growth on agar medium (Figure 1). Some fungal cells contain globules that are stained by Sudan Black B (Figure 2). At least three different genera of fungi were isolated. Two were identified by their reproductive structures as Fusarium sp. (Figure 3) and Phomopsis sp. The third genus was unidentified.

Abbreviations used in the figures of this study: A = aperture; AP = appressorium; B = pit border; F = fiber; H = hyphae; L = lipid bodies; M = margo of pit membrane; P = parenchyma cell; PA = perforation in end wall of vessel element; T = torus; VT = vascular tracheid. All wood anatomy images are of Osmanthus armatus, except for Figure 6, which is from O. americanus.

Water-Conducting Tissue

The water-conducting tissue, which is called wood or secondary xylem, is a complex tissue containing fibers, parenchyma cells, and tracheary elements (Figure 4). Tracheary elements conduct water and are of two types: vascular tracheids (Figure 4) and vessel members (Figure 5). Figure 4 shows a vascular tracheid with helical sculpturing. This cell type is identified by an elongate shape, absence of cytoplasm at maturity, and helical sculpturing. A vessel member has these same three features but in addition has perforated end walls (Figure 5). In both cell types, lateral contact is maintained by bordered pit pairs. It is with these pit pairs that we are primarily concerned.

Bordered Pit Pairs

Figures 6-8 show light microscope and SEM images of bordered pit pairs in some detail. Figure 6 is from a related species (O. americanus) and was previously published in this journal (Dute et al. 2012), but the anatomy is the same as O. armatus. 'X' in Figure 6 indicates a sectional view through a bordered pit pair. Water travels from the lumen of one cell, through the aperture on one side of the bordered pit membrane, through the membrane, and then out of the aperture on the other side into the lumen of another cell. 'Y' in Figure 6 shows the pit pair in oblique view. In this image the aperture and pit membrane are visible. There are two parts to the latter: the margo and torus. 'Z' shows the pit membrane in face view as the upper aperture and pit border have been cut away. The centrally located torus appears darker than the surrounding margo due to its greater thickness and different chemistry (Figure 6). Figures 7 and 8 are scanning electron micrographs of face views of bordered pits with the pit membrane removed to show the underlying pit border and aperture (Figure 7) and with the pit membrane present (Figure 8). Note that the torus diameter is greater than that of the aperture (Figure 6 at Y). Therein lies the secret to the function of the bordered pit pair possessing a torus. The margo is permeable to water, but the torus is not. When air embolisms encounter the pit membrane, the latter is deflected or aspirated so that the torus covers one of the apertures. In the aspirated condition, the pit membrane prevents the movement of air embolisms into functioning (i.e. water-conducting) tracheary elements.

Fungal Presence and Growth in Wood Fibers and Parenchyma Cells

Figure 9 shows a longitudinal section of wood whose cells contain septate, branched hyphae of different diameters. A wood fiber cell containing a hypha is indicated by an asterisk.

Fungal hyphae appear more numerous in parenchyma cells, both ray parenchyma and axial parenchyma, than in the other cell types, perhaps because parenchyma cells typically contain stored food. By the time we investigated the wood, the stored food and the cytoplasm of the parenchyma had largely disappeared. In some cells, only a plasmolyzed cell membrane remained (Figure 10, unlabeled arrow). SEM provides a more three-dimensional image of parenchyma cells than does light microscopy (Figures 12 and 13 versus Figures 10 and 11) and shows the density and branching of the hyphae.

Hyphal growth from one parenchyma cell to another was commonly observed during bright field light microscopy (Figures 10 and 11). In some cases, what we interpret as an appressorium is formed by a hyphal strand (Figure 10). Many instances were observed of hyphal growth directly through the aperture and pit membrane of simple pit pairs from one parenchyma cell to another (Figure 11).

Fungal Growth in Water-Conducting Cells

It is not uncommon to find hyphae growing along the length of both vessel members (Figure 14) and vascular tracheids. The perforations in the end walls of vessel members provide a path whereby hyphae can move vertically from one vessel member to the next (Figure 14). Vessel members are dead at maturity so hyphae are not gaining nutrition, but rather the fungal strands can spread rapidly and colonize the wood.

Lateral spread of hyphae through both types of water-conducting cells occurs through the bordered pit pairs (Figures 15-17). Such growth is preceded by branching of the hypha, growth of the branch through an aperture (Figures 15 and 16), penetration of the torus of the pit membrane (Figure 17), and growth through the other aperture into the lumen of the laterally adjacent cell (Figure 17).

DISCUSSION

A drawback to this study is the small sample size, but there is no reason to believe that the progress of the fungal infection in this specimen was unique. In fact, it appeared quite the opposite. An earlier study from this laboratory (Dute et al. 2002) dealt with the introduction of fungi into the wood of Cercis canadensis by the Asian ambrosia beetle and the resulting fungal growth. Various types (genera) of fungi were involved, and they infested all cell types within the wood by growing "directly through the cell walls, by penetrating pit membranes, and by traversing perforations" (Dute et al. 2002). The same manner of hyphal growth was found in the present study.

Other studies have suggested that in torus-bearing pit membranes, the thickened torus is more resistant to damage than the surrounding margo. Wheeler (1983) suggested that the torus thickening in pit membranes of Ulmus and Celtis might keep the displaced pit membrane from rupturing and spreading air embolisms. Experimental evidence for this hypothesis was obtained by Dute & Rushing (1987) when they chemically removed the torus from aspirated pit membranes of Osmanthus americanus. The treated pit membranes tore at the site where the torus had been removed. A later study involving 17 different species of Osmanthus (Dute et al. 2010) showed that not all species had tori. In those that did not, the aspirated pit membrane tore under the heat of the SEM's electron beam where it (the membrane) overlay the aperture. Those species with the thickened torus remained intact at that same site. A similar situation was found to exist in torus-bearing vs non-torus-bearing species of Daphne (Dute et al. 1990). Taking this information into account, hypothesizing that torus thickening might also inhibit passage of fungal hyphae does not seem unreasonable, although the hypothesis did prove untrue.

In all instances, hyphal penetration of the pit aperture of a bordered pit pair was preceded by the formation of a small branch that grew directly into the pit aperture (q.v. Figures 15 and 17). The mechanism for the attraction of the hyphal branch toward the pit aperture is unknown.

ACKNOWLEDGMENT

The authors wish to thank the Department of Biological Sciences Fund for Excellence for its support.

LITERATURE CITED

Dute, R. R. 1994. Pit membrane structure and development in Ginkgo biloba. IAWA Journal 15: 75-90.

Dute, R. R. 2015. Development, structure, and function of torus-margo pits in conifers, Ginkgo and dicots. In: U. Hacke (ed.), Functional and Ecological Xylem Anatomy: Chapter Three. Springer International Publishing.

Dute, R. R., Bowen, L. A., Schier, S., Vevon, A. G., Best, T. L., Auad, M., Elder, T., Bouche, P., and Jansen, J. 2014. Pit membranes of Ephedra resemble gymnosperms more than angiosperms. IAWA Journal IS: 217-235.

Dute, R. R., and Elder, T. 2011. Atomic force microscopy of torus-bearing pit membranes. IAWA Journal 32: 415-430.

Dute, R., Hagler, L., and Black, A. 2008. Comparative development of intertracheary pit membranes in Abies firma and Metasequoia glyptostroboides. IAWA Journal 29: 277-289.

Dute, R. R., Hubbard, Z. S., and Patel, R. V. 2012. Intervascular pit membranes in roots of two species of Osmanthus (Oleaceae). Journal of the Alabama Academy of Science 83: 819.

Dute, R. R., Miller, M. E., Davis, M. A., Woods, F. M., McLean, K. S. 2002. Effects of ambrosia beetle attack on Cercis canadensis. IAWA Journal 23: 143-160.

Dute, R., Rabnaey, D., Allison, J., and Jansen, S. 2010. Torus-bearing pit membranes in species of Osmanthus. IAWA Journal 31:217-226.

Dute, R. R., and Rushing, A. E. 1987. Pit pairs with tori in the wood of Osmanthus americanus (Oleaceae). IAWA Bulletin new series 8: 237-244.

Dute, R. R., Rushing, A. E., and Freeman, J. D. 1992. Survey of intervessel pit membrane structure in Daphne species. IAWA Bulletin new series 13: 113-123.

Dute, R. R., Rushing, A. E., and Perry, J. W. 1990. Torus structure and development in species of Daphne. IAWA Bulletin new series 11: 401-412.

Johansen, D. A. 1940. Plant Microtechnique. McGraw-Hill Book Co.

Pittermann, J., Sperry, J. S., Hacke, U. G., Wheeler, J. K., Sikkema, E. H. 2005. Torusmargo pits help conifers compete with angiosperms. Science 310: 1924.

Ruzin, S. E. 1999. Plant Microtechnique and Microscopy. Oxford University Press.

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Abigail L. Schweig (1), Kathy Lawrence (2), and Roland R. Dute (1),

(1) Department of Biological Sciences

(2) Department of Entomology and Plant Pathology, Auburn University Auburn, AL

Correspondence: Roland R. Dute (duterol@auburn.edu)
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Author:Schweig, Abigail L.; Lawrence, Kathy; Dute, Roland R.
Publication:Journal of the Alabama Academy of Science
Article Type:Report
Date:Jul 1, 2014
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