The Massaria Disease of Plane Trees: Its Wood Decay Mechanism* Uwe Schmitt1,**, Benjamin Lüer2, Dirk Dujesiefken3 and Gerald K
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SchmittIAWA et Journalal. – Massaria 35 (4), disease2014: 395–406 in Platanus 395 THE MASSARIA DISEASE OF PLANE TREES: ITS WOOD DECAY MECHANISM* Uwe Schmitt1,**, Benjamin Lüer2, Dirk Dujesiefken3 and Gerald Koch1 1Thünen Institute of Wood Research, Leuschnerstraße 91, D-21031 Hamburg, Germany 2Department of Wood Science, University of Hamburg, Leuschnerstraße 91, D-21031 Hamburg, Germany 3Institute of Arboriculture, Brookkehre 60, D-21029 Hamburg, Germany **Corresponding author; e-mail: [email protected] *Dedicated to our dear colleague Dr. Adya P. Singh on the occasion of his 70th birthday ABSTRACT Branches of Platanus × hispanica with distinct symptoms of the Massaria disease were investigated by light and transmission electron microscopy and cellular UV- microspectrophotometry. The samples collected in the city of Mannheim, Germany, were infected in vivo with the fungus Splanchnonema platani and showed various degrees of wood decay. The investigations were focused on the decay pattern of cell walls in the different cells, i. e., fibres, vessels as well as ray and axial parenchyma cells. The following results were obtained. Hyphae of the ascomycete fungus Splanchnonema platani penetrated from cell to cell through the pits and not through the cell wall middle lamella, by the formation of thin perforation hyphae. During this process, the 1–5 µm thick hyphae became narrower without attacking the wall around the pit canal. After penetration through a pit, the hyphae again enlarged to their original diameter. This is true for all pit pairs connecting the various cell types. Late decay stages did not show a decay of cell corner regions and middle lamellae of fibres as well as vessel and parenchyma cell walls. Phenolic deposits in parenchyma cells were still present in severely attacked xylem tissue. These features point to a low lignolytic capacity of the fungus. The frequently found microscopic decay pattern with the formation of oval or spherical cavities in the S2 layer of the secondary wall with an often structurally intact S3 layer is a characteristic of soft- rot decay. This classification is also supported by the remaining cell corner and middle lamella regions in advanced decay stages. As a consequence of this decay type, branches fracture in a brittle mode. Keywords: Platanus × hispanica, branch, Massaria disease, wood decay, soft-rot, fine structure, topochemistry. INTRODUCTION Since 2003, plane trees (Platanus spp.) in Germany increasingly show symptoms of the so-called Massaria disease. This disease is externally characterised by initially pinkish discolorations on the upper side of branches which turn into dark brown and black with © International Association of Wood Anatomists, 2014 DOI 10.1163/22941932-00000074 Published by Koninklijke Brill NV, Leiden Downloaded from Brill.com10/08/2021 06:49:26PM via free access 396 IAWA Journal 35 (4), 2014 progressing disease. Lower branch surfaces appear without any visible modifications. In the xylem attached to those bark discolorations, decay develops and spreads very fast into the inner xylem. With increasing decay, branches finally break. Kehr and Krau- thausen (2004) identified the ascomycete fungus Splanchnonema platani (Ces.) Barr (syn. Massaria platani Ces.) as the causal agent of the Massaria disease. Splanchnonema is known as a common weak parasite of plane trees growing in the Mediterranean and in North America (Nalli 1981; Ciccarone 1988; Grosclaude & Romiti 1991). Sutton (1980) as well as Sinclair and Lyon (2005) identified this fungus as a bark inhabiting organism of dead branches. Splanchnonema platani presumably spread out from the Mediterranean to southern Germany, where the disease was first observed in the mid 1990s. By 2005 the disease had spread to entire central Europe (Dujesiefken & Kehr 2008). These authors also suggested that increasing summer temperatures during the last 15–20 years were responsible for this spread. In the xylem of affected branches, S. platani causes a severe and fast spreading decay of the cell walls to the effect that branches may already break within some months after infection. In some cases, a period of only few weeks might be sufficient for developing a high risk of branch breakage (Dujesiefken et al. 2005; Stuffrein 2012). Especially in urban areas, the Massaria disease poses a serious danger as breaking branches may endanger pedestrians or damage parking cars. Therefore, for public safety early rec- ognition of this disease through regular tree inspections is of utmost necessity. Little is known about the decay mechanism of this fungus at the cellular level. Dujesiefken et al. (2011) found some evidence for a decay mechanism resembling that of soft-rot fungi. Fine structural details of the action of S. platani in the xylem of plane branches were studied by light and electron microscopy to reveal the decay pattern on the cellular level and thus contribute to a better understanding of the decay mechanism in the xylem. Furthermore, topochemical analyses using cellular UV-microspectrophotometry provided detailed information on the delignification of individual wall layers in cells of affected xylem portions. Material AND METHODS In September 2009 and February 2010, 16 branches from Massaria-affected plane trees (Platanus × hispanica) growing in the city of Mannheim, Germany were either harvested or collected after breakage. The disease symptoms on the upper branch surfaces were pinkish, brown or black discolorations with various stages of decay in the xylem as recorded visually on transverse surfaces (Fig. 1, 2). Outer (with bark still attached) and inner xylem portions with and without decay were dissected with a saw and reduced in size with a razor blade for microscopy. A 15–20 year old healthy plane tree grown in the city of Hamburg served as control. For light microscopy, samples from the upper and lower sides of all 16 branches with final dimensions of 10× 5 × 5 mm3 were fixed in neutral buffered formaldehyde (mixture of 20 ml 37% formaldehyde, 1.3 g K2HPO4 and 0.8 g KH2PO4, 180 ml de- mineralised water) for 1–2 days, dehydrated in a graded series of propanol (30–100% in 10% steps) and embedded in Technovit 7100. Sections of 5 µm thick prepared with a rotary microtome were stained for two hours with a standard Giemsa solution (azur Downloaded from Brill.com10/08/2021 06:49:26PM via free access Schmitt et al. – Massaria disease in Platanus 397 1 2 Figure 1 & 2. Massaria-affected branches of plane trees (Platanus × hispanica). – 1: Pinkish/ brownish discolorations on upper branch surface. – 2: Transverse surfaces with severe (left) and beginning xylem decay (right). B/eosin/methylene blue) (Giemsa 1904). A parallel set of samples was prepared and embedded for transmission electron microscopy (TEM) as described below. The sec- tions for light microscopy were cut with an ultramicrotome and a diamond knife to a thickness of 1 µm and stained with 1% (w/v) toluidine blue. Sections were examined in transmission and polarising mode with an Olympus BX51 microscope. For TEM, samples from the same xylem portions used for light microscopy were trimmed to a final size of about 5 × 1 × 1 mm3, fixed overnight in a mixture of 5% (v/v) glutaraldehyde and 4% (w/v) formaldehyde (Karnovsky 1965), washed in a 0.1 M cacodylate buffer, postfixed in 1% (w/v) aqueous osmium tetroxide, again washed in buffer, dehydrated in a graded series of acetone and embedded in Spurr’s epoxy resin (Spurr 1969). Ultrathin sections with a thickness between 80–100 nm were prepared with an ultramicrotome using a diamond knife and stained with either a 1% (w/v) aqueous potassium permanganate solution containing 0.1% (w/v) sodium citrate according to Donaldson (1992), or with the conventional combination of 1% (w/v) aqueous uranyl acetate and 8% (w/v) lead citrate (e.g. Hayat 2000). A Philips CM12 transmission electron microscope was used at accelerating voltages of 60 or 80 kV. A parallel set of samples with the same origin and size as described for TEM was prepared for cellular UV-microspectrophotometry (UMSP). Except postfixation with osmium tetroxide, they were embedded in the same way as for TEM. Semi-thin sections of 1 µm were also cut with an ultramicrotome using a diamond knife, mounted on quartz slides, immersed in a drop of non UV-absorbing glycerine and covered with quartz Downloaded from Brill.com10/08/2021 06:49:26PM via free access 398 IAWA Journal 35 (4), 2014 cover slips. UMSP was carried out with a Zeiss UMSP 80 microspectrophotometer equipped with a scanning stage for the determination of image profiles at a constant wavelength of 278 nm (absorbance maximum of hardwood lignin) using the software APAMOS® (Zeiss). The profiles were recorded with a local geometrical resolution of 0.25 × 0.25 µm2 and a photometric resolution of 4096 greyscale levels which were then converted into 14 basic colours representing the measured absorbance intensities (for more details see Koch & Kleist 2001; Koch & Grünwald 2004). Results AND DISCUSSION Massaria-affected branches with typical pinkish and/or brown discolorations on the upper side (Fig. 1, 2) were selected for the current investigation. Those branches were colonised by the ascomycete fungus Splanchnonema platani which was already identi- fied in 2004 by Kehr and Krauthausen for plane trees in Germany as the causal agent of the Massaria disease. Splanchnonema platani hyphae first invade branches through the bark and subsequently colonise the xylem tissue. Dujesiefken et al. (2011) found that in xylem tissue hyphae preferably use rays and vessels for the very fast spread of the disease. This is in agreement with earlier observations on soft-rot attack by various fungal species (e.g. Liese 1964, 1970; Daniel 1994), but also for the early stages of brown and white rot decay (review: Wilcox 1970). Whenever hyphae were detected in fibres, the microscopic studies revealed that they were able to grow through bordered pits (Fig.