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

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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. 3) (see also Dujesiefken et al. 2011) which likely contributes to the fast spread of the disease within the xylem. Penetrations of pits by hyphae are variously described as the primary passageways of soft-rot fungi (e.g. Corbett 1965; Greaves & Levy 1965; Levi 1966; Wilcox 1970). Neither perforation hyphae between two adjacent fibres nor T or L branching within the secondary wall of a fibre were observed in the analysed material. These features are mostly associated with typical soft-rot decay (Corbett 1965; Liese 1966; Ünligil & Chafe 1974; Schwarze et al. 1995). Light microscopy of the healthy branch (control) showed the characteristic anatomical features of fibres, vessels and parenchyma cells with regard to shape, size and wall thickness (Fig. 4). In Mas- saria-diseased branches, light microscopy re- vealed distinctly decayed fibres, whereas vessels and parenchyma cells appeared largely intact (Fig. 5, 6). Liese (1970) reported for the soft-rot fungus Bisporomyces sp. that vessels are typically less degraded, an observation also reported for brown-rot fungi (Wilcox 1970). In the present study, vessel walls remained completely intact in all investigated specimens.

Figure 3. Transmission electron micrograph. Hypha in the lumen of xylem fibers growing through a bor- dered pit.

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Figure 4. Light micrograph of healthy tissue with xylem, cambium and phloem.

Figure 5 & 6. Light micrographs of Massaria-affected branch xylem. – 5: Secondary walls of fibers are distinctly degraded, whereas walls of vessels appear without decay. – 6: Severe decay of numerous fibers; secondary walls are nearly completely degraded, middle lamella portions remain.

Electron microscopic investigations on transverse sections of fibres from sound wood (Fig. 7) and fibres from early decay stages showed a few, mostly rounded cavities of different size, preferably in the S2 secondary wall layers; S1 layers as well as intercorner middle lamellae and cell corner regions (Fig. 8) appeared largely unmodified. The cavi- ties did not contain any remnants of degradation products. Singh (2009) assumed that this feature points to a complete degradation of all wall components, which was also reported by Levi (1966) for soft-rot decay of beech wood by Chaetomium globosum. S3 layers along S2 cavities often remained structurally intact, but were not seen in

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Figure 7. Transmission electron micrograph of sound branch xylem. Fiber walls with typical layering and warty layer on the inner surface.

Figure 8 & 9. Transmission electron micro- graphs of Massaria-affected branch xylem. 8: Early decay stage with cavities in secondary wall S2 layers of fibers, S1 layers remain un- modified; vessel walls do not show decay. 7 9: Advanced decay with complete degradation of some secondary wall portions; vessel walls without decay symptoms.

8 9 advanced stages of secondary wall degradation (Fig. 8). Fibres at advanced stages of decay were characterised by either a degradation of major parts of secondary walls or a complete loss of all secondary wall layers. Such a decay pattern is commonly attributed to soft-rot decay, while brown rot decay is known to leave degradation products in attacked wall portions (Bauch et al. 1976). S3 layers were not present any more along

Downloaded from Brill.com10/08/2021 06:49:26PM via free access Schmitt et al. – Massaria disease in Platanus 401 those heavily decomposed secondary wall parts. Intercorner middle lamellae and cell corner regions, however, remained unaffected, also during advanced stages. These structural details confirm the commonly higher resistance of lignin-rich wall portions against fungal attack which was described by several authors (e.g. Corbett 1965; Liese 1970; Nilsson & Daniel 1983; Daniel 1994). Increased resistance of lignin-rich wall portions against soft-rot decay was also shown for hardwood fibres with polylamellate secondary walls composed of alternating broad and narrow layers, the narrow ones having a distinctly higher lignin content than the broad ones. During soft-rot attack, broad layers are preferably degraded as reported for fibre walls of kempas (Koompas- sia malaccensis) attacked by the soft-rot fungus Chaetomium globosum (Schmitt et al. 1996). This decay pattern was also observed for polylamellate fibre walls of monocoty- ledonous woody bamboo species (Schmidt & Wei, personal communication), where higher amounts of lignin were observed in the narrow lamellae (Parameswaran & Liese 1985; Murphy & Alvin 1992; Lybeer & Koch 2005). When comparing the decay patterns of brown-rot and soft-rot, the fact that middle lamella portions and partly the S3 layers remain intact even in advanced decay stages represents a specific and often described structural feature for soft-rot decay regularly found in decayed softwoods (= soft-rot type 1: Corbett 1965; Wilcox 1970; Nilsson 1976). Soft-rot decay in hardwoods, however, may develop less cavities and more erosion type decay with a simultaneously degraded S3 layer also in early decay stages (= soft-rot type 2: Liese 1970; Wilcox 1970; Nilsson 1976). Corbett (1965) differentiated already between two types of soft-rot decay. As seen in longitudinal sections, the cavities in the plane wood with their characteristic pointed ends are usually aligned along the microfibril angle of the S2 wall layer (Fig. 14). Soft-rot decay type 2 normally produces well developed lumen erosions which might form V-shaped decay holes in the secondary wall or trough-like structures. V-shaped decay holes were not observed in the analysed Massaria-diseased plane branches, whereas some of the more heavily decayed fibres occasionally showed some evidence of erosion troughs (Fig. 9). The co-occurrence of soft-rot cavities and erosion troughs in Massaria-diseased plane trees is in agreement with observations with the white-rot fungus Inonotus hispidus facultatively producing soft-rot characteristics of type 1 and 2 (Schwarze et al. 1995). With progressing decay, numerous fibres in the diseased plane branches complete- ly lose their secondary wall layers; however, middle lamella portions and cell corner regions remain still visible (Fig. 9). The lack of hyphae in advanced decay stages as shown in Figure 8 and 9 may be due to autolysis or drying of those branch portions before as well as after breakage. In the xylem of advanced decay stages, vessels and parenchyma cells still possess unmodified cell walls (Fig. 6 and 9). The findings of higher resistance of vessel walls are probably due to their generally higher lignin content (Takabe et al. 1992; Daniel 1994; Donaldson 2001) and the occurrence of strongly conjugated guaiacyl lignins as compared to fibre walls with a lower lignin content predominantly composed of syringyl lignins (Daniel & Nilsson 1998; Nilsson et al. 1989). Accordingly, amount and type of lignin obviously play an important role in determining decay resistance of cell walls.

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Figure 10–13. UV-microscopic scanning micrographs of branch xylem as two-dimensional (left) and three-dimensional (right) profiles; color scale indicates different UV-absorbance intensities at a wavelength of 278 nm. – 10: Sound xylem with typical wall layering and higher absorbance of the vessel (v) wall, i.e., higher lignin content. – 11: Early decay stage with cavity formation in secondary wall portions. – 12: Fibers of sound xylem with highest absorbance in cell corner regions. – 13: Fibers with advanced decay, secondary walls partly totally degraded, middle lamella and cell corner portions without reduced absorbance.

Figures 10–13 show typical UV scanning profiles of sound and decayed xylem tis- sue at a wavelength of 278 nm. These images demonstrate the higher lignin content in vessel walls as evidenced by higher absorbance intensities in a range of abs278nm 0.40–0.45 as compared to the measured values of the fibre walls (abs278nm 0.2–0.25; Fig. 10, 11). No gelatinous fibres with a normally prominent G-layer were found in sam-

Downloaded from Brill.com10/08/2021 06:49:26PM via free access Schmitt et al. – Massaria disease in Platanus 403 ples taken from upper branch tissue. The described pattern of decay as observed by electron microscopy was confirmed by the UMSP analyses. The scanned cell walls of fibres and vessels of the healthy tissue (control) reveal the typical absorbance behav- iour of lignified cell walls with distinct differences between the cell corners (abs278nm 0.45–0.60), compound middle lamellae (abs278nm 0.30–0.35) and secondary walls (abs278nm 0.15–0.25) as described for several hardwoods (Koch et al. 2003, 2006; Prislan et al. 2009). The purple towards green colour pixels of the illustrated compound middle lamella (CML) represent distinct lignification with the highest absorbance values in the region of the cell corners. The absorbance values of the adjacent secondary walls show significantly lower intensities on either side of the CML. For a more detailed il- lustration, the selected scanning areas are also presented as a three-dimensional image profile where the CML region stands out as a highly absorbing band with the highest UV-absorbance at the cell corners. The three-dimensional presentation allows a better evaluation of the topochemical distribution of lignin within the individual cell wall layers, based on more than 30,000 measuring points. The three-dimensional UV-image profile revealed that the average lignin concentration in the secondary wall is less than in the CML. The absorbance values of the cell corners are about three times higher than those in the secondary wall of fibres. For comparison, the scanned cell walls of the Massaria-affected xylem show strongly delignified local cell wall areas within the S2 of fibres which are characterised by a distinct decrease of the UV-absorbance values. The S2 layers of fibres display an inhomogeneous pattern with completely degraded as well as unchanged cell wall areas (Fig. 11). These spectral changes may indicate a selective depolymerisation of the ligno-cellulosic cell wall matrix of the S2 by the fungus. However, the cell wall of vessels with a higher concentration of conjugated guaiacyl lignin as well as the wood rays with deposits of phenolic extractives are unaf- fected and do not show any detectable decrease of the UV-absorbance values. Equally, cell corners and CML regions do not show significant changes of the UV-absorbance intensities indicating a high selectivity of the lignin depolymerisation.

Figure 14. Polarised light micrograph of decayed xylem fibers, longitudinal section. Note the “pointed ends” of cavities typical for soft-rot decay type (arrowheads).

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Massaria-diseased plane wood also shows considerable differences to brown-rot decay in the absorbance behaviour of degraded secondary fibre walls at 278 nm (ab- sorbance maximum of hardwood lignin). Bauch et al. (1976) reported that brown-rot fungi such as Coniophora puteana produce an increasing absorbance in decaying cell walls during the first weeks of attack which is caused by the selective decay of cellu- lose and hemicelluloses. The same authors, however, describe a gradually decreasing absorbance for soft-rot decay by Chaetomium globosum in oak xylem. The analysed cell walls of Massaria branches do not show an increased UV-absorbance in degraded portions, also in the vicinity of cavities, but a gradually decreasing absorbance in fibre walls during decay (Fig. 12, 13). Such a comparison between brown and soft-rot clearly supports the assumption that the Massaria fungus Splanchnonema platani is producing a soft-rot rather than a brown-rot decay type. Microscopic studies also exclude white and brown-rot type for the Massaria disease, because soft-rot specific cavities in the S2 wall were formed and the remnants of S3 layers along cavities are not found in white and brown-rotted samples.

CONCLUSIONS

When comparing a typical soft-rot decay of gymnosperm wood (e.g. Liese 1970; Wilcox 1970) with the decay pattern of the investigated plane branches, there are sev- eral similarities as revealed by microscopy and spectroscopy techniques. First, in Mas- saria-diseased branches of plane trees, the fungus Splanchnonema platani forms cavi- ties in the S2 layers of fibre walls with characteristic “pointed ends” at their tips, a phenomenon also described for soft-rot in hardwoods (Corbett 1965). Second, the topo- chemical analyses of attacked plane fibres clearly show that in the vicinity of second- ary wall cavities no such chemical modifications occur as usually observed for brown and white-rot fungi. Finally, heavily degraded xylem portions display numerous fibres with degradation of their entire secondary walls, while the middle lamella portions and cell corner regions remain unmodified. According to these microscopic and micro- spectrophotometric observations in the xylem of Massaria-diseased plane branches, S. platani hyphae are able to produce a typical soft-rot decay; the decay pattern in the areas with distinct secondary wall cavities is attributed to soft-rot type 1 (for a review of decay types see also Blanchette 1995) which is known to also form erosion troughs.

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Accepted: 7 May 2014

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