Portal de Periódicos da CAPES

Blue stain fungi infecting an 84‐million‐year‐old conifer from South Africa

2021; Wiley; Volume: 233; Issue: 3 Linguagem: Inglês

10.1111/nph.17843

1469-8137

Christine Strullu‐Derrien, Marc Philippe, Paul Kenrick, Robert A. Blanchette,

Plant Pathogens and Fungal Diseases

Fossil fungi are frequently observed in association with fossil plants in a geological record that dates back over 400 Myr to the beginning of the Devonian Period (e.g. Strullu-Derrien et al., 2014, 2018; Taylor et al., 2015; Krings et al., 2018; Krings & Harper, 2019). Despite this lengthy fossil record, there are relatively few studies of wood colonizing fungi (e.g. Pujana, et al., 2009; Feng et al., 2015; McLoughlin & Strullu-Derrien, 2015; Harper et al., 2017; Gnaedinger & Zavattieri, 2020; Scaramuzza dos Santos et al., 2020; Tian et al., 2020). Nowadays, fungi that induce white, brown, or soft rots are recognized as the major groups capable of degrading wood, but not all to the same level: only white rot fungi have the potential to degrade the entirety of the wood structure. Each decay type produces diagnostic micromorphological signatures (Blanchette, 1995, 2000; Schwarze, 2007). Features such as these can be preserved in petrified woods along with the fungi that gave rise to them. One group of wood colonizing fungi that do not destroy lignocellulose, but which often hasten the death of trees attacked by insects, are blue stain fungi. These are a polyphyletic group taxonomically placed in the Ascomycota (e.g. Ophiostoma, Grosmannia, Ceratocystiopsis, Ceratocystis and others) (Seifert et al., 2013; de Beer et al., 2014), the spores of which are frequently dispersed by bark beetles and other wood inhabiting insects (Leach et al., 1934; Peris et al., 2021). Once established, their hyphae extend into the sapwood of trees. Some are pathogenic, infecting living cells, whereas others are more saprobic, subsisting on resins and lipids. They have been observed in the parenchyma cells and resin ducts of conifers, and inside vessels, fibres, and parenchyma cells of angiosperms (Ballard et al., 1983; Eriksson et al., 1990; Blanchette et al., 1992; DiGuistini et al., 2011). Typical for blue stain species is the generation of dark-coloured melanin within their hyphal cell walls, which provides a measure of protection against UV light, water stress, and host tree resistance factors (Lundell et al., 2014). Blue stain fungi also have unique patterns of colonization and characteristics that can be used to identify them in wood. These include septate hyphae that are pigmented, preferential colonization of ray parenchyma cells and resin ducts, and the ability to produce holes in plant cell walls that allow them to move through said walls (Hubert, 1931; Eriksson et al., 1990; Blanchette et al., 1992). The mechanism used for penetrating the plant cell wall appears to be physical force, where an appressorium-like structure penetrates the wall with a small diameter hyphal peg. Once through the cell wall, the fungus resumes normal growth within the cell lumen (Ballard et al., 1983). Here we describe a fungus colonizing wood of the extinct conifer Agathoxylon Hartig from the Upper Cretaceous Mzamba Formation of Pondoland (South Africa), and we draw comparisons with a blue stain fungus colonizing the wood of extant Pinus strobus. Our new observations lead us to conclude that this fossil represents the first documented occurrence of blue stain fungi in the geological record. The Mzamba Formation in Pondoland, South Africa, is restricted to a small area in the Mzamba River Estuary (south of Port Edward) and a series of discontinuous outcrops in a narrow coastal strip extending northwards to Richards Bay in KwaZulu-Natal (Johnson et al., 2006). The sediments are interpreted as a submarine mass flow deposit (Smith & Guastella, 2018). Logs were most likely originally deposited on the palaeocoast by the proto-Mzamba River and subsequently transferred below the storm wave base by submarine mass flows (Smith & Guastella, 2018), where they became silicified. This palaeoenvironmental interpretation is consistent with the presence of crustacean burrows assigned to the ichnogenus Ophiomorpha, which is generally considered to be an indicator of high-energy marine palaeoenvironments (Leaman et al., 2015). An ammonite-based study dated the Mzamba formation as Middle Santonian to Early Campanian stages (c. 84–80 million years ago (Ma)) of the Upper Cretaceous, the basal wood-yielding beds being Middle Santonian (c. 84 Ma) (Klinger & Kennedy, 1980). The material studied consists of three thin sections housed in the collections of the Senckenberg Museum in Frankfurt (Germany) under the numbers SMB 9665/1 to 9665/3. These were made from an original sample of silicified wood in the collections of the South African Museum, Cape Town, numbered SAM 9547, that was borrowed by Richard Kräusel during a visit in 1953–1954 (Schultze-Motel, 1966). According to Schultze-Motel the material was returned, but it has not been possible to find it in the collections of the South African Museum. Observations of the fossil fungus were made using a Wild Heerbrugg (St-Gall, Switzerland) Makroskop M420; photographs were taken using a Leitz Metallux 3 camera (Solms, Germany) and the LAS suite (https://www.leica-microsystems.com/). The images shown in Figs 1-3 are untouched, except for some adjustment of contrast or light level. Eastern white pine (Pinus strobus L.) wood with blue stained sapwood was sectioned and observed using a Nikon (Melville, NY, USA) E600 compound light microscope, and photographs were taken using a Nikon DXM1200F digital camera. Xylological analysis of 27 fossil wood specimens from this locality led Schultze-Motel (1966) to describe a new genus, Dammaroxylon Schultze-Motel. Due to its poor preservation, he suggested that specimen SMB 6965 should be left in open nomenclature as Dammaroxylon sp. Dammaroxylon is nowadays considered to be a junior taxonomic synonym of Agathoxylon (Philippe, 1995; Rößler et al., 2014). The three slides that we examined show a homoxylous wood. Intertracheary pits are uniseriate and contiguous, in a typical araucarian pattern (Philippe & Bamford, 2008). Cross-field pits are only locally preserved, and cross-fields are of the araucarioid type. What can be observed of the anatomy of this wood does not allow it to be assigned more precisely than to Agathoxylon sp. in accordance with Schultze-Motel's conclusions. A late Cretaceous wood with such anatomy can be considered with a relatively high degree of certainty to belong to the family Araucariaceae. In most modern trees, two types of wood can be recognized: sapwood is the outer, pale-coloured wood, and heartwood the inner, mostly darker wood (Githiomi & Dougal, 2012). Heartwood does not differ structurally from sapwood (Taylor et al., 2002), but they do differ functionally. Sapwood is the principal conducting tissue within the trunk and it also plays a role in carbohydrate storage. Sapwood contains a variety of cell types, some of which are living and physiologically active, whereas heartwood no longer contains living cells (Lehnebach et al., 2017). In gymnosperms, another feature that is important in heartwood formation is bordered pit aspiration, which occurs as wood moisture levels drop, causing the pit membrane to move across the pit chamber and into contact with the pit border, where it remains. This happens in the innermost sapwood or in the outer part of the transition zone (Kozlowski & Pallardy, 1997). The small sample of fossil wood we studied did not allow us to observe either a change of color or aspiration of pits. The main feature that would point to a sapwood identity is the fungal colonization of the ray parenchyma cells, which implies that the substrate for the fungus is carbohydrates and other nutrients within the cytoplasm of living cells. The Agathoxylon wood is altered by fungal colonization. It appears to have undergone a colour change, becoming dark and taking on a reddish hue (Fig. 1a). In the radial section, hyphae are growing throughout the cells, from ray parenchyma cells to other cells (Figs 1b,c, 2a,d). Many appear to be unbranched, growing perpendicularly (Fig. 1b,c) or parallel to the tracheids (Fig. 2). Within the rays, where hyphae occur preferentially, they are more densely packed and occasionally branched (Fig. 1c,d). The hyphae can be seen to penetrate the cell wall without eroding it (Figs 1b,c, 2a,d). A hyphal peg was not observed, but the hyphae have clearly penetrated several cell walls as they grew across the cells. To do this without cell wall erosion, it is likely that the fungus produced penetration pegs. Hyphae are septate, 3–5 µm in diameter, and have well delimited dark walls (Fig. 2b,c). Swellings at the end of some filaments might represent chlamydospores (Fig. 2d). Histological studies of blue stain fungi colonizing wood have previously been published (Liese, 1970; Ballard et al., 1983; Eriksson et al., 1990; Blanchette et al., 1992) but additional examination was done on modern wood (Fig. 3a) to provide micrographs to compare with the fossil. Radial sections of pine wood showed a preferential colonization of the ray parenchyma cells. Pigmented hyphae were able to grow in the lumina of some tracheids but were concentrated in the ray parenchyma cells (Fig. 3b–d), Hyphae move from one tracheid to another by forming appressoria-like structures that penetrated the cell wall (Fig. 3c). A thin penetration peg traversed the cell wall, and hyphae resumed normal sized growth once in the adjacent cell lumen. Hyphae often penetrated through many tracheids without causing cell wall erosion. Some cell wall degradation of nonlignified ray parenchyma cells was apparent. Hyphae colonizing the wood cells are septate (Fig. 3b). The fossilization of the Agathoxylon wood involved transportation by water currents and burial in a submarine mass flow deposit before petrifaction. Under these circumstances, the surfaces of logs become abraded, so it is very unlikely that fruiting bodies such as perithecia or synnemata would be preserved. Our identification of the fossil fungus was therefore based entirely on micromorphological signatures preserved within the xylem. The occurrence of septate hyphae enables us to place this new fossil fungus in the Ascomycota. The fossil exhibits several features that are very similar to blue stain fungi. The dark and thick walls of the hyphae are suggestive of pigmentation, an inference that finds further support in the darkened and reddish hue of the wood. The hyphae are particularly abundant in the ray parenchyma, suggesting preferential colonization of this tissue. Furthermore, they do not appear to erode or decompose the tracheid cell walls. Although we have not observed penetration pegs or appressoria directly, the observed passage of hyphae through neighbouring cells walls and cell lumens without disruption is again suggestive of hyphal growth in blue stain fungi. We conclude that this suite of features provides strong evidence of an affinity with blue stain fungi. Early molecular phylogenetic studies showed that blue stain fungi are a polyphyletic assemblage of genera within the Sordariomycetes (Ascomycota) (Spatafora & Blackwell, 1994). Recent research based on much larger samples of species provides further phylogenetic insights into the genera previously placed in this group. The relationship between Ceratocystis and Ophiostoma was confirmed to be a distant one, and it is now established that Ophiostoma belongs to the Ophiostomatales (Sordariomycetidae) and Ceratocystis to the Ceratocystidaceae (Microascales; Hypocreomycetidae) (Réblová et al., 2011; de Beer et al., 2013). Ceratocystis sensu lato was further segregated into 14 genera, each with distinctive morphological features and ecological roles (de Beer et al., 2014; Mayers et al., 2020). In addition to blue-stain fungi, Ophiostomatales and Ceratocystidaceae contain plant pathogens, wound colonizers and ambrosia fungi. The ambrosia fungi form a mutualistic relationship with ambrosia beetles, which cultivate the fungus in galleries within the less nutritious heartwood. The fungus is the principal source of food for their larvae (Peris et al., 2021). The sap stain species of conifers are placed in the genus Endoconidiophora. Notable similarities between Ophiostoma and Ceratocystis sensu lato, including similar ascomata with globose bases, generally long necks, and ascospores exuded in slimy masses, are most probably convergent adaptations to their insect vectors (de Beer et al., 2014). Because of the lack of taxonomically diagnostic characters in the fossil, we are unable to make further comparisons with extant genera. The Sordariomycetes are clearly an ancient group of fungi. Calibrated molecular phylogenetic trees provide a range of dates for their divergence from other Ascomycota that span the Late Palaeozoic to the earliest Mesozoic (i.e. Middle Mississippian to Lower Triassic) (Hongsanan et al., 2017, and references cited therein). A recent phylogenomic study gave a slightly younger stem-based age for the group of c. 240 Ma (Middle Triassic) (Shen et al., 2020). L. Le Renard et al. (unpublished) show that Spataporthe taylorii (Bronson et al., 2013) from the Lower Cretaceous of Vancouver Island in Canada represents the oldest unambiguous fossil Sordariomycete. The Ophiostomataceae crown group origin was calibrated to c. 90 Ma (130–50 Ma confidence interval; Hyde et al., 2017), placing the mean calibration in the Lower Cretaceous (132–129 Ma). Based on a larger sample of ingroup species and a genomic scale analysis, Vanderpool et al. (2018) calibrated the Ophiostomatales crown to 101 Ma (±25 Ma; Mid Cretaceous), and Mayers et al. (2020) estimated a median age of the Ceratocystidaceae crown of c. 80 Ma (54–116 Ma confidence interval; Upper Cretaceous). The first fossil evidence of an ambrosia fungus of likely ophiostomatoid affinity has been described from filamentous sporodochia adjacent to a wood-boring beetle preserved in mid Cretaceous (110–97 Ma) amber from Myanmar (Poinar & Vega, 2018). This fossil and the molecular calibrations of living species indicate that the geological record of blue stain fungi could extend as far back as the latter part of the Mesozoic Era, which is consistent with our fossil finding. Calibrating the fungal tree of life is especially problematic because of the lack of fossil evidence that can be placed with confidence and precision into the phylogenetic scheme. For example, Paleopyrenomycites devonicus from the Rhynie chert (Taylor et al., 1999, 2005) is widely used in dating the fungal tree of life, and an affinity with Ascomycota is generally accepted. However, the idea that P. devonicus represents an extinct clade of stem group Ascomycota has recently been suggested (Berbee et al., 2020). The critical and careful placement of fossils within the fungal tree of life is essential to establishing a better justified calibration. Blue stain fungi are dispersed by bark beetles in the weevil subfamily Scolytinae. These beetles have developed an intricate relationship with the fungi that has evolved over millions of years of interaction and co-evolution (Peris et al., 2021). The bark beetle lifestyle is thought to have originated in woody angiosperms, to be followed by multiple later transitions to conifer feeding (Pistone et al., 2018). Our new fossil indicates that a shift to conifers had already occurred in Gondwanan Araucariaceae by the Santonian Stage of the Upper Cretaceous. The Scolytinae have a lengthy geological record. The oldest fossil comes from Lebanese amber of the Lower Cretaceous (c. 125 Ma) (Kirejtshuk et al., 2009). Another potentially significant early find was a fossil attributed to the extant genus Microborus in amber from Myanmar (c. 100 Ma) (Cognato & Grimaldi, 2009), but further study cast doubt on the provenance and age of the amber from which Microborus has been described (Clarke et al., 2019). Recent molecular calibrations give ranges for the origin of crown group Scolytinae that span the uppermost part of the Cretaceous and early Paleogene (Shin et al., 2017) through to firmly within the latter part of the Mesozoic Era: 82 Ma (±16 Ma; Upper Cretaceous) (Gunter et al., 2016) and 112 Ma (±4 Ma; mid Cretaceous) (Pistone et al., 2018). Our finding of a blue stain fungus in wood of the Upper Cretaceous (Santonian Stage; c. 84 Ma) is therefore more consistent with the older molecular calibrations of its primary insect dispersal agent, suggesting that this important ecological association could have first developed during the latter part of the Mesozoic Era. The quality and diversity of the fossil record of fungi is beginning to gain recognition, but it still remains a largely untapped treasure trove. Petrified woods are a prime example of an important but barely used source of information on fossil fungi. They are common in museum collections world-wide, where they were originally collected and prepared with the purpose of studying fossil plants. As we have shown here and elsewhere, they are also an important source of information on the microorganisms that use plants as a food resource. Through re-examination of a piece of petrified wood that was collected and described in botanical terms over 50 years ago, we document the first fossil evidence of blue stain fungus infecting wood. We further suggest that an ecological association with bark beetles as a dispersal agent plausibly originated during the latter part of the Mesozoic Era. CS-D thanks the Fondation ARS Cuttoli-Paul Appell/ Fondation de France for supporting her work on fossil fungi (grant no. 00103178). MP received support from the SYNTHESYS+ Project (www.synthesys.info/), which is financed by the European Commission via the H2020 Research Infrastructure programme. Sarena Govender, Iziko Museums of South Africa, is acknowledged for having searched for the original sample in the collections of the South African Museum (Cape Town, South Africa). CS-D and RAB conceived of the study. MP and RAB acquired specimen photographs. CS-D, RAB and MP analysed the data. CS-D and RAB wrote the first draft, helped by PK. All authors contributed to the final version of the manuscript. Data available on request from the authors.