Geosmithia species in southeastern USA and their affinity to beetle vectors and tree hosts
Introduction
Members of Geosmithia (Ascomycota: Bionectriaceae) are globally distributed, ubiquitous fungi that are commonly associated with bark and ambrosia beetles (Coleoptera: Scolytinae), especially with the phloem-feeding species (Kolařík et al., 2007, 2017; Lin et al., 2016; Pitt, 1979). Other wood-boring insects such as the Bostrichidae and Curculionidae may also vector Geosmithia species (Juzwik et al., 2015; Kolařík et al., 2017). Geosmithia species are predominantly isolated from beetles from woody materials, although they have been documented from a few other substrates including soil (Kolařík et al., 2004), seed-feeding beetles (Huang et al., unpublished), animal skin (Crous et al., 2018), indoor environment (Crous et al., 2018), insect-free plant tissues (McPherson et al., 2013), and food materials (Pitt and Hocking, 2012). Spores of Geosmithia are presumably transmitted by adhering to the exterior surfaces of their beetle vectors and are not known to be carried in specialized fungal transport organs (mycangia) as is known for many mutualistic fungal associates of bark and ambrosia beetles. Despite their associations with bark beetles, the ecological roles of most Geosmithia species in the symbiosis remain obscure. Some species serve as a food source or supplementary nutrition for the beetles (Kolařík and Kirkendall, 2010; Machingambi et al., 2014), but most are probably commensals with minimal or no benefit to the beetle. Some Geosmithia species exhibit extracellular antimicrobial metabolites but without a known ecological implication (Stodůlková et al., 2009). Geosmithia species are found almost exclusively on branch- and twig-dwelling bark beetles but rarely on trunk-infesting bark beetles (Kolařík and Jankowiak, 2013; Jankowiak et al., 2014). Given the fact that trunk-infesting beetles behave as pests more commonly than twig boring beetles, their fungal flora, i.e. the ophiostomatoid fungi, has received much more research attention. Branch- and twig-infesting bark beetles are equally common and diverse, but the intriguing mycobiota associated with them remains understudied.
One Geosmithia species is known to contribute to a significant tree disease: the canker-causing G. morbida (Kolařík et al., 2011). Following high density colonization by its beetle vector, the walnut twig beetle (WTB, Pityophthorus juglandis), in the phloem of walnut (Juglans spp.) or wingnut (Pterocarya spp.) trees, the fungus causes numerous small lesions and the disease is termed Thousand Cankers Disease (TCD) (Tisserat et al., 2009; Kolařík et al., 2011; Hishinuma et al., 2016). TCD has been reported in western and northeastern USA and recently in Europe (Tisserat et al., 2009; Grant et al., 2011; Hadziabdic et al., 2013; Montecchio et al., 2014). While originally G. morbida was considered an invasive species in most of the USA, the population structure of the fungus suggests that it is a native and a widespread species, albeit rare (Zerillo et al., 2014). The emergence and the disappearance of the Thousand Cankers Disease in the eastern US is, therefore, most likely a result of environmental stress on the trees, not of a pathogen invasion. Another species, Geosmithia sp. 41, was reported to induce dieback symptom on coast live oak (Quercus agrifolia) (Kolařík et al., 2017; originally reported as G. pallida by Lynch et al., 2014). These two mildly pathogenic species were thought to assist the colonization of beetle vectors by suppressing the defense system of tree hosts, however, this “immunosuppressing hypothesis” has been challenged (see Six and Wingfield, 2011).
Fungal communities associated with phloem-infesting bark beetles are shaped by multiple biotic and abiotic factors. The tree host is one of the most important factors. Several studies have shown that beetle species infesting the same tree species share similar fungal assemblages of ophiostomatoid fungi (Kirisits, 2004; Linnakoski et al., 2012; Jankowiak et al., 2017a). Other factors affecting the fungal community structure include beetle ecology, the surrounding host tree community, and climatic factors (Six and Bentz, 2007; Jankowiak et al., 2017b). These factors also influence the communities of Geosmithia, most notably by the fact that different beetles co-infesting the same host tree have similar Geosmithia assemblages (Kolařík et al., 2008; Machingambi et al., 2014). Several Geosmithia species inhabit living tree as endophytes, but their effect on the resulting Geosmithia community has not been evaluated (McPherson et al., 2013).
The specificity of the association between Geosmithia, the beetle vectors and the host trees is variable. Geosmithia species range from generalists to specialists for both beetle vectors and host trees (Kolařík et al., 2008, 2017; Kolařík and Jankowiak, 2013). For example, Geosmithia ulmacea is vectored solely by bark beetles infesting Ulmus species, Geosmithia sp. 12 is vectored by Hylesinus spp. from Fraxinus spp., Geosmithia morbida is vectored by Pityophthorus juglandis from Juglans and Pterocarya spp., and G. sp. 34 and 44 occurring exclusively on beetles from Calocedrus decurrens and Pinus spp. (Kolařík et al., 2017). In contrast, some generalist Geosmithia, e.g. members in the G. pallida species complex (GPSC), can be recovered from varied beetle vectors from varied hosts. It remains unclear whether the host tree specialist Geosmithia are also specific to particular beetle vectors. Some Geosmithia species are found almost exclusively on beetles that are specific to a limited range of tree species. The specificity observed could be an artefact of specificity of some bark beetles to host trees, or beetle-selected microenvironment.
The question of vector specificity is important for our understanding of the economically important Thousand Cankers Disease. Surveys of the G. morbida in North America have revealed that P. juglandis is the predominant vector, but some generalist beetles such as Xylosandrus crassiusculus (Curculionidae, Scolytinae), Xyleborinus saxesenii (Curculionidae, Scolytinae), and Stenomimus pallidus (Curculionidae, Cossoninae) emerging from J. nigra can also harbor G. morbida propagules (Juzwik et al., 2015, 2016). A broad, systematic survey of alternative vectors of G. morbida is therefore needed, considering the possibility of spread of the fungus beyond the original vector.
Geosmithia studies in North America have focused on the causal agents of TCD (i.e. G. morbida) and mostly conducted in the West and Northeast, where black walnuts are prevalent (Burns and Honkala, 1990). The community of Geosmithia species in the Southeast, however, has never before been systematically addressed. The two Geosmithia community surveys in North America have hinted at what appears to be a large species diversity with many undocumented species and new Geosmithia-beetle-tree associations (Kolařík et al., 2017; Huang et al., 2018). The Southeastern region hosts the highest diversity of tree species and the highest diversity of bark beetles (Atkinson, 2018), and therefore it may be the center of the Geosmithia diversity in North America.
We conducted a culture-based survey of Geosmithia associated with bark beetles in North Florida and Georgia. Our replicated and phylogenetically informed sampling design allowed us to ask three questions: (1) Are the causal agents of TCD (the fungus and the beetle) present in the surveyed region? (2) If G. morbida is present, are there any alternative beetle vector or tree hosts that accommodate this fungus? and (3) What is the specificity of the Geosmithia association with its beetle vectors and tree hosts?
Section snippets
Sampling and isolation
We sampled host trees that represent the local diversity of Juglandaceae: black walnut (J. nigra), pignut hickory (Carya glabra), and pecan (C. illinoinensis), and six tree species that are phylogenetically divergent but common in the Southeast: red cedar (Juniperus virginiana), loblolly pine (Pinus taeda), laurel oak (Quercus laurifolia), American sweetgum (Liquidambar styraciflua), sugarberry (Celtis laevigata), and white ash (Fraxinus americana). To characterize the Geosmithia-beetle-tree
Identification of Geosmithia species and their occurrence on trees and beetles
In total, 195 beetle specimens were excised from 45 exposed branch units (Table 1), of which 23.1% (n = 45) yielded Geosmithia species resulting in 55 isolates. We did not find Pityophthorus juglandis, the known vector of TCD, in the assayed branch baits.
Among the 55 Geosmithia isolates, 14 species were determined based on the morphological characteristics and molecular makers (Fig. 2). We did not recover G. morbida in our isolates. Species in the G. pallida species complex (GPSC, i.e. pallida,
Discussion
This is the first study to quantitatively examine the symbiotic relationship of Geosmithia species with their beetle vectors and host trees in the southeastern USA. We systematically deployed branch sections from targeted species to be colonized by bark beetles that potentially carry Geosmithia species, and quantitatively described the Geosmithia community in relation to their beetle vectors and host trees.
We considered beetle individuals as a quantitative unit for studying their association
Conclusion
The association among fungi, bark beetle vectors, and host trees is often thought to be complicated, but continued sampling is beginning to explain the patterns in these relationships. This dataset suggests that the distribution of Geosmithia among beetle vectors is primarily driven by the encounters of the beetles and the fungi in the hosts tree substrates, and in some cases by phylogenetic relatedness between the fungi, but the fungus-beetle associations are flexible. Moreover, as the beetles
Acknowledgement
We thank John and Katherine Ewel for access to their pecan farm. This work was supported by the United States Department of Agriculture Forest Service, USDA APHIS Farm Bill section 10007, Florida Department of Agriculture and Consumer Services – Division of Plant Industry, and the National Science Foundation (DEB 1556283).
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