Introduction

Bark and ambrosia beetles (Coleoptera: Curculionidae: Scolytinae and Platypodinae) are associated with a diverse set of ecto and endosymbionts, classified among the prokaryotes, filamentous fungi, yeasts, and microinvertebrates. Fungal symbionts are the most studied, and their dependency on the insect vector ranges from obligatory, in strictly entomochoric fungi, to incidental ones, acquired from the environment. Fungal symbionts interact with the host insect and tree, forming mutualistic, commensal, or antagonistic interactions (Beaver 1989; Six 2013; Hofstetter et al. 2015). The best-studied fungal symbionts of bark beetles belong to ophiostomatoid fungi (Ascomycota: Ophiostomatales) and Microascales. However, beetle galleries frequently harbor many other fungal groups, and many of the non-ophiostomatalean have been historically ignored (Kirschner 2001; Kirschner et al. 2001; Kolařík et al. 2006; Jankowiak and Kolarik 2010). Filamentous fungi placed in the genus Geosmithia (Ascomycota: Hypocreales, Bionectriaceae) used to be sporadically reported as plant or soil saprobes (Pitt 1979; Pitt and Hocking 2009). The very first record of Geosmithia from the bark beetle niche, and a suggestion of its phytopathogenicity, was from fir-infesting bark beetle species in the USA by Wright (1938), but the fungus was misidentified as Spicaria anomala (Kolařík et al. 2017). The regular association of Geosmithia fungi with bark beetles was simultaneously discovered in Germany (Kirschner 1998, 2001) and Czechia (Kubátová et al. 1999, 2004).

During the first decade of the new millennium, the question of the tightness of the association of Geosmithia with bark beetles was not yet settled. The reasons for this are varied. The species that had been identified in early studies, such as G. putterillii, have been known from various non-specific substrates such as soil or cereals (Kolařík et al. 2004; Pitt and Hocking 2009). In addition, the generic concept of Geosmithia before 2012 included species of Hypocreales (Geosmithia in the current definition) but also Eurotiales, which have no connection to insects (Houbraken et al. 2012). Furthermore, Geosmithia species strongly resemble certain species of Penicillium, Paecilomyces, or Mariannaea, which are common and also widely ignored residents of bark beetle galleries. Geosmithia produces masses of dry spores, a typical feature of airborne fungi, but does not form slimy spores, a typical entomochoric adaptation. In addition, Geosmithia is typically found on hardwoods and conifers of the cypress family, associated with little-studied secondary bark beetles of minor economic importance. Finally, Geosmithia is highly sensitive to cycloheximide, an antifungal agent often used to isolate Ophiostomatales, which are resistant to the compound, from bark beetles. This means Geosmithia and their associations with bark beetles were often missed, and there was a skepticism about the significance of the association.

In the more recent years, however, many independent studies confirmed Geosmithia species as stable and often dominant symbionts of many bark beetles worldwide, forming fungal communities specific to the host trees frequented by the vector beetles. The subsequent discovery of a phytopathogenic species G. morbida (Kolařík et al. 2011), and also species living as primary ambrosia fungi (Kolařík and Kirkendall 2010), resulted in the recognition of Geosmithia as a genus containing regular bark beetle symbionts with possible mutual coevolution.

Here, we summarize and interpret Geosmithia biology based on a review of more than 140 publications (Fig. 1). This review, for the first time, synthesizes the taxonomy, diversity, ecology, biogeography, and biotechnological potential of the genus Geosmithia, including a description of the history of research and an outline of future directions. The paper also addresses the question of why some bark beetles are associated with Geosmithia and other species are not.

Fig. 1
figure 1

Upper part. Chronology of important events related to Geosmithia taxonomy (below) and ecology, host range, and biogeography (above). Lower part. Overview of publications on hypocrealean Geosmithia species over the last 20 years (2001–2021), with a breakdown of papers focusing on thousand cankers disease (orange bar) and on other aspects (blue bar). The graph is based on articles extracted from the Scopus database and additional important papers. The chart does not include the numerous papers that focus primarily on the biology of the walnut twig beetle, a TCD vector. The graph also presents the increase of described species within the genus (pink line)

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History of the genus and the main differentiation features

Like in other morpho-genera of anamorphic fungi, the Geosmithia generic concept has undergone dramatic changes (Fig. 1). In the current concept, its characteristics include the following: absence of a sexual state, variable colony colors but never green (which diagnoses it as distinct from Penicillium), the cylindrical shape of phialides without a prominent neck and with roughened walls, elliptical to cylindrical conidia produced in chains, and the specific initials and base of the conidiophore (Kolařík et al. 2004). The discovery of morphologically unique ambrosia fungi in Geosmithia (the identity determined by using gene sequencing) expands this morphological concept to also include solitary and globose conidia (Kolařík and Kirkendall 2010). The colony color ranges from white to cream, to various shades of yellow, brown, rusty, or red. Geosmithia produces the Penicillium-like conidiophores, or conidiophores can be much more complex, irregularly and repeatedly branched. Besides macronematous conidiophores with enteroblastic phialides, semimicronematous or microcronematous conidiophores can also be formed on aerial or substrate mycelium (Kolařík et al. 2004). Whereas Penicillium-like conidiophores produce columns of dry conidia, microcronematous conidiophores form holoblastic, solitary conidia in slimy droplets. This conidial type, referred to as substrate conidia, is another feature found in related genera such as Gliocladium and Nalanthamala (Schroers et al. 2005). Another typical Geosmithia feature is the conidiophore base, making a so-called peg foot with smooth cell walls and curved shapes (Kolařík et al. 2004) (Fig. 2).

Fig. 2
figure 2

Morphological features of Geosmithia. A Colony morphology on MEA can range from brown (G. funiculosa), lilac (G. carolii), white to cream (G. putterillii), and yellowish (Geosmithia sp. 11). B, C A yeast-like stage is present in some species during the initial growth phase. Geosmithia carolii on MEA, 1 day, 24 °C. D Oblong and catenate conidia of G. carolii. E Globose and multinucleate conidia of G. eupagioceri stained by propidium iodide and observed under confocal microscope. F Long conidial chains of Geosmithia sp. 8 CCF4528. G Solitarily produced conidia of G. microcorthyli. H Substrate conidia of G. carolii. I Penicillate conidiophore in G. putterillii. J The complex branched conidiophore of G. eupagioceri. K A simple conidiophore in Geosmithia sp. 31. Scale bars B 500 µm, C–E, H, K 10 µm; I, J 20 µm

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The history of Geosmithia taxonomy is linked to the morphologically similar Penicillium, and the type species was first described as Penicillium putterilli (Thom 1930). Species later classified in Geosmithia were first aggregated into the series P. pallidum in section Asymmetrica–Funiculosa that was established for P. pallidum, P. putterillii, P. lavendulum (now in Geosmithia), and P. namyslowskii (now in Penicillium, Eurotiales) (Raper and Thom 1949). John Pitt (1979) proposed a new genus Geosmithia, named in honor of George Smith, to include species from the P. pallidum series and some species now classified in Eurotiales. Although at first the concept was not accepted by some authors (Ramirez 1982; Stolk and Samson 1986), it was soon solidified in taxonomic lists (Pitt and Samson 1993; Pitt et al. 2000), and other authors begun to use the name Geosmithia for newly discovered species of similar morphology (Pitt and Hocking 1985; Yaguchi et al. 1993, 1994, 2005). The first studies utilizing molecular data showed that some of the species, including the type species G. putterillii, belonged to Hypocreales, whereas others belonged to the Eurotiales (Ogawa et al. 1997; Ogawa and Sugiyama 2000; Peterson 2000; Iwamoto et al. 2002). An eventual revision resulted in the creation of the monophyletic Geosmithia within Hypocreales and placed other species into the genera of Penicillium, Rasamsonia, and Talaromyces within Eurotiales (Houbraken et al. 2012). These changes also affected the classification of Rasamsonia argillacea, a fungus of clinical importance (Giraud et al. 2013), which is still sometimes incorrectly identified by the old name Geosmithia argillacea (Giordano et al. 2021).

Taxonomy and diversity

The genus possesses relatively high phylogenetic diversity, with over 67 phylogenetic species, of which 32 have been formally described (Fig. 1, Table 1). Most of the remaining species have been studied to a degree that allows diagnosis to the species level, but they have not been described formally. These species are informally identified by numbers. This numbering system originated in Kolařík et al. (2007, 2008), and species thus labeled are frequently used in literature (Table 1).

Table 1 List of the recognized Geosmithia species with geographical distribution and substrate origin. The host spectrum is expressed as a list of host plant families from which the insect vector was collected. Only data confirmed by molecular data are shown
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Before 2004, only two species, G. lavendula and G. putterillii (incl. its synonym, P. pallidum), were formally accepted. Later, G. putterillii was found to be a complex of three species, G. putterillii, G. pallida (it itself consisting of five phylogenetic species), and G. flava (Kolařík et al. 2004). Three other species, G. fassatiae, G. langdoni, and G. obscura were described from bark beetles in Europe (Kolařík et al. 2005). A large survey of Geosmithia in Europe and the Mediterranean basin recognized other 23 undescribed species marked as Geosmithia spp. 1–5, 8–13, 16, 19–31 (Kolařík et al. 2007, 2008; Kolařík and Jankowiak 2013). Subsequent surveys in the USA revealed other 20 species, classified as Geosmithia spp. 32–48 (Kolařík et al. 2017; Huang et al. 2019) or described as G. morbida (Kolařík et al. 2011), G. proliferans, and G. brunea (Huang et al. 2017). Recently, several numbered species were formally described: G. ulmacea (sp. 13) and G. omnicola (sp. 10) (Pepori et al. 2015), G. xerotolerans (sp. 21), G. carolliae (sp. 19) (Crous et al. 2018) and G. longistipitata (sp. 28) (Strzałka et al. 2021). Some of the previously recognized taxa (sp. 2—G. pumila, sp. 3, 23—G. pulverea, sp. 20—G. granulata) and others newly found (G. luteobrunnea, G. radiata, G. brevistipitata, G. bombycina, G. subfulva, and G. fusca) were described from China (Zhang et al. 2022), Israel, and Europe (G. cupressina, G. fagi, and G. pazoutovae) (Strzałka et al. 2021; Meshram et al. 2022)). Four species, G. eupagioceri, G. microcorthyli, G. rufescens, and C. cnesini, were described from ambrosia beetles in Costa Rica (Kolařík and Kirkendall 2010; Kolařík et al. 2015). Other five tentative and undescribed species were recognized during the surveys on bark beetles in South Africa and Israel (Machingambi et al. 2014; Dori-Bachash et al. 2015) or on other substrates (Deka and Jha 2018; Sun et al. 2018) (Table 1). The species G. tibetensis (Wu et al. 2013), described from soil in Tibet, may not be a true Geosmithia; no molecular data were provided, and its morphology fits that of Eurotiales.

The methods used to characterize Geosmithia species follow those used in studies on the genus Penicillium and Aspergillus. The most commonly used culture substrates are two nutrient-rich media, Malt extract agar (MEA) and Czapek Yeast Autolysate Agar (CYA), and the basal medium Czapek Dox Agar (CZD), the combination of which provides good resolution between most species. Regarding the cultivation temperature, growth at 24–25 °C, the optimal temperature for perhaps all species, and 37 °C, tolerated by few species only (e.g., G. lavendula and G. morbida) is studied.

The ITS rDNA marker, commonly used to delimit species across fungi, is used to characterize Geosmithia species, but it has its limits, especially among closely related species. Therefore, alternative markers are needed for better resolution in some species complexes. Other commonly used markers include the genes for the RNA polymerase II second largest subunit (RPB2, region defined by the primers fRPB2-5F/fRPB2-7R), β-tubulin (TUB2, primers T10/Bt2b) and translation elongation factor 1-α (TEF-1α) including the large exon part (primers EF1-983F/EF1-2218R) and the intron area (EF1-728F/EF1-986R). The latter shows by far the greatest variability among Geosmithia species (Strzałka et al. 2021). The discriminatory power of the alternative markers can be assessed by studying groups of species that are clearly distinguishable morphologically and ecologically, but have identical ITS sequences, such as G. microcorthyli (Kolařík and Kirkendall 2010), G. longistipitata (Strzałka et al. 2021), Geosmithia sp. 24 (Dori-Bachash et al. 2015), Geosmithia sp. 16 (Kolařík and Jankowiak 2013), and G. langdonii species complexes (Kolařík et al. 2017).

Host range and intimacy of the association with bark beetles

Geosmithia species are most commonly isolated from the subcortical niche created by bark beetles. The materials which yield most colony-forming units are the internal surfaces of galleries, particularly the pupal chambers, but also the surface of eggs, larvae, and adults, and the gallery detritus. Adults captured outside of galleries, prior to the gallery initiation or after emergence from pupation, also frequently yield Geosmithia cultures. Geosmithia fungi are usually isolated from all the gallery throughout the insect’s life cycle and can be visually conspicuous, particularly in pupal chambers and detritus in larval passages (Fig. 3).

Fig. 3
figure 3

Geosmithia on native samples and cultivations plates A G. lavendula (lilac) and G. radiata (white) in Hypoporus ficus galleries (Ficus, Croatia). B G. lavendula in bostrichid gallery (Toxicodendron, California). C G. microcorthyli in galleries of ambrosia beetle Microcorhyllus sp. (Costa Rica). D G. flava in galleries of Ernoporus tiliae (Tilia, Czechia). E G. flava in pupal chamber of Cryphalus piceae (Abies, Czechia). F Necrosis caused by G. morbida in the phloem of Juglans. G G. flava and Ophiostoma novo-ulmi (white droplets) growing on agar plated with Scolytus multistriatus adults (Ulmus, Czechia). H Agar plate with Geosmithia colonies obtained from H. ficus galleries (Ficus, Croatia). I. Pityophthorus pityographus adult and detritus from the gallery overgrown by yeasts and Geosmithia sp. 24 (Pinus, Czechia)

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Each of the above substrates requires a different approach for optimal Geosmithia recovery. Spores attached to surfaces of beetle adults and larvae are cultured using a wash on standard agar media MEA and PDA, and spore load is quantified by serial dilution. Fungi from gallery detritus or walls can be cultured by directly spreading this material onto agar plates. This method readily yields Geosmithia, but it is not quantitative. To reduce the ubiquitous, non-indigenous fast-growing molds, a rinse in a modified White solution can be used, particularly when isolating from beetles captured in the environment outside of the galleries (Barras 1972; Kolařík et al. 2008). Geosmithia communities can be documented without culturing by using DNA metabarcoding with the standard ITS rDNA primers (Morales-Rodríguez et al. 2021).

Since the pioneering work of Wright (1938), 153 species of subcortical insects (Curculionidae, Scolytinae, and Platypodinae: 140; other Curculionidae: 5; Cerambycidae: 2; Bostrichidae: 6) have been studied for the presence of Geosmithia; this fungus was found on 119 of them (Table 2). Within scolytine beetles, it was common on phloem-feeding species (111 out of 140 species) but also on ambrosia beetles (10 species out of 14). It has also been found on seed-feeding Coccotrypes species (Scolytinae). Geosmithia vectors from other beetle groups include the Bostrichidae (6 out of 6 studied species) and Cerambycidae (2 of 2 studied species). It was absent in conifer-associated weevils of the genera Hylobius and Pissodes, but it was isolated from another subcortical weevil, Magdalis armigera from elm. Surveys focused specifically on Geosmithia, or comprehensively documenting fungal communities of subcortical beetles, have been carried out mainly in Europe, the Mediterranean basin, and North America, with fewer studies from the rest of the world, such as from South America, South Africa, China, and Taiwan (Fig. 4, Tables 1 and 2).

Table 2 Summary of the insect vectors studied for Geosmithia presence and the strength of Geosmithia-vector association
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Fig. 4
figure 4

Map showing the locations where Geosmithia species spectrum and diversity has been studied and indicates the total number of species found in each area and the number of species not yet found outside that area (“endemic” species). The map is based on Table 2

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The degree of Geosmithia association with tree hosts or with beetle vectors can be determined by various quantitative approaches. Unfortunately, different approaches have been used by different authors, including nonstandard definitions of a sample and of sample size, making it difficult to compare studies. We recommend using basic measures such as the proportion of gallery systems (e.g., Kolařík et al. 2017), of numbers of insect bodies (adults, larvae), or gallery segments (e.g., Jankowiak et al. 2014; Dori-Bachash et al. 2015) with Geosmithia present, out of all sampled. A more quantitative estimate of prevalence is the percentage of CFU counts belonging to Geosmithia within the entire fungus community (Skelton et al. 2018).

Already Roland Kirschner (2001) noted that bark beetles differ in their degree of association with Geosmithia. Subcortical insects (mostly bark beetles) can be divided into those with whom Geosmithia is associated strongly, moderately, or not at all (Table 2, Fig. 5). Strongly associated vectors include beetles infesting broad-leaved shrubs and trees, with two exceptions: (1) beetles on Betula and Alnus and (2) beetle species preferring trunk bases. Beetles on conifers with thin bark are also strongly associated vectors, particularly the many twig- and branch-beetles on Pinaceae and Cupressaceae. Most conifers within Cupressaceae support diverse communities of Geosmithia fungi, with the exception of Calocedrus. Isolations from the Calocedrus-specific beetle Phloeosinus fulgens typically yield a low abundance of Geosmithia strains and mostly Pinaceae-specific species. This may reflect the larger size of the tree and a more humid environment than in most other Cupressaceae (Table 2). Other than bark beetles, additional beetle vectors of Geosmithia include several subcortical non-scolytine weevil subfamilies (Cossoninae and Mesoptiliinae) and Bostrichidae. Wood borers, which occur under bark only as larvae but not as adults (Cerambycidae and Buprestidae), do not serve as reliable vectors and, therefore, are not typically associated with Geosmithia.

Fig. 5
figure 5

Schematic presentation of the Geosmithia association across bark beetles of different organ preference. The abiotic factors of different beetle substrates are shown

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Kirschner (2001) also noted that beetles frequently transmitting Geosmithia tend to carry lower frequency and diversity of ophiostomatoid fungi and called Geosmithia an ecological replacement for ophiostomatoids. Subsequent studies confirmed this pattern. Geosmithia are rare or absent on insects colonizing large limbs and trunks of Pinaceae and Betula. Within Pinaceae, Geosmithia abundance and diversity are negatively correlated with the thickness of the wood substrata preferred by the insects (Kolařík and Jankowiak 2013; Jankowiak and Bilanski 2018). Similarly, on Betula, the bark beetle Scolytus ratzeburgi feeds in a very moist substrate, under the impermeable bark, and hosts an abundance of ophiostomatoid fungi, but no Geosmithia (Linnakoski et al. 2008). On most other hardwoods, such as Fraxinus, Ulmus, and woody plants from the Rosaceae family, vectors specific to the trunk show much less frequent association with Geosmithia. This pattern is replicated all around the world, but the factors responsible for it remain unclear. One of these factors could be the relatively greater tolerance to desiccation and osmotolerance in Geosmithia, as is known in G. xerotolerans (Crous et al. 2018), and greater competitiveness under drought conditions, as found in G. morbida (Williams and Ginzel 2021). Other abiotic variables, such as oxygen level and resin concentration, on the growth of Geosmithia should be tested, as they have been identified as distinguishing the growth of Edoconidiophora polonica, living in the fresh phloem of the tree trunks, from Ophiostoma species living in the dead phloem and in thinner tree parts (Solheim 1991).

Vector specificity, community composition, and biogeography

The recent 20 years of research on Geosmithia worldwide have finally enabled the first attempt at a synthesis of the ecology and distribution of these fungi (Tables 1 and 2, Fig. 4). Geosmithia can be divided into generalist species that are common across vectors worldwide and can also be found outside of the subcortical habitat, such as in decaying wood, soil, cereals and foodstuffs (Kolařík et al. 2004; Labuda and Tancinová 2006; Pitt and Hocking 2009), sea sediments (Ameen et al. 2014; Sun et al. 2018), cave environment (Bastian et al. 2009; Crous et al. 2018), or as plant endophyte (Sakalidis et al. 2011; McPherson et al. 2013; Deka and Jha 2018) (Table 1). This is typical of species in the G. pallida complex (G. pumila, G. pulverea), then G. fassatiae, G. flava, G. granulata, G. langdonii, G. obscura, G. omnicola, G. putterillii, G. xerotolerans, and Geosmithia sp. 1. In contrast, specialists species occur on vectors sharing the host plants of the same plant family. These include species that are restricted in occurrence to Pinaceae hosts, then G. morbida (Juglans, Europe, North America), and G. ulmacea (Ulmus, Europe, North America). Sometimes, the host preference is maintained in the particular geographical area with occurrence on other hosts in different areas (e.g., Geosmithia sp. 12—Fraxinus, G. sp. 32—Cupressaceae, G. sp. 11—Olea, G. carolliae—Ficus). Some species common in some areas (e.g., G. sp. 41 in North America, G. funiculosa in Europe) are absent in others, suggesting biogeographical patterns independent of the tree host and vector distribution (Fig. 4, Table 1). We do not have enough data for ambrosial Geosmithia species, but a high vector specificity can be expected. This appears to be the case with G. eupagioceri, which has so far only been found on the beetle Eupagiocerus dentipes in two separate collections in Central America (Kolařík and Kirkendall 2010, J. Hulcr, unpublished).

Geosmithia communities in any given locality are structured by the influence of the local host tree availability, biogeographical limits, and the presence of suitable vector beetles. The strongest predictor is the host plant. Increasingly, it is becoming evident that the beetle vectors are passive (with exception of primary ambrosia species), not actively involved with Geosmithia, and that these apparent fungus-beetle associations are derived from the underlying patterns of the tree host use by the respective Geosmithia species. As Geosmithia fungi depend on host trees for development, and on beetles frequenting those trees for transmission, their ecological specialization is best understood on the level of tree-beetle networks. Consequently, insect vector species that regularly co-occur in the same tree part consequently have similar communities of fungi without being able to actively select them. For example, Pinus trees support the same community across different vectors beetles, and the community is different from those associated with Picea (Kolařík and Jankowiak 2013; Jankowiak et al. 2014) and Abies (Jankowiak and Kolarik 2010; Jankowiak and Bilanski 2018) and even further distant from those specific to angiosperms and Cupressaceae (except of Calocedrus). As an example, polyphagous vectors such as Pityogenes chalcographus and Pityophthorus pityographus carry fungi specific either to Pinus or to Picea, depending on the substrate from which they were collected (Jankowiak et al. 2014). This same pattern of Geosmithia community structure has been observed in angiosperms in temperate Europe and the USA (Kolařík et al. 2008; Huang et al. 2019; Strzałka et al. 2021). Community composition is also shaped by the degree of specificity of the bark beetle vectors available in a given area. If there are only host-specific beetles on a given host plant, more sharply delimited Geosmithia communities are formed. Conversely, polyphagous beetle vectors create more diffuse fungus communities (Kolařík et al. 2007, 2017). In turn, this regional species pool and its dynamics also influence the richness of Geosmithia species in individual beetle galleries: in mixed forest, Geosmithia communities are more diverse and then in conifer monocultures (Jankowiak et al. 2014).

Some Geosmithia species may be primarily endophytic and only secondarily associated with bark beetle galleries. In California, G. langdonii was isolated from both bark beetle galleries as well as an endophyte, whereas two other species were only isolated from bark beetle galleries (McPherson et al. 2013).

Geosmithia interactions with host insect and plant

It is not completely clear how adult bark beetles transport Geosmithia. The majority of known and reliable vectors lack mycangia or any other organs adapted to fungus dispersal, and propagules appear to be transmitted passively in the gut or opportunistically attached to crevices and punctures of the exoskeleton. Several reports show Geosmithia presence in mycangia (Six et al. 2009; Belhoucine et al. 2011; Kolařík et al. 2017). Phoretic mites are also able to vector Geosmithia fungi (Machingambi et al. 2014).

Bark beetle-associated fungi are known to have diverse symbiotic (i.e., mutualistic, neutral, or antagonistic) interactions with their beetle vectors. The most straightforward is commensalism, in which the fungus benefits from the beetle’s ability to invade fresh plant tissues, which enables the fungus to exploit these nutrient sources, but the fungus does not necessarily benefit the beetle vector (Six and Wingfield 2011; Six 2020). Ambrosial Geosmithia species are mutualistic, as they provide nutrition to the beetle hosts, but it remains unknown whether the non-ambrosia Geosmithia also provide any benefit. Most species are good degraders of hemicellulose, and some are able to also degrade cellulose and lignin, which may benefit the beetle directly or indirectly. Some are able to utilize uric acid as nitrogen sources (Veselská et al. 2019), and thus recycling of nitrogen from the beetle waste product may be a benefit to their beetle associates that has not been tested. Geosmithia fungi can further interact with the insect via volatile chemicals. Volatiles of G. morbida attract its insect vector and may synergize beetle aggregation (Blood et al. 2018).

Geosmithia spp. also interact with other fungi in the beetle galleries. For example, mycoparasitism by Geosmithia species was observed on Ophiostoma novo-ulmi, the fungus responsible for the Dutch elm disease (Pepori et al. 2018). Geosmithia spp. produce a variety of biologically active compounds, through which they can interact with the ambient microbial community. Antibiosis towards fungi and bacteria has been found in many Geosmithia species (Veselská et al. 2019) and tested most extensively in G. lavendula (Stodůlková et al. 2009; Malak et al. 2013a; Hadj Taieb et al. 2019) and G. pallida KU693285 (Deka and Jha 2018). Machingambi et al. (2014) have suggested that mites (bark beetle parasites) were unable to feed or reproduce in the presence of Geosmithia associates; the miticidal potential of Geosmithia should be studied in detail. G. lavendula and other species produce hydroxylated anthraquinones (hAQs) with many bioactive properties (Hilker and Köpf 1994; Poche 1998; Ganapaty et al. 2004; Stodůlková et al. 2009); the role of hAQs in the bark beetle ecosystem has not been evaluated.

Evolution and biology

The reconstruction of phylogenetic relationships among Geosmithia should has been conducted primarily using protein-coding genes. Ribosomal DNA markers, typically used in other fungi, have several limitations in Geosmithia. Specifically, Geosmithia sp. 26 is a species complex that has rDNA sequences very different from other species and a very low GC content, preventing a quality alignment (Kolařík et al. 2017). Subsequently, phylogenies inferred from rDNA and from protein-coding genes are in conflict (Veselská et al. 2019). The rapid rDNA sequence evolution and GC content deviation in Geosmithia sp. 26 may be a consequence of fluctuations in the effective population size and bottlenecks, possibly related to the switch between free-living to host-associated life strategy (see Kolařík and Vohník 2017; Kolařík et al. 2021).

Geosmithia species feature many life history traits distributed across the phylogeny, making the genus an ideal model for studying the evolution of individual life styles and associated phenotypic traits (Veselská et al. 2019). Basal Geosmithia lineages are generalists, with broad host ranges across angiosperms and gymnosperms, and are sometimes found also outside the bark beetle habitat. At least six lineages convergently evolved specificity to the Pinaceae (Veselská et al. 2019; Strzałka et al. 2021; Zhang et al. 2022). The shifts were accompanied by losses of metabolic capacity and by genome size inflation. In vitro, this is apparent by the inability to grow on basal CZD agar, which lacks important nutrient supplements such as vitamins (in particular the B group). Three other derived lineages converged on the ambrosia strategy, providing nutrition to specific beetle vectors. This was accompanied by the cell and genome size inflation and the production of large amounts of oleic fatty acid, likely associated with the nutritive function (Veselská and Kolařík 2015; Veselská et al. 2019). The ambrosia species also produce large conidia, a phenotype seen in other ambrosia fungi (Kolařík and Kirkendall 2010; Kolařík et al. 2015).

One lineage, G. morbida, became a plant pathogen, with the unique ability to digest all components of lignocellulose, a feature that can serve as its virulence factor (Veselská et al. 2019), as in other plant pathogenic fungi (Doehlemann et al. 2017; Jagadeeswaran et al. 2021). In general, specialists, such as those on Pinaceae and the Juglans-specific G. morbida, have a reduced metabolic breadth in comparison to generalists.

The genome size in Geosmithia spp. correlates with cell size (e.g., conidia), as in most eukaryotes (Gregory 2001), and is related to the ecology of the species. Specifically, species specialized to a narrow host range (including G. morbida) have relatively large genomes, compared to generalists. The largest genomes are present in the ambrosial species (Veselská and Kolařík 2015).

Relatively little research has been done on the genetics and mating behavior of these fungi. As with other filamentous ascomycetes, there is a system of vegetative incompatibility that leads to some isolates making mycelial fusions with each other but not with others. In G. morbida, this is manifested by non-coalescent necrotic lesions in the host tree tissues (Montecchio et al. 2015). The sexual stage has never been observed, and the only population genetics study in the genus suggested the absence of recombination in G. morbida (Zerillo et al. 2014). Both mating gene idiomorphs (MAT1-1, MAT1-2) are present across the genus (M. Kolařík, unpublished), and targeted crossing experiments should be carried out to induce the sexual stage, as has been done in other molds where the sexual stage was unknown (O’Gorman et al. 2008). In Geosmithia, a cleistothecial type of sexual state can be expected, as is the case with related fungi such as Nigrosabulum, Mycoarachis, or Hapsidospora (Rossman et al. 1999; Plishka et al. 2009). The genome size, determined by flow cytometry, is 20.5 to at least 54 Mb. The largest genomes are those of ambrosial species, probably due to the ancient polyploidization (Veselská and Kolařík 2015). The genome size values, measured by flow cytometry in G. morbida, G. flava, and G. putterillii (24.4–24.7, 25.5–25.8, 26–26.3 Mbp), agree with those measured by whole genome sequencing (26.5, 29.6, 30.0 Mbs) (Veselská and Kolařík 2015; Schuelke et al. 2017). The number of genes is around 6000, and only 73–146 were found to be species-specific. Between 300 and 400 (349–403) protein-coding genes belong to secreted proteins. There are few genes involved in secondary metabolism compared to related taxa such as Acremonium chrysogenum and Stanjemonium grisellum. In G. morbida, 26 genes have homologs with known involvement in interactions with the plant host and thus a potential role in pathogenesis (Schuelke et al. 2017).

Geosmithia fungi, like other Dikarya, have hyphae coated with hydrophobins, small proteins that form a hydrophic membrane and play a crucial role in interactions with hydrophobic substrates such as plant or insect cuticle. Species of Geosmithia have class II hydrophobins, called GEO1 (Bettini et al. 2012; Frascella et al. 2014). They possess an intragenic tandem repeat sequence involved in the rapid generation of variation and subsequent adaptation. GEO1 is also under strong selection pressure, suggesting that the capacity for adhesion is important in the evolution of the genus. Phylogenetic relationships reconstructed from GEO1 and those from classical gene barcodes are highly incongruent. This suggests that GEO1 evolves through multiple horizontal transfers and/or by birth–death evolution (Frascella et al. 2014). The same mechanisms are known to affect other genes under high selection pressure, such as secondary metabolism gene clusters in the genus Fusarium (Proctor et al. 2018). There is also evidence that at least six Geosmithia species obtained another hyrophobin, cerato-ulmin, by a horizontal transfer from Ophiostoma novo-ulmi. Cerato-ulmin is involved in the virulence of O. novo-ulmi, a causal agent in Dutch elm disease of elms, and is present only in Geosmithia strains from elms (Scala et al. 2007), but not in those from other tree hosts (Bettini et al. 2014).

Phytopathogenic potential and thousand cankers disease

While most Geosmithia spp. appear to be saprophytes, the pathogenicity capabilities of some species deserve closer scrutiny. Already the first study on Geosmithia (Wright 1938) showed the infectious potential of Geosmithia from Scolytus praeceps and S. subscaber. When inoculated into a live plant host, these strains were able to cause significant necrosis in the cambium of Abies concolor trunk. Based on the morphology, the strain used in the study probably belonged to Geosmithia sp. 33 or sp. 34 (Kolařík et al. 2017), and the pathogenicity observations, while convincing, require further verification.

Pathogenicity has been studied by inoculating the phloem of seedlings or adult tree branches in more than 20 Geosmithia species and mostly showed no evidence of pathogenicity. In particular, no pathogenic effect was found in Geosmithia sp. 16 on Abies alba (Jankowiak and Kolarik 2010), two species from Geosmithia sp. 24 species complex on Pinus spp. in Israel (Dori-Bachash et al. 2015), four species (G. cupressina, G. langdonii, G. omnicola, G. sp. 708) on Cupressus (Meshram et al. 2022), five species (G. flava, G. omnicola, G. pumila (= G. sp. 2), G. sp. 8, G. sp. A) on Virgilia (Machingambi et al. 2014), six Geosmithia spp. (G. fagi, G. flava, G. langdonii, G. ulmacea, G. pulverea (= sp. 3), and G. funiculosa (= sp. 5)) on Acer, Fagus, Quercus, Tilia, and Ulmus (Strzałka et al. 2021; Crous et al. 2022), G. luteobrunnea on Liquidambar (Gao et al. 2021; Zhang et al. 2022) and 11 Geosmithia strains originating from Czechia, Korea, Vietnam, China, Papua New Guinea, Taiwan on Quercus shumardii, and Q. virginiana (Li et al. 2022).

A few Geosmithia species do induce phloem necrose, however, and several are involved in plant diseases. Pathogenicity assays performed using the excised shoot method showed ability of tissue lesion formation in G. granulata (= sp. 20), G. lavendula, G. omnicola, and G. pallida on Pistacia vera (Hadj Taieb et al. 2019). However, testing pathogenicity on detached shoots is questionable, as the results cannot be extrapolated to natural field conditions. Mild, but significant, lesions were created by Geosmithia sp. 12568 (Cryphalus piceus, South Korea) on Pinus spp. (Li et al. 2022) and by Geosmithia sp. on an artificially inoculated Olea europea trunk (van Dyk et al. 2021). Čížková et al. (2005) have shown that G. pumila (= sp. 2) and G. langdonii inhibited the growth of garden cress Lepidium sativum.

Several tree diseases are caused by bark beetles which carry Geosmithia species, and the fungi may form discolored areas around the beetle galleries, but are not pathogenic themselves. In the so-called Foamy Bark Canker of Quercus agrifolia in California (USA), the disease appears to be caused by an infestation by the bark beetle Pseudopityophthorus pubipennis. The beetle vectors Geosmithia sp. 41 and other species (Kolařík et al. 2017). This fungus produces significant lesions on artificially inoculated excised oak shoots (Lynch et al. 2014), but both the disease and its causal agent need further study. Large mortality of American sweetgum (Liquidambar styraciflua) planted in China caused by the bark beetle Acanthotomicus suncei also involves several species of Geosmithia, most commonly G. luteobrunnea, around the beetle galleries, but again, the fungus is not pathogenic on its own (Gao et al. 2021; Zhang et al. 2022). Similarly puzzling is the presence of the Dutch elm disease pathogenicity factor cerato-ulmin in Geosmithia spp. (Scala et al. 2007; Bettini et al. 2014), while no active role of the respective Geosmithia species in the disease has been demonstrated yet.

The only species that creates significant necroses, the accumulation of which may kill the host plant, is Geosmithia morbida. Together with its vector, the walnut twig beetle Pityophthorus juglandis, the two organisms contributed to the phenomenon of the thousand cankers disease (TCD) of black walnut, Juglans nigra (Tisserat et al. 2009; Kolařík et al. 2011). G. morbida and its vector beetle P. juglandis are native to the West of Northern America, and recently, they dispersed to other parts of the continent, as well as to Italy (reviewed in Daniels et al. 2016) and France (Saurat et al. 2023). For several years following this expansion and a drought, there was a notable dieback of black walnut across the USA. The dieback has recently subsided, with the exceptions of locations where black walnut is planted outside of its typical growing conditions (i.e., California), suggesting that the disease has been largely a symptom of drought. While temporary, the impact of TCD spurred research on Geosmithia and its symbiosis with bark beetles (Fig. 1). Research on G. morbida involved its genetic variability (Hadziabdic et al. 2014a; Zerillo et al. 2014), host tree range and virulence (Sitz et al. 2017, 2021; Hefty et al. 2018), vectors (Chahal et al. 2019), migration (Hadziabdic et al. 2014b; Montecchio and Faccoli 2014; Moricca et al. 2020; Marchioro and Faccoli 2022), detection (Stackhouse et al. 2021), eradication (Dal Maso et al. 2019; Seabright et al. 2019; Juzwik et al. 2021), and competition with co-occurring fungi (Gazis et al. 2018). The walnut twig beetle transmits other Geosmithia species (Kolařík et al. 2017), as can be seen from preliminary results with infection experiments with G. obscura (Pietsch et al. 2022).

A cross-phylogeny comparison of pathogenic and non-pathogenic species at the genome (Schuelke et al. 2017) and phenotype level has shown that G. morbida is unique among Geosmithia species in producing an enzyme that breaks down both cellulose and lignin (Veselská et al. 2019). This capacity can be considered one of the virulence factors responsible for the ability to necrotize the phloem of walnut (Veselská et al. 2019). An interesting avenue of research is the study of the presence of viruses in G. morbida that may modulate virulence (Montecchio et al. 2015).

Secondary metabolite production and biotechnological potential

The order Hypocreales is known for the ability to produce a variety of secondary compounds, including toxins. Even crude extracts from Geosmithia show the potential for antibacterial and antifungal activity across the genus (Deka and Jha 2018; Veselská et al. 2019). Aside from common fungal metabolites, 48 secondary metabolites were found uniquely in Geosmithia (Table 3). Prominent yellow, orange, and red pigments produced by G. lavendula represent more than twenty different hydroxylated anthraquinones, often novel to science, several with antibacterial or anti-inflammatory activity (Stodůlková et al. 2009, 2010; Malak et al. 2013c) and with the potential as persistent textile dyes or mordants (Flieger et al. 2009). Geosmithia pallida complex strain FS140 (Table 2) produced 12 different thiodiketopiperazines, including three previously unknown ones (Sun et al. 2018). A single strain identified morphologically as G. langdonii yielded 14 metabolites, including four new ones (Malak et al. 2013b, 2018). Their biological activities include antimicrobial, cytotoxic, angiotensin-converting enzyme inhibitory, antileishmanial, or nematicidal properties (Table 3).

Table 3 List of secondary metabolites reported from Geosmithia
Full size table

While these first studies on secondary metabolites in Geosmithia yielded a large proportion of novel compounds and broad biological activity, the chemical arsenal is rather limited in terms of biosynthetic pathways, yielding mostly low molecular weight, structurally simple metabolites. The three species studied—G. morbida, G. putterilli, and G. flava—have only 14 to 19 secondary metabolite gene clusters, which contrasts with related filamentous fungi having four times higher numbers of similar gene clusters (Schuelke et al. 2017). However, the genetics of secondary metabolite production was explored in these three species only, all belonging to a single phylogenetic lineage, and the novelty of these products bids for further bioprospecting.

Conclusion and future research

Geosmithia has been in the spotlight only for the last decade, and so it is not surprising that many questions, long studied in ecologically similar taxa, are still unanswered. The broad evolutionary direction towards long-term and stable adaptation to the beetle-tree ecosystem observed in Geosmithia is in many aspects similar to that observed in ophiostomatoid fungi. In both groups, association with beetles led to the evolution of ambrosia lineages from phloem inhabiting ancestors and a coevolutionary response from the beetles, e.g., by the evolution of mycangia (although on a greater scale in the ophiostomatoid lineages). What benefits Geosmithia spp. present to their vectors needs further study in light of similar studies on ophiostomatoid fungi. Another question is the spatial distribution of Geosmithia spp. around galleries. Due to their hyaline hyphae and lack of pigment production, it is unclear how far they penetrate the surrounding tissues and whether they thus contribute to wood decomposition or interact with fungal species that decompose dead wood, as is known for ophiostomatoid fungi (Skelton et al. 2020).

One of the most important paradigms that has emerged from the surge of studies on Geosmithia is that these fungi are an ecological complement to the ophiostomatoid fungi (Kirschner 2001). We suggest here the terms Geosmithia-type and ophiostomatoid fungi-type association. Both fungal groups are dependent on bark beetle vectors for their dispersal and reproduction. However, Geosmithia is almost exclusively found in phloem that is drier, and typically more advanced in decay, and as a result are associated with bark beetle communities utilizing upper and thinner parts of trees. Ophiostomatoid fungi (both lineages), in turn, dominate phloem and sapwood, which retains moisture longer, and therefore are associated with bark beetles in the trunk and roots.

In terms of pathogenicity, some Geosmithia cause discoloration of the phloem around the beetle vector gallery, but bona fide pathogenicity in the absence of the beetle is rare, truly present only in G. morbida. Several species, such as those on fir in North America, are good candidates for verification of possible weak pathogenicity.

The other major lineages of fungi associated with bark beetles—the Ophiostomatales, Microascales, and several groups within Fusarium—also include a range of specificity, from plant pathogens, to soil saprobes, to obligate ambrosia fungi, and sometimes closely related species display dramatically different ecology. Geosmithia shows a similar pattern and is an excellent model for the study of adaptive traits related to species interactions. The evolutions of these traits in Geosmithia have been documented at the phenotypic level (Veselská et al. 2019), and the next step needs to include a deeper, genomic level.

The main lesson learned from the recent surge of interest in the study of Geosmithia is that these fungi are woefully undersampled geographically. A few species are cosmopolitan generalists, but many show considerable specificity to hosts and locations (Fig. 4). Continued studies on this genus need to emulate the methods from better-studied taxa such as Penicillium, Aspergillus, or Fusarium. More variable DNA markers are needed to answer taxonomic, evolutionary, and molecular biology questions. Similarly, a broader array of differentiated media (DG18, G25N, MY70S, CREA) is needed for morphological and metabolic characters. The whole genus still lacks sufficient genomic data, as only three genomes have been published to date.

Given the many new chemicals isolated from Geosmithia, these fungi deserve research also for their biotechnological potential. They do not produce structurally complex substances, and also, the diversity of secondary metabolites and biosynthetic pathways is modest. However, the known substances show no or very little cytotoxicity to animal cells, and at the same time, they have a number of biological activities. The bioactivity is highly selective, i.e., the fungi do not harm insects, while showing antibacterial antibiosis. Their potential to interact with organisms that are pathogens of bark beetles, such as nematodes and mites, should be tested. Ambrosia species are a promising target for fungus-based food research, since they provide a complete nutrition to their animal vectors concentrated in enlarged conidia rich in proteins and oils, while being entirely non-toxic and non-melanized (Veselská et al. 2019, M. Kolařík unpublished).