Geosmithia species associated with fir-infesting beetles in Poland

Geosmithia species (Ascomycota: Hypocreales) are common ectosymbionts of scolytine bark and ambrosia beetles that feed on coniferous and deciduous trees in different forest ecosystems. Geosmithia morbida is the canker pathogen that causes extensive mortality of Juglans nigra. Because little is known regarding the Geosmithia species on European silver fir (Abies alba), we have investigated the diversity and abundance of these fungi associated with insects infesting European silver fir in Poland. Samples associated with eight beetle species were collected from three fir forests. Fungi were isolated from beetles and galleries. Isolates were identified based on morphological characteristics, DNA sequence comparison for three gene regions (ITS, ßT, TEF1-α), and phylogenetic analyses. Geosmithia was detected in 33% of the total 531 beetle samples obtained from A. alba. Two undescribed species of Geosmithia were distinguished, Geosmithia sp. 9 and Geosmithia sp. 16. Associations of Pityokteines spp. with Geosmithia fungi were recorded for the first time. Pityokteines vorontzowi and Pityophthorus pityographus appear to be regular vectors for Geosmithia sp. 9 and Geosmithia sp. 16, respectively. Pityokteines curvidens and Cryphalus piceae were associated with Geosmitha sp. 9 at lower frequencies.

In this survey, eight beetle species and their galleries were collected from three different sites in the silver fir stands of Poland.The main objective of this study was to provide insight into the diversity of Geosmithia species associated with fir-infesting beetles in Poland.

Study area and sampling of bark beetles and galleries
The beetles and galleries were collected during the study of Jankowiak et al. [28].The bark beetles and weevils were collected at three sites in Poland.These research sites were composed mainly of European silver fir (Tab.1).
Newly emerged beetles were collected according to the procedure described by Jankowiak et al. [28].The adults feeding on fir were excised from their galleries with sterilized forceps about 2-4 weeks after the main flight period and placed individually in sterile Eppendorf tubes (1.5 mL) and stored at 4°C for 1-2 days until the fungal isolations were performed.One to two beetles were collected from each gallery, and each collected beetle was placed individually into a sterile Eppendorf tube.
The beetle galleries, except P. vorontzowi, were collected from windblown trees naturally infested by insects, and from trapping logs (2 m long, 0.2 m in diameter) or branches lying stacked in stands from May-July in 2013 and 2014.The galleries of P. vorontzowi were collected from May till June 2009.Complete galleries, including sapwood up to 2 cm away from the tunnel, were removed from the wood and placed in separate paper bags.
Due to differences in the number of collected beetles and their galleries, the galleries and the beetles were treated as similar substrates (e.g., one beetle of P. curvidens + one gallery of P. curvidens = two beetle samples of P. curvidens).A total of 531 beetle samples were collected.

Fungal isolations and morphological grouping
For fungal isolations, each beetle was removed from its storage microtube with sterilised tweezers, morphologically identified with taxonomical keys [29,30], and squashed onto the surface of malt extract agar (MEA; 20 g Biocorp malt extract, 20 g Biocorp agar, and 0.2 g tetracycline, per liter of distilled water) in Petri dishes.Fragments of discolored sapwood underneath each gallery up to a depth of 10 mm into the sapwood (two to six fragments per gallery; 4 × 4 mm size) were collected without disinfection, and plated on MEA medium in Petri dishes.This was performed according to the isolation procedures described by Jankowiak et al. [28].The plates were than incubated at 25°C for 2-6 weeks and observed daily for fungal growth.When necessary, cultures were purified by transferring small pieces of mycelium or spore masses from individual colonies to fresh 2% MEA.Purified cultures were grouped according to culture morphology by using a Nikon Eclipse 50i microscope (Nikon Corporation, Tokyo, Japan) and an Invenio 5S digital camera (DeltaPix, Maalov, Denmark) with Coolview 1.6.0software (Precoptic, Warsaw, Poland).Representative isolates of fungi were deposited in the culture collection of the Department of Forest Pathology, Mycology and Tree Physiology, Hugo Kołłątaj University of Agriculture, Krakow, Poland (Tab.2).

DNA extraction, amplification, and phylogenetic analyses
DNA was extracted using the Genomic Mini AX Plant Kit (A&A Biotechnology, Gdynia, Poland) according to the manufacturer's protocol.Partial gene sequences were determined for the internal transcribed spacer regions (ITS1 and ITS2) including the Tab. 2 Cultures examined in this study and their GenBank accession numbers.* According to [9].** All isolates originated from Abies alba at Rozpucie site.
All sequences were aligned online with the MAFFT v6 [36], using the E-INS-i option with a gap-opening penalty of 1.53 and an offset value of 0.00.Phylogenetic analyses were performed using the maximum likelihood (ML) and Bayesian inference (BI) methods according to the procedures of Jankowiak and Bilański [27] and Jankowiak et al. [37].For the ML and BI analyses, the best fitting substitution models for each data set were estimated using the corrected Akaike's information criterion (AICc) in jModelTest 0.1.1 [38].The best evolutionary substitution models for ITS and βT datasets were respectively GTR+I+G and HKY+I+G.
ML searches were conducted in PhyML 3.0 [39] via the Montpelier online server (http://www.atgc-montpellier.fr/phyml/) with 1,000 bootstrap replicates.BI analyses based on a Markov Chain Monte Carlo (MCMC) were carried out with MrBayes v3.1.2[40].The MCMC chains were run for 10 million generations using the best fitting model.Trees were sampled every 100 generations, resulting in 100,000 trees from both runs.The burn-in value for each dataset was determined in Tracer v1.4.1 [41].All sequences generated in this study were deposited in NCBI GenBank (Tab.2) and are presented in the phylogenetic trees (Fig. 1 and Fig. 2).Acremonium alternatum Link was used as an outgroup.

Collections of bark beetles and fungal isolations
In the collected material eight subcortical beetle species were found (Tab. 1 and Tab.3).A total of 218 Geosmithia isolates were obtained from 531 bark beetle individuals and their galleries.Altogether, 16 were selected for molecular identification (Tab.2).The presence of Geosmithia in galleries of P. curvidens is presented in Fig. 3.

Fungal identification and DNA sequence comparisons
Based on our preliminary morphological investigation, the obtained isolates were separated into two Geosmithia groups.Both groups presented penicillium-like asexual stages.Genotype analyses confirmed that groups corresponding to the genera Geosmithia were in the Hypocreales (Fig. 1).
The partial βT gene was used to identify isolates at the species level (Fig. 2).Due to the lack of reference sequences for the TEF1-α gene for Geosmithia in GenBank, the phylogenetic analysis for this gene was not been performed in this study.
Analyses of the ITS sequences revealed that our isolates belong to the Geosmithia genus.ITS sequence analysis showed also that two morphological groups of Geosmithia could be divided into two clades: the first clade containing Geosmithia sp.16, 25, and 29-31, and the second clade represented Geosmithia sp. 9 (Fig. 1).The ßT tree for isolates from the first clade supported the identification of Geosmithia sp.16, while the remaining isolates grouped with the reference sequences of Geosmithia sp. 9 (Fig. 2).

Prevalence of different fungal species
The frequencies of occurrence of the Geosmithia species varied between beetle species (Tab.3).Several bark beetle species were commonly found in association with Geosmithia fungi, which were isolated from ≥70% of the sampled beetles and galleries.The following beetle species appeared to have rather strict associations with Geosmithia fungi: P. pityographus and P. vorontzowi.Geosmithia fungi were also present in 24-37% of the   The Geosmithia species that was found in association with the broadest vector spectrum (four beetle species) was Geosmithia sp. 9.This fungus was the most frequently encountered fungus on P. vorontzowi.Geosmithia sp. 9 was also commonly found on C. piceae and P. curvidens (Tab.3).Less widespread species included Geosmithia sp.16 isolated from two vectors.This fungus was commonly found on P. pityographus and sporadically on C. piceae (Tab.3).

Discussion
Associations of Geosmithia spp. with various tree hosts and vectors are relatively well known in Europe.In general, Geosmithia is more frequent on hardwoods than on conifers, although Geosmithia is commonly found in association with pine-infesting bark beetles in Central Europe [2,4,5,6,9,12,15,17,18].Our survey demonstrated that Geosmithia fungi are also regular associates of phloembreeding bark beetles on European silver fir.This association was previously documented for C. piceae in Poland [19].
The European silver fir weevil (P.piceae) and the striped ambrosia beetle (T.lineatum) were not associated with Geosmithia in our study.Similar results have recently been reported from the Western USA [11].According to a study conducted by Kolařík et al. [11], Geosmithia spp.are carried by insects that enter the phloem and xylem as adults, but not by woodborers (e.g., weevils) Tab. 3 Frequencies* (%) of Geosmithia species obtained from eight beetle species collected from Abies alba in Poland.A Samples included beetles and galleries.B Samples included only beetles.* The frequency of occurrence was calculated according to the following formula: F = (NS/NTs) × 100; where F represents the frequency of occurrence (%) of the fungus, NS represents the number of samples from which a particular fungus was isolated, and NTs represents the total number of samples.** According to [9].where adults do not enter these tissues.Although, there are many studies confirming associations between ambrosia beetles and Geosmithia fungi [8,10,11,14,16], our study showed that T. lineatum does not transmit Geosmithia.There was also no evidence that this ambrosia beetle can transmit Geosmithia fungi in pine habitats [9].The genus Geosmithia currently includes 44 recognized taxa with only 16 described to date, most of which are associated with phloem-breeding bark beetles [4,5,[7][8][9][10][11][12][13][14][15][16].In the present study, we identified two distinct Geosmithia operational taxonomic units (OTUs) based on morphological and molecular characterizations.They were closely related to previously revealed species, and included Geosmithia sp. 9 and Geosmithia sp.16 [9,12].Although Geosmithia sp. 9 is known from P. pityographus on Picea abies (L.) H. Karst.and Pityogenes chalcographus (L.) on P. sylvestris and P. abies [9,12], there was evidence that this fungus is mainly associated with C. piceae on A. alba [19].Our results, therefore, support those of Jankowiak and Kolařík [19] who often found Geosmithia sp. 9 from C. piceae.In addition, Geosmithia sp. 9 was especially associated with Pityokteines species, especially P. vorontzowi.This suggests that Geosmithia sp. 9 is a specialist that is mainly limited to A. alba and fir-infesting bark beetles.Other examples of Geosmithia specialists include G. ulmacea (Ulmus) or G. morbida (Juglans) [7,15,18].Geosmithia sp.16 is mainly known from Pityogenes bidentatus (Herbst) and P. pityographus on P. sylvestris [9,12,17].Geosmithia sp.16 was also found to be associated with P. pityographus in the present study.Our findings suggest that this fungus has a high affinity for P. pityographus.However, the host range of Geosmithia sp.16 appears to be wider than Geosmithia sp. 9, taking into account that P. pityographus as a polyphagous species colonizes many conifers.

Pissodes
According to Jankowiak et al. [12], Geosmithia spp.seem to be associated mainly with insects breeding under thin bark, such as on branches.In contrast, beetles that feed under thick bark (e.g., the trunk) more frequently transmit ophiostomatoid fungi.Our results support this hypothesis because P. vorontzowi and P. pityographus had the strongest relationship with Geosmithia, while C. piceae and P. curvidens have shown less frequent association with Geosmithia.Pityokteines vorontzowi and P. pityographus colonize thin branches as compared to C. piceae and P. curvidens, which attack trunks or thicker branches [42].

Fig. 1
Fig.1Phylogram obtained from analyses of the ITS data for the Geosmithia spp.Sequences obtained during this study are presented in bold type.Presented phylograms were obtained from maximum likelihood (ML) analyses.The bootstrap values (>75%) for ML and posterior probabilities (>75%) that were obtained from Bayesian (BI) analyses are presented at nodes as follows: ML/BI.* Bootstrap values <75%.

Fig. 3
Fig.3Example of the association between Pityokteines curvidens and Geosmithia sp. 9. (A) Adult (with attached mite, white arrow) walking in a gallery containing conidiophores of Geosmithia sp. 9. (B) Larva in a gallery colonized by Geosmithia sp. 9.

Tab. 1
Features of the study areas, list of samples, and characteristics of the beetle species.