New insights into the role of phytopathogenic fungi vectored by pine bark beetles in pine decline

1 Kateryna Davydenko – Corresponding Member of Forestry Academy of Sciences of Ukraine, PhD, (Agricultural Sciences), Senior Researcher, G. M. Vysotsky Ukrainian Research Institute of Forestry & Forest Melioration, 86 Pushkinska st., Kharkiv, 61024, Ukraine. Visiting Researcher at the Department of Forest Mycology and Plant Pathology, Uppsala BioCenter, Swedish University of Agricultural Sciences, P.O. Box 7026, SE75007, Uppsala, Sweden. Tel.: +38-098-66-755-26. E-mail: kateryna.davydenko74@gmail.com ORCID http://orcid.org/0000-0001-6077-8533 1. БІОЛОГІЧНІ АСПЕКТИ РОСЛИННИХ УГРУПОВАНЬ

Introduction. Bark beetles (Coleoptera: Curcu lionidae, Scolytinae) are insects known all over the world that are among the most economically and ecologically important pests that are very dangerous for forests, especially for stressed and weakened trees. One of the most distinguished characteristics of bark beetles is the ubiquitous association with different fungi that were recognized more than one hundred years ago (Six, 2003;Six & Wingfi eld, 2011). Bark beetles are wellknown for promoting the spread of these fungi into the living trees as well as to untreated timber and wood products (Linnakoski, 2011). There are a few dramatic examples of the invasion of bark beetle species and vectored fungi into new areas where they became aggressive and caused threats to forests after they had been accidentally introduced (Linnakoski 2011 ;Bezos, Martinez-Alvarez, Diez, & Fernandez, 2015;Hulcr et al., 2020).
Bark beetles comprise a highly diverse group of insects with a worldwide distribution. The overwhelming majority of bark beetle species are capable to attack weakened or dying trees when their populations are low in abundance, but a mass-attack on large numbers of healthy trees can take place once bark beetle populations are numerous (Hlásny et al., 2019).
Among aggressive bark beetles that have developed adaptations to colonize trees are mostly from the Pinaceae family. Despite the strong mechanism of tree resistance, these insects are economically and ecologically dangerous as during their outbreaks, they can devastate both managed and natural forests (Raffa, Bonello, & Orrock, 2020). The abundance of windthrown or coniferous trees weakened by drought, as e.g. Norway spruce (Picea abies L.) and Scots pine (Pinus sylvestris L.), can also trigger the growth of the bark beetle population. In case of the lack of fallen or stressed trees with low level of resistance, bark beetles can attack healthy trees (Linnakoski, 2011(Linnakoski, , 2012Bezos et al., 2015;Hulcr et al., 2020).
Moreover, bark beetles from the genera Hylurgus (Hylurgus ligniperda) and Hylastes (H. angustatus and H. ater) are the serious pests causing extensive damages to young plantations, stands and timber of Pinus sylvestris. Hylurgus ligniperda is among the most common bark beetles in Ukraine (Meshkova & Davydenko 2012;Davydenko et al., 2014), Davydenko, 2021). Hylurgus ligniperda breeds in logging residues including stumps, roots and logs of Pinus sylvestris trees while larvae of H. ligniperda may also feed on roots and butts of healthy-looking and diseased seedlings and saplings (Davydenko et al., 2014).
Material and methods. Field study and sample collection. During the 2012-2018 period, to examine the presence of ophiostomatoid fungi, samples of beetles and blue-stained wood were taken from pine stands located in the different regions of the foreststeppe zone of Ukraine. The sample plots are given below (Tab. 1).
Stands at all sites were ca. 50-70 year-old mostly monoculture plantations of Scots pine. Sampling of bark beetles has been carried out randomly from standing trees of Pinus sylvestris. Trees were living but slightly or severely weakened by bark beetle and drought. The time of sampling coincided with the period when the dispersal fl ight period of young generation has completed and their galleries have already been built. Samples of bark with bark beetle galleries, phloem and sapwood were taken from the relevant part of standing trees using sterilized forceps, individually placed in sterile Ependorff tubes and transported to the laboratory. Half of the beetles from each site were stored at 4°C for fungal culturing and the other halfat -20°C for direct DNA sequencing of fungi.
Study of fungal communities of bark beetles. Cultural method. The half of bark beetles of each species and each site were used for fungal isolation. The isolation from bark beetle and wood from their galleries target to gain as much as possible ophiostomatoid fungi for further pathogenicity tests as well as other fungi (Davydenko et al., 2014(Davydenko et al., , 2017. Fungal cultures were divided into groups based on their morphology and, for species identifi cation, representative cultures from each group were subjected to sequencing of the internal transcribed spacer regions of the fungal ribosomal RNA (ITS rRNA). The isolation of DNA, amplifi cation and sequencing were performed according to methods described by Davydenko (Davydenko, 2021). The amplifi cation by PCR was done using primers ITS1F and ITS4. In addition, β-tubulin gene and the partial elongation factor 1-alpha (EF1-a) gene were amplifi ed and sequenced for fungi from the genera Ophiostoma, Graphilbum and Grosmannia. The β-tubulin gene was amplifi ed using the primers Bt2a and Bt2b (and EF1-a gene was amplifi ed using the primers EF1F and EF2R. Databases at GenBank were used to determine the identity of ITS rRNA sequences. The criteria used for the identifi cation were as follows: sequence coverage > 80%; similarity to species level 98-100%, similarity to genus level 94-97%. Study of fungal communities of bark beetles. Direct sequencing. While the conditions vary signifi cantly in phloem compared with nutrient media, the cultural method is selective for fast-growing fungi and is biased towards those fungal species that are able to utilize successfully artifi cial media. The use of molecular methods circumvents this problem and allows fi lling a gap by extracting DNA directly from bark beetles and then amplifying it using nested PCR reactions (Persson et al., 2009). The fungal specifi c primers NLC2 (GAGCTGATTCCCAAACAACTC) and NSA3 (AAACTCTGTCGTGCTGGGGATA) were applied fi rst (Davydenko, 2021), then, in a second (nested) PCR ribosomal internal transcribed spacer (ITS) region has been targeted as the main primer for the identifi cation of a fungal community (Davydenko et al., 2014(Davydenko et al., , 2017Davydenko, 2021). After sequencing, taxonomic identifi cation of derived sequences was carried out by comparing with known sequences in databases such as GenBank. Pathogenicity test. As mentioned above, some ophiostomatoid species are more or less pathogenic to their host plants. Therefore, to confi rm pathogenicity and defi ne pathogenic extent, the reappearance of the original symptoms after artifi cial inoculation by tested species is defi ned. For this, pathogenic ophiostomatoid fungi are inoculated into phloem by making wounds to similar bark beetles damage as decribed in our previous studies (Davydenko et al., 2017;Davydenko & Baturkin, 2020;Davydenko, 2021). Further, the fungal inoculation results in the formation of necrotic lesions in the phloem and colonization of sapwood, and in some cases, in the tree mortality (Davydenko & Baturkin, 2020). Thus, the virulence of the fungi was evaluated by measuring lesion sizes and monitoring the mortality of host plants after inoculation (Jankowiak, & Bilański, 2013;Repe, Bojovic, & Jurc, 2015).
Statistical analyses. Statistical analysis was carried out using the statistical software package PAST: Paleontological Statistics Software Package for Education and Data Analysis (Hammer et al., 2001). All the obtained data were tested for adherence to the normal distribution using the Kolmogorov-Smirnov test.
Sorensen similarity indices were used to characterise the diversity and composition of fungal (Magurran, 1988). The data from the inoculation and vector tests were analysed using analysis of variance (ANOVA). Signifi cant treatment differences were further evaluated by Fisher's Fisher's exact test followed by Tukey`s HSD post hoc test. The signifi cance was evaluated at the 0.05 p-level.
Results and discussion. Ophiostomatoid and other fungi associated with bark beetles infesting Pinus sylvestris in Ukraine were investigated in this study. Among countries neighboring Ukraine, the most comprehensive and detailed review of fungi associated with bark beetles infesting many conifers and hardwoods, including coniferous living trees, cut down, fallen trees and logs, has been done in Poland by Jankowiak (Jankowiak & Hilszczanski, 2005;Jankowiak & Kolarik 2010;Jankowiak, 2013;Jankowiak & Bilanski, 2013). These studies indicate numerous ophiostomatoid fungi associated with a bark beetle in Poland, the country nearest to Ukraine. Another Ukraine neighboring country is Slovakia, where only one research work has been recently published by researchers, focused on ophiostomatoid fungi and pine bark beetle (Pastircakova et al., 2018).
The use of both cultural sequencing methods of bark beetle discovered species-rich fungal communi ties associated with pine bark beetle in Ukraine, encom passing different groups of the fungal kingdom and representing several different ecological roles. The overall fungal community was composed of 83.5% Ascomycota, 9.5% Basidiomycota and 3% Mucoromycotina, 4% of species remaining unidentifi ed. The most commonly detected groups for all species were saprotrophic fungi (28.1%), ophiostomatoid fungi (18.8%) and pathogens (17.3%), while the frequency of the other ecological group vary from 0.8 to 7.02 % (Fig. 1)  In particular, Ascomycota species were common to be associated with all insects at all sites. Among ophiostomatoid fungi, only Ceratocystis ips and Pesotum piceaе (syn. Ophiostoma piceaе) have been detected for all species and sites while Grosmannia penicillata and Leptographium sosnaicola were associated exclusively with Ips sexdentatus and Ophiostoma pallidulum with Ips acuminatus (Fig. 2). Moreover, Grosmannia penicillata, G. olivacea, Graphium sp., Ophiostoma canum O. minus. Ophiostoma pallidulum, Graphilbum rectangulosporium and Leptographium sosnaicola were found fi rst in Ukraine and described to be associated with bark beetles in the present study. Other ophiostomatoid fungi Ceratocystis ips and Pesotum piceae were mentioned as associated with Pinus sylvestris logs in Ukraine. Among the basidiomycetes, species belonging to Polyporales, species within Tremellomycetes, Corticiales, Agaricales and Russulales were also detected. Within Basidiomycota group, wood-decaying fungi (Bjerkandera adusta, Fomitopsis pinicola, Heterobasidion annosum, Phlebiopsis gigantea), mycorrhizal fungi (Hebeloma sp.) as well plant pathogen species (Cryptococcus sp.) and nutritional fungus Entomocorticium sp. were found.
Heterobasidion annosum s.l. has also previously been isolated from Hylurgus lighiperda (Davydenko et al., 2014), Hylastes ater and H. angustatus (Meshkoba & Davydenko, 2012) but not from Ips acuminatus (Davydenko et al., 2017), which can be explained by the localization of bark beetles in the bottom stem and root rot mycelia grown into the wood and therefore the different fungal species can appear on the body surface of insects.
Entomocorticium sp. was the most common fungus associated with Ips acuminatus (24,5%) and Ips sexdentatus (12,33%), while no nutritional fungi had been found with Hylurgus ligniperda (Davydenko et al., 2014). In both cases Entomocorticium sp., was detected by direct sequencing fi rst in association to Ips acuminatus and Ips sexdentatus. This is mycangial fungus, which was the most commonly detected fungus in the present study, has previously only been reported in association with Dendroctonus ponderosa Hopkins, (Coleoptera: Curculionidae) and Dendroctonus frontalis Zimmermann (Coleoptera: Curculionidae) (Klepzig et al., 2004). Entomocorticium sp. may provide nutritional benefi ts to larvae of bark beetles while Ophiostoma minus results in poorly developed larvae, which often fail to reach the adult stage (Klepzig et al., 2004). Entomocorticium sp. may provide some protection for larvae of bark beetles against the negative impact of O. minus. Ophiostoma minus is a high virulent fungus in our study, apparently, also antagonistic fungal symbiont, that threatens normal development of larvae because it can stop the increase of nutrient concentrations of the phloem as mycangial fungi do. In contrast, Ambrosiella macrospora (Franke-Grosm.), which is primarily known as a food source for larvae and thought to be non-pathogenic and which is commonly associated with Ips acuminatus in Italy (Villary et al., 2012), but was not detected in our study. The latter examples demonstrate that Ips acuminatus and Ips sexdentatus may vector rather different fungal communities in different parts of Europe including unique nutritional fungus serving an additional source of nutrition for both adults and larvae (Jankowiak, 2013  Sphaeropsis sapinea has been shown to be associated with Ips acuminatus in high frequency (24,0%), second after Entomocorticium sp., but quite rare isolated pathogens from the Ips sexdentatus and never from Hylurgus ligniperda in our study which can be attributed to the absence of H. ligniperda and I. sexdentatus maturation feeding in the crown.
Therefore, our study, provides evidence for the fi rst time that S. pinea is commonly associated with Ips acuminatus and Ips sexdentatus in Ukraine. Generally, complete fungal diversity associated with bark beetle species is better estimated with studies sampling by pooling data for all sites, as can be illustrated by the fact that diversity indices are higher for the Ips sexdentatus and Ips acuminatus than those for Hylurgus ligniperda, Hylastes ater and H. angustatus.
To determine if the insects actively spread the fungi requires more investigation, but it is clear from this study that fungi can be present on the body surface of insects or in their stomachs and could be vectored by bark beetles. We observed that almost all attacks of pine bark beetles were effi cient for some bluestain fungi which were able to colonize sapwood. Some species (as Ophiostoma minus) impair the development of bark beetle larvae whereas other species facilitate bark beetle attack by overcoming tree resistance. This suggests that when established in phloem at the early-stage, ophiostomatoid species competitive abilities are important mechanisms infl uencing the fungal species community. In term of these abilities of ophiostomatoid fungi, the study of their pathogenicity and degree of virulence seems important for awareness of their infl uence on forest health as well as for understanding partnerships in bark beetle-fungi symbiosis, which contributes to most of the success and diversity of these insects.
Several methods can be used by researchers to assess and compare the virulence of the ophiostomatoid fungi associated with bark beetles. The most common method is to measure the size of the lesions forming after the inoculation. Moreover, typical symptoms and damage to the tree would be also useful for assessment. Pathogenicity test revealed different degrees of virulence of different fungal species together with colonization patterns of the plant tissue (Tab. 2). There was a mortality of the inoculated seedlings (75, 58.33 and 45% respectively) after inoculation causing decline of the rest of them (Tab. 2) which was expressed in wilting of the new shoots of the current's years shoots and needle yellowing. Grosmannia penicillata, G. olivacea and Ophiostoma bicolor caused the dead of 33.33, 16.67 and 8.33 7% seedlings respectively and also decline of survivors. Inoculation with Graphilbum rectangulosporium resulted only declining 10% of seedlings (See Tab. 2). Ceratocystis ips, Ophiostoma canum, O. pallidulum and Pesotum piceae did not cause any decline or death of the pine seedlings. No plant dieback or decline was observed in the control.
All testing fungi resulted in lesions of different sizes (See Tab. 2) that were signifi cantly higher than sterile inoculated control plants (p = 0.000). Leptographium sosnaicola and O. minus induced signifi cantly larger necrotic lesions than other ophiostomatoid fungi.
Therefore, all inoculated fungi showed capability to infect plant tissues and cause lesions of different sizes (See Tab. 2). The lesions were generally covered with resin and extended vertically in both directions from the point of inoculation.
Ophiostoma minus, Leptographium sosnaicola and Graphium sp. showed signs of being the most virulent in this study as well as G. olivacea, G. penicillata and O. bicolor which also caused pine decline and mortality. These results support other studies that have found Grosmannia and Leptographium species could show high virulence to conifers in Europe and Scandinavia where they are well-known to exist in symbiosis with spruce bark beetles (Linnakoski, 2011;Jankowiak, 2013). Grosmannia olivacea appears to be signifi cantly less virulent compared with Grosmannia penicillata, while inoculation with Ceratocystis ips, Ophiostoma canum, O. pallidulum and Pesotum piceae resulted in relatively small lesions indicating that these species were weak pathogenic although lesion reactions were longer and deeper compared to control seedlings.
Finally, our results and conclusions corroborate a few previous fi ndings, suggesting that pathogenicity is not specifi c ability and differences in virulence exist between the fungi (Linnakoski, 2011;Jankowiak, 2013;Zhao et al., 2019).
Conclusions. To sum up, eleven ophiostomatoid fungi (Graphium sp., Leptographium sosnaicola, Grosmannia olivacea, Grosmannia penicillata, Ophiostoma bicolor, O. ips, O. canum, O. piceae, O. minus, O. nigrocarpum Graphilbum rectangulosporium) were found in association with fi ve bark beetles (Hylurgus ligniperda, Hylastes ater, Hylastes angustatus, Ips acuminatus and Ips sexdentatus) in Ukraine on Scots pine trees infested by bark beetles. It can be concluded that the studied bark beetles are vectors for different functional groups of fungi including aggressive pathogens, and that ophiostomatoid fungi are the most closely associated symbionts. Moreover, high virulence fungi can help bark beetles to overcome the tree resistance and may signifi cantly contribute to tree mortality.