PINE ENGRAVER BEETLE IPS ACUMINATUS AS A POTENTIAL VECTOR OF SPHAEROPSIS SAPINEA

The pine engraver beetle Ips acuminatus Gyll. is a potential vector of the Sphaeropsis tip blight pathogen according to Leach’s postulates. The specimens of I. acuminatus were associated with numerous fungi species, namely Sphaeropsis sapinea (Fr.) Dyko & B. Sutton and ophiostomatoid species. The association between opportunistic pathogen S. sapinea and I. acuminatus has been confirmed for 62.9 % of all branches (44 % of needle samples and 82 % of wood samples). The presence of S. sapinea in the galleries and on the surface of the beetle indicates that I. acuminatus may transport the pathogen and later introduce it into healthy trees. The bark beetle can transfer pathogenic fungus during maturation feeding on the shoots of healthy pine crowns and into the branches during making galleries. K e y w o r d s : Sphaeropsis tip blight, insect-fungus interaction, Scots pine.

). Unfortunately, no data about the spread of S. sapinea in Ukraine are known, excluding some regions in the Left-Bank Ukraine (Davydenko 2018, Meshkova et al. 2012. Moreover, this pathogen was found to be in close relation with Ips acuminatus (Davydenko et al. 2017). Previously, associations between S. sapinea and other bark beetles, namely Tomicus piniperda, Hylastes attenuates, Hylurgops palliates, have been reported from northern Spain (Bezos et al. 2015). Furthermore, an association between the exotic insect Leptoglossus occidentalis and S. sapinea has been revealed in Italy on pine cones (Luchi et al. 2012).
Тhe etiologies of bark beetle and associated tree diseases are usually identified as they become apparent. To a great extent, that is due to the fact that causal agents are often easy to isolate and the completion of Koch's postulates is straightforward in practice (Ploetz et al. 2013). However, the regular incidence of secondary and other additional beetle species in damaged trees, which play no role in the development of tree diseases, makes it unclear about the roles of the respective beetle in tree disease transmission (Ploetz et al. 2013). Leach (1940) reviewed insects which vector plant pathogens and developed four rules to confirm that an insect is the vector of a respective pathogen. He pointed out that it is necessary to demonstrate four postulates: (1) a close association of the insect with diseased plants; (2) regular visits of healthy plants by the insect; (3) an association of the pathogen with the insect; and (4) the development of the disease in healthy plants after interaction with pathogen-infected insects. Therefore, our study also provides evidence that S. sapinea is commonly associated with I. acuminatus in Ukraine, contributing to the decline and dieback of P. sylvestris.
The aim of the study was to determine whether the pine engraver beetle, Ips acuminatus, is a vector for the pathogen S. sapinea. To confirm this, Leach's postulates (Leach 1940, Bezos et al. 2015 were tested: (1) a close, although not constant, the association between I. acuminatus and trees affected by Diplodia tip blight; (2) regular visit by I. acuminatus to healthy Scots pine forest; (3) the presence of the pathogen on the insect in nature; and (4) whether I. acuminatus can successfully vector the pathogen to disease-free host material under controlled conditions. Materials and Methods. The field study was carried out in 2016. Association between bark beetles and healthy/infested crowns of pine trees has been studied by different authors (Ploetz et al. 2013, Bezos et al. 2015, Lieutier et al. 2015 and different methods were used for testing Leach's postulates (Bezos et al. 2015). In the present study, we tried to demonstrate the capability of I. acuminatus to infest symptomless green crowns of P. sylvestris in plots affected by S. sapinea. To confirm an association between I. acuminatus and diseased trees (postulate 1), an inspection of cut Scots pine trees attacked by I. acuminatus was carried out to find trees infested by bark beetles and S. sapinea. Field study sites were pure pine forest stands located in Sumy Region in Ukraine (Ohtyrske Forest Enterprise). To make sure that S. sapinea is present in sites, visual symptomatic branches have been collected ( Figure 1). The stands at all sites were ca. 60-80-year-old plantations of Scots pine (Pinus sylvestris) where S. sapinea was found according to morphological symptoms using wet chamber and light microscopy ( Figure 2). Simultaneously, examination and sampling of Scots pine trees attacked by I. acuminatus were carried out in disease-free stands (postulate 2). To determine whether I. acuminatus regularly visits healthy Scots pines, also fallen branches and shoots of P. sylvestris affected by pine engraver beetle were collected and analysed for the presence of the pathogen.
Adults of I. acuminatus were collected individually from infested trees of P. sylvestris from all sites (disease-free and with the presence of S. sapinea) and stored singly until analyses for associated fungal pathogens (postulate 3). For this, 192 individuals from four sites were collected randomly and analysed.
Fungal culturing and molecular identification. To determine whether the pathogen occurs on the insects in nature (postulate 3), sampled adults of I. acuminatus were checked for the pathogen presence. Samples for fungal isolation were placed on 2 % malt extract agar (MEA, Difco, BD, Franklin Lakes, NJ, USA) containing 200 ppm of cycloheximide and 300 ppm of streptomycin (Sigma-Aldrich), to be selective for Diplodia and Ophiostoma species and avoid the growth of bacterial isolates and fast-growing fungi such as Trichoderma spp, Penicillium spp. etc. Obtained cultures were used to get pure isolates by transferring mycelium from the edges of single colonies to fresh 2% MEA. Cultures were incubated at 22 °C for 10-12 days and grouped according to the morphological characteristics of colonies and conidiophores, and single spore cultures were prepared from germinating conidia of isolates representing morphological groups of different sites (Linnakoski et al. 2012).
The morphological identification of S. sapinea was based on the macro-and microscopic characteristics of the isolates. Specimens were observed both under a stereomicroscope and a light microscope after anamorph fruiting structures were mounted on glass slides in cotton blue.
DNA was extracted from the single spore cultures of the isolates representing morphological groups of different sites. Approximate DNA concentrations were determined at 260 nm using the Nano-drop 2000 spectrophotometer (Nano-drop Technologies, Wilmington, DE, USA), and extracts were diluted to 10 ng μl−1 in double-distilled water (Sigma-Aldrich, St. Louis, MO, USA). The presence of S. sapinea was verified using the specific primer pairs DpF and BotR described by Stanosz et al. (2007). PCR was performed in a final volume of 50 μl. Each tube contained: 0.8 lM forward primer (Sigma-Aldrich, Schnelldorf, Germany); 0.8 lM reverse primer (Sigma-Aldrich); 12.5 μl TaqManTM AmpliTaq Gold PCR Master Mix (Applied Biosystems, California, USA); 5-10 ng fungal DNA. Each DNA sample was assayed in duplicate. Negative controls (sterile water) and DNA from reference strain were included in all reactions. A Biometra T1 Thermocycler (Whatman Biometra, Gottingen, Germany) was used for the PCR with the following cycler protocol: 95°C for 5 min, 30 cycles of 95°C for 1 min, 53°C for 1 min and 72°C for 1 min with a final extension of 72°C for 5 min. PCR fragments were analysed by agarose gel electrophoresis with 0.7 g in 100 ml 1 9 Tris-boric acid-EDTA buffer (TBE) and visualized by SYBR Safe (Life Technologies, Milan, Italy) staining.
Vector tests. To confirm the vector of the pathogen by I. acuminatus, the disease was produced experimentally under controlled conditions (postulate 4) on healthy shoots which were attacked by artificially inoculated specimens of I. acuminatus. For this, ten adults of I. acuminatus insects were inoculated with a conidial suspension of S. sapinea. Each insect was inoculated with 50 μl of the suspension by micropipette. The suspension was obtained from a pure culture of S. sapinea growing on MEA and forming typical spores. Scots pine branches (5-7) with diameters 13-18 mm were put in glass receptacles with water to avoid the desiccation. These branches were checked preliminary for disease-free (S. sapinea) by molecular methods to be sure that latent infection of S. sapinea absents (Davydenko 2018). Branches together with inoculated specimens of I. acuminatus were placed onto plastic containers for maturation feeding and colonization for 45 days. Afterward, all branches were visually checked to find symptoms of Spaheropsis shoot blight (SSB). Moreover, 3-5 pieces of wood tissue ca. 1 cm length and 3-5 needles from each shoot with visible entry holes and maturation feeding were removed and plated on MEA containing antibiotics in order to reisolate S. sapinea using classical phytopathological methods (Davydenko 2018).
Statistical analyses. All data were tested for adherence to the normal distribution using the Kolmogorov -Smirnov test. The differences between the insect and wood samples in relation to the presence/absence of S. sapinea were analysed by Fisher's exact test and by the analysis of variance (ANOVA) followed by Tukey's HSD post hoc test. The significance was evaluated at the 0.05 plevel. Statistical analysis was carried out using the statistical software package PAST: Paleontological Statistics Software Package for Education and Data Analysis (Hammer et al. 2001).
Results and Discussion. Postulate 1&2: the association between I. acuminatus and P. sylvestris in both infested by S. sapinea and disease-free sites.
In general, 120 samples of branch, shoots and needles of Scots pine attacked by I. acuminatus were collected from 12 randomly selected trees at four sites. A sampling of needles and wood resulted in 197 morphological groups of fungal cultures. Molecular analyses of fungal morphological groups using S. sapinea specific primers demonstrated the absence of S. sapinea in free-disease sites attacked by I. acuminatus (Figure 3).

Fig. 3 -UV visualization of PCR on agarose gel electrophoresis (staining 1% TBE buffer)
All shoot samples in disease-free areas where I. acuminatus was present demonstrated the absence of S. sapinea infection (Table 1).
However, two from 30 needle samples collected from Site 2 disease-free demonstrated the presence of S. sapinea, probably in the latent stage, because no symptoms of SSB has been observed. However, Z-test showed that a single case is not significantly different from the sample (Z = -1.6202, p-value (two-tailed) = 0.10519), so we can assume that postulate 1 has been proved. All symptomatic samples of shoots and needle collected at sites 3 and 4 showed the presence of SSB pathogen (Table 1). F-test showed a significant difference between presence/absence of SSB at diseases-free and disease-presence sites (F = 72.25, df = 15, p-value =1.744E-09), that is a crucial proof for postulate 2. Furthermore, F-test illustrates a significant difference (F = 36, df = 7, p-value = 0.0009645) between the infection rate into needles and wood (shoots), but probably, this does not prove the general applicability of results to spread the infection. We consider that different infection rates could be explained by the various qualities of samples for DNA extraction and using species-specific primers. Therefore, our results only reaffirmed the possibility for I. acuminatus to visit both infested and not infested by SSB areas (postulate 1 and 2). Postulate 3 association of the pathogen with the insect: Samples of specimens of I. acuminatus and shoots with signs of maturation feeding or breeding galleries were analysed aiming to identify fungal phytopathogens, in particular ophiostomatoid fungi and S. sapinea. The most abundant fungal phylum was Ascomycota for all samples accounting for an average of 87.8% of the total species. The most commonly detected fungi from pure culture from the insects were Sphaeropsis sapinea (39.58%) and ophiostomatoid fungi (Table 2). A total of 96 breeding galleries were sampled from symptomatic trees, and 16.67 % of them gave rise to S. sapinea colonies when plated on MEAs. The S. sapinea pycnidia were also observed on samples placed in wet chambers. Our study demonstrated that S. sapineawas found to be associated with I. acuminatus (Table 2) that has already been confirmed by our previous study (Davydenko et al. 2017, Davydenko 2019. A few specimens of I. acuminatus collected in diseasefree sites were identified to be associated with S. sapinea (6.3% of all beetles). No S. sapinea was found in the galleries of I. acuminatus in disease-free trees. Other pathogens such as Lophodermium species were found to be associated with I. acuminatus, that confirmed our previous results (Davydenko et al. 2017, Davydenko 2018, 2019. Postulate 4: Vector test under control condition. To confirm that the bark beetles of I. acuminatus can vector pathogen S. sapinea during maturation feeding or making breeding galleries into branches with I. acuminatus, the disease was produced experimentally under controlled conditions. Individuals of bark beetles were collected from dying Scots pine trees infested by bark beetles (Ohtyrske Forest Enterprise). Our study showed that the most common species was Ips acuminatus (82.8% of all samples), while 17.3% were Tomicus piniperda and Ips sexdentatus that colonize the lower part of the stem with thick bark; only a few specimens of Tomicus minor were found which colonize the upper part of the stem with thin bark, that had already been revealed in the previous study (Meshkova & Zinchenko 2013).
Scots pine branches were cut from disease-free trees and checked preliminary by molecular analyses with Diplodia-specific primers randomly. SSB-free Scots pine branches (5-7) with diameters 13, 15, and 18 mm together with inoculated by S. sapinea specimens of I. acuminatus were placed into plastic containers. Results of check of branch samples in the presence/absence of S. sapinea demonstrate the capacity of I. acuminatus to vector S. sapinea (Figure 4) as well as other fungal pathogens, e.g. ophiostomatoid fungi (Davydenko et al. 2017, Davydenko 2019. In general, 62.9% of all branches (44% of needle samples and 82% of wood samples) showed the presence of S. sapinea, while any confirmation of the S. sapinea presence in control samples was not found and all groups showed a significant difference comparing with control one (χ 2 f = 9.49, p-value is 0.0132 Chi-square at Cochran -Mantel -Haenszel test). According to SSB data for different branch diameter, the chi-squared test statistic is 1.20 with an associated p < 0.1371, so the null hypothesis is not rejected, since p > 0.05, and a conclusion is made that branch diameter is not associated with SSB infection. As no data from the Chi-square test indicate that the probabilities of the two variables are related, we cannot consider a relationship between variables. Therefore, our results confirm postulate 4 which indicates the development of the disease in healthy plants after their interaction with pathogen-infested insects. Undoubtedly, not all the samples with S. sapinea showed SSB typical symptoms. However, this could be explained by the existence of the latent phase of S. sapinea that has been confirmed by various authors worldwide (Davydenko 2019), so the development of typical symptoms could slow down.
The association between I. acuminatus and P. sylvestris trees affected by S. sapinea was observed during field sampling in 2015-2016 and during other field studies. Bark beetles and breeding galleries collected from symptomatic trees were positive for S. sapinea. This may indicate that the larvaes/beetles were already infected by SSB when making a gallery or that the crown and bark was already infected with S. sapinea. The probability of I. acuminatus being contaminated with the pathogen would be increased by the insects excavating their breeding galleries in diseased trees (Bezos et al. 2015, Davydenko et al. 2017.
Conclusion. Our study confirmed that Ips acuminatus is probably a vector of Sphaeropsis sapinea, according to Leach's postulates.
Our study demonstrates that specimens of I. acuminatus were associated with numerous fungi species, which were generally dominated by tree pathogens, namely Sphaeropsis sapinea and ophiostomatoid species. The association between opportunistic pathogen S. sapinea and I. acuminatus is of considerable importance to forest health, particular to drought-stressed Scots pines. The presence of S. sapinea in the galleries and on the surface of the beetle indicates that I. acuminatus may transport the pathogen and later introduce it into healthy trees. Moreover, the shoots are most likely to become infected with the pathogen during maturation feeding. However, further studies are required for a better understanding of the relationship between the life cycles of the I. acuminatus and the S. sapinea. For this tree disease, improved detection, prediction, and management should be the major goals of future research.