Features of scots pine mortality due to incursion of pine bark beetles in symbiosis with ophiostomatoid fungi in the forest-steppe of central Siberia

Citation: Barchenkov, A.; Rubtsov, A.; Safronova, I.; Astapenko, S.; Tabakova, K.; Bogdanova, K.; Anuev, E.; Arzac, A. Features of Scots Pine Mortality Due to Incursion of Pine Bark Beetles in Symbiosis with Ophiostomatoid Fungi in the Forest-Steppe of Central Siberia. Forests 2023,14, 1301. https:// doi.org/10.3390/f14071301 Academic Editors: Qing-He Zhang and Giacomo Alessandro Gerosa Received: 10 May 2023 Revised: 15 June 2023 Accepted: 21 June 2023 Published: 24 June 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Article Features of Scots Pine Mortality Due to Incursion of Pine Bark Beetles in Symbiosis with Ophiostomatoid Fungi in the Forest-Steppe of Central Siberia Alexey Barchenkov 1,2, Alexey Rubtsov 1, Inna Safronova 3, Sergey Astapenko 3,4, Kseniia Tabakova 1, Kristina Bogdanova 1, Eugene Anuev 1and Alberto Arzac 1,5,* 1Institute of Ecology and Geography, Siberian Federal University, 660036 Krasnoyarsk, Russia; [email protected]u (A.B.); [email protected] (A.R.); [email protected] (K.T.); [email protected] (K.B.); [email protected] (E.A.) 2V.N. Sukachev Institute of Forest, Siberian Branch of the Russian Academy of Science, 660036 Krasnoyarsk, Russia 3Forest Protection Centre of Krasnoyarsk Krai, Branch of the FFA FBI RCFH, 660036 Krasnoyarsk, Russia; [email protected]u (I.S.); [email protected] (S.A.) 4Center for Forest Pyrology, Branch of the FBI “ARRISMF”, 660062 Krasnoyarsk, Russia 5EiFAB, Universidad de Valladolid, 42004 Soria, Spain *Correspondence: [email protected]u; Tel.: +7-902-979-96-95 Abstract: Forest decline is a significant issue affecting critical ecosystem processes worldwide. Here, we describe mortality in Pinus sylvestris L. monitored trees caused by the inhabitation of pine bark beetles (Tomicus minor Hart.) in symbiosis with ophiostomatoid fungi (Ophiostoma piceae (Munch) H. et P. Sydow) infection in the forest-steppe of central Siberia. Stem sap flow (Q) and stem diameter fluctuations (dRc) were monitored in eight pine trees during seven consecutive growing seasons (2015–2021). In addition, microcore sampling every ten days allowed the determination of stem wood formation in monitored trees in the 2021 growing season. During 2020 and 2021, two cases of Qtermination were recorded among the monitored trees, with microcores revealing no cambium formation. Thus, the seasonal Qonset matches the beginning of the beetle dispersal period when they attack and inhabit tree stems. The decline of circumferential stem size began 10–12 days after Q onset, during the massive inhabitation of beetles into the stems. The disturbance of Qin trees occurred in 21–23 days, and total cessation of Qwas observed 23–26 days after the Qonset at the beetle’s egg development phase. The timing of dRc disturbance and Qcessation observed directly coincides with the beetle life cycle. Thus, the phenology of pine trees and T. minor beetles is driven by seasonal weather conditions, particularly the cumulative air temperature (>0 ◦C). Keywords: dieback Pinus sylvestris; stem sap flow; water transport; dendrometer; tree-growth; wood anatomy 1. Introduction Forests decline due to biotic and abiotic disturbances is a significant problem on a global scale, with important consequences on carbon dynamics and other ecosystem services. The dieback of tree species in the Northern Hemisphere, linked to insect invasions and pathogenic diseases [ 1 , 2 ], has accelerated during the last decades [ 3 ]. Becoming widespread in forest loss [ 4 ], with the boreal forest as the focus of the alert [ 1 , 2 ]. Siberia represents ca. 60% of the world’s boreal forests (Kayes and Mallik, 2020), and in recent decades, has been severely affected by the Siberian silk moth (Dendrolimus sibiricus Chetverikov; Lepidoptera: Lasiocampodae) [ 5 , 6 ] and the four-eyed bark beetle (Polygraphus proximus Blandford) outbreaks [ 7 , 8 ], usually acting in symbiosis with associated fungi [ 9 , 10 ]. Moreover, the pine bark beetle (Tomicus minor Hartig; Coleoptera: Curculionidae: Scolytinae) has also been recently recognized as an invasive species in the Russian Northwest [ 11 ]. Forests 2023,14, 1301. https://doi.org/10.3390/f14071301 https://www.mdpi.com/journal/forests Forests 2023,14, 1301 2 of 15 Moreover, insect activity (i.e., phenology, migration, and population dynamic) is environmentally driven [ 5 ], as well as climate extremes may also lead to drought-induced pine forest mortality [ 12 , 13 ]. Therefore, all forest decline causes should be comprehensively studied and descriptively reported for better actions in future forest protection. The number of publications about forest pathogens is growing, but the mechanisms of pest-induced harmful influence on trees remain unclear. Fungi of the ophiostomataceae family, a group of wood blue-stain fungi [ 14 ], represent the first stage of fungal succession in plant tissues of conifers when bark beetles inhabit living trees. Their primary way of dispersion is by entomochory—the fungi spores easily stick to the exoskeletons of xylophagous insects [ 15 ]. Thus, the beetles penetrating under the bark of trees transfer the fungi spores to the phloem layer, from where the mycelium actively spreads into the conductive tissues (xylem and sapwood) and clogs them. As a result, the transport of water and nutrients along the trunk is disrupted, leading to tree stress and death [16]. Enlarging a worldwide network of research sites for long-term environmental and ecophysiological monitoring of terrestrial ecosystems increases the possibility of studying forest vulnerability to pest-related impacts on the level of individual tree responses. Although different methodologies have been applied in multiple forest health monitoring and assessment studies (e.g., [ 17 – 19 ]), here we focused on dendrometry, sap flow, and wood anatomy tools as indicators of vital physiological processes in trees. We aimed to analyze and compare water/growth-related processes in three conifer species of trees [ 20 ], for what a combination of in situ monitoring (i.e., dendrometers and sap flow measurements) was established; however, while monitoring the trees, we recorded the dieback of two studied Pinus sylvestris L. (Pinales: Pinacea: Pinoidae) trees caused by the inhabitation of pine bark beetles in the area during 2020 and 2021. Although similar cases in the USA showed how pine trees died due to pine beetles incursion in terms of physiological processes either from dendrometer records [ 21 ] or combined with sap flow measurements [ 22 ], to the best of our knowledge, this is the first time in which tree dieback is instrumentally monitored in Siberia, providing a great opportunity to study this phenomenon. Moreover, additional proxies such as wood anatomy may be used to detect early signs of pine mortality in the xylem and phloem at the upper parts of trees [ 23 ], and also, when applied in combination with dendrometer measurements, became a promising approach in tree growth studies [ 24 – 27 ]. Here, we combined evidence from in situ physiological monitoring (i.e., circumferential stem variations and sap flow measurements), dendrochronological measurements, seasonal growth monitoring, entomology, and mycology to increase our understanding of P. sylvestris dieback induced by pine bark beetles’ outbreak during 2020 and 2021. We theorize that (1) an environmental factor may lead to the weakening of trees allowing the infestation by the beetle-fungi symbiosis; (2) the cessation of sap flow and cambial activity in infested trees will be linked to the pine bark beetle life cycle, which in turn will be influenced by weather conditions. 2. Materials and Methods 2.1. Study Area The study was carried out at the Pogorelsky bor experimental field station (56 ◦ 22 0 N, 92 ◦ 57 0 E, 247 m a.s.l.; Figure 1a) located in the forest-steppe zone of Central Siberia, 37 km northward from Krasnoyarsk (Russia). A research plot of about 450 m 2 in size with 50-year-old Scots pine (P. sylvestris) adjoins with cultivated Larix provenance trial plot [ 28 ]. The climate of the region is cold continental subarctic (Dfc) [ 29 ] with cold winters, warm summers, and relatively low precipitation (Figure 1b). The site shows a mean annual temperature of 1.18 ◦ C and total annual precipitation of 440 mm during the 2005–2017 period. The average height of pine trees within the research plot was 21 m, the mean tree diameter at breast height was 25 cm, and the stem basal area of the plot was 58 m2/ha. Forests 2023,14, 1301 3 of 15 Forests 2023, 14, x FOR PEER REVIEW 3 of 15 Figure 1. Description of the research site: (a) site location (red circle); (b) mean monthly air temperature (°C, red line), relative air humidity (%, green line), and precipitation (mm, blue columns) for the 2005–2017 period; (c,d) photographs of the studied trees (photo by A. Rubtsov). 2.2. Instrumental In situ Measurements Physiological monitoring was carried out in eight trees over the 2015–2021 period, with an increasing amount of instrumentally measured pines from three trees in 2015– 2016 to eight trees in 2020–2021 (Figure 1c,d; see Table S1 for further details). Precise stem circumference length changes (dRc, mm) at 1.8 m were obtained from DR-26 band dendrometers (EMS, Brno, Czech Republic). Stem upward sap flow rate (Q, kg/h/cm) measurements at 1.3 m were performed following the trunk segment heat balance (THB) method [30] with SF-51 and SF-81 (Microset 8x, EMS, Brno, Czech Republic) sensors . In addition, local micrometeorological variables (i.e., precipitation, air temperature, relative air humidity, and photosynthetic radiation (EMS QTHi)) and soil variables (i.e., moisture, temperature, and water deficit) were recorded with 10 min temporal resolution. Further, all initial data were preprocessed in a few steps, including verification, correction of errors, and noise reduction by averaging to hourly mean values. Preprocessing raw sap flow data also included correction of Q values considering heat losses from the point of measurements to the environment, known as sap flow baseline [31], and averaging to hourly time series. In 2020, after the death of tree PP1, monitoring instruments were installed in tree PP10. 2.3. Dendrochronological Measurements and Seasonal Growth One diametral wood core was taken at breast height using a 5 mm diameter increment borer per monitored tree in the summer of 2022 (1 August). Cores were polished using a belt sanding machine with successive sandpaper (up to 800 grit), and their surfaces were scanned at 3200 dpi with an Epson Perfection V800 scanner (Epson, Suwa, Japan). Tree-ring width (RW) was measured on the scanned cores using CooRecorder version 9.3 (Cybis Elektronik & Data AB, Saltsjöbaden, Sweden). A master chronology previously produced for the area (including 17 trees sampled at 700 m from the research plot [32,33]) was used to accurately cross-date the trees, verified using COFECHA [34]. Individual raw Figure 1. Description of the research site: ( a ) site location (red circle); ( b ) mean monthly air temperature ( ◦ C, red line), relative air humidity (%, green line), and precipitation (mm, blue columns) for the 2005–2017 period; (c,d) photographs of the studied trees (photo by A. Rubtsov). 2.2. Instrumental In Situ Measurements Physiological monitoring was carried out in eight trees over the 2015–2021 period, with an increasing amount of instrumentally measured pines from three trees in 2015–2016 to eight trees in 2020–2021 (Figure 1c,d; see Table S1 for further details). Precise stem circumference length changes (dRc, mm) at 1.8 m were obtained from DR-26 band dendrometers (EMS, Brno, Czech Republic). Stem upward sap flow rate (Q, kg/h/cm) measurements at 1.3 m were performed following the trunk segment heat balance (THB) method [ 30 ] with SF-51 and SF-81 (Microset 8x, EMS, Brno, Czech Republic) sensors. In addition, local micrometeorological variables (i.e., precipitation, air temperature, relative air humidity, and photosynthetic radiation (EMS QTHi)) and soil variables (i.e., moisture, temperature, and water deficit) were recorded with 10 min temporal resolution. Further, all initial data were preprocessed in a few steps, including verification, correction of errors, and noise reduction by averaging to hourly mean values. Preprocessing raw sap flow data also included correction of Qvalues considering heat losses from the point of measurements to the environment, known as sap flow baseline [ 31 ], and averaging to hourly time series. In 2020, after the death of tree PP1, monitoring instruments were installed in tree PP10. 2.3. Dendrochronological Measurements and Seasonal Growth One diametral wood core was taken at breast height using a 5 mm diameter increment borer per monitored tree in the summer of 2022 (1 August). Cores were polished using a belt sanding machine with successive sandpaper (up to 800 grit), and their surfaces were scanned at 3200 dpi with an Epson Perfection V800 scanner (Epson, Suwa, Japan). Tree-ring width (RW) was measured on the scanned cores using CooRecorder version 9.3 (Cybis Elektronik & Data AB, Saltsjöbaden, Sweden). A master chronology previously produced for the area (including 17 trees sampled at 700 m from the research plot [ 32 , 33 ]) was used to accurately cross-date the trees, verified using COFECHA [ 34 ]. Individual raw series were standardized using ARSTAN [ 35 ]. Pearson’s correlations between monthly climatic data (temperature, precipitation, and water balance) and RW residual chronologies were Forests 2023,14, 1301 4 of 15 used to evaluate the pine growth response to climate for the 1979–2021 period (this period covers the age of sampled monitored trees). Correlations were performed with a temporal resolution ranging from the previous year, July, to the growth year, September. The water balance (WB) was estimated as WB = P − PET, where P is the precipitation, and PET is the potential evapotranspiration calculated by the Thornthwaite equation [36]. Microcores for seasonal wood formation monitoring were collected every ten days, from 14 May 2021 to 30 September 2021, with a “Trephor” [ 37 ] and placed into Eppendorf tubes (four mL) containing an ethanol–glycerol solution and stored at five degrees ( ◦ C) for further processing. Due to the small size of the microcores, thick barks were removed from the trees before the sampling to extract only living tissues following the methodology proposed by Rossi et al. [ 37 ]. Next, microcores were dehydrated with ethanol in different consecutive concentrations (70%, 90%, and 96%) and cleared with Sub-X previous infiltration (Leica TP1020 tissue processor, Wetzlar, Germany) and embedding (Leica HistoCore Arcadia, Wetzlar, Germany) in paraffin. Finally, 10 µ m thick cross-sections were prepared with a rotary microtome (Leica RM2235, Wetzlar, Germany), stained with a safranin-alcian blue solution, fixed with Eukitt, and digitized with a NanoZoomer slide scanner (Hamamatsu, Shizuoka, Japan). 2.4. Beetles and Fungi Monitoring Boring dust, the first sign of beetles’ attacks on the trees, was seen one week after the vegetation season began in two of the monitored years (2020 and 2021). In situ inspections of the dead pines, performed in October 2020, June and October 2021, and May 2022, allowed the recognition of the past activity of the pine bark beetle T. minor. Considering the presence of multiple entrance and exit holes in the bark, abundant longitudinal egg galleries with perpendicular (to the stem) larval tunnels, and the occasional dead beetle bodies in the phloem layer. Therefore, different approaches were followed to determine the severity of T. minor beetle activity: (1) A quantitative estimation of the T. minor seasonal population after the emergence of beetles from stems was carried out in October 2021 on two of three colonized trees, including PP6 (Figure 1c, the tree died in 2021). Firstly, the total number of beetle entrance and exit holes was accounted for in the bark, on the lower part of the stems, at 1.5 m height in segments with an area determined by a height of 25 cm multiplied by the circumference length of the trunks. Secondly, the bark was removed, and the numbers of mother galleries and larvae tunnels of beetles were counted and converted to pcs/dm 2 . (2) A collateral estimation of T. minor population density considering their intensity of maturation (secondary) feeding in pine shoots was carried out in May 2022 by counting damaged shoots that fell on the ground after the 2021 season. The falling shoots pruned by T. minor were calculated in five random plots with a size of 1 m 2 within the research plot. (3) Determination of intraseasonal phenological phases of beetles development and activity in the research plot was based on the calculation of accumulated positive air temperature (Taccu), calculating daily average (Tavg) from 10 min air temperature records for every single day and progressively summed positive Tavg values, skipping Tavg < 0 ◦ C, starting from 1 January for the years 2020 and 2021), matching the Taccu values with previously reported ranges accordingly to A.I. Yakovenko [ 38 ]. The calculated timing of T. minor intra-annual life cycle periods consisted of the following phases: flight, egg layout, egg development, feeding of larvae, pupae development, young beetles under the bark, secondary feeding, and hibernation, with the division of each phase period to the intensity stages—onset, massive, and ending. Samples of fine and thick roots, bark, and pieces of underlying stem wood (approximately 4 × 5 × 6 cm) were taken to determine the species of fungi infecting and its dispersion rate in the pine wood. In October 2020, from PP1 and two control trees (all the trees died in 2020), and in June 2021, from PP6 and another no monitored dead tree, PPx, (both trees died in 2021). Cultivation and determination of fungi species from the bark and stem sapwood samples containing the galleries of T. minor were carried out in the laboratory. First, the initial presence of fungal sporulation in the samples was checked Forests 2023,14, 1301 5 of 15 under magnification (Micros MC100 with digital camera Levenhuk C1400NG, Gewerbezone, Austria). Then, isolation of pure spores of fungi culture was performed with a sterile bacteriological needle from beetle galleries into Petri dishes with filter paper impregnated with custom medium made of unhopped beer wort agar diluted to a sugar concentration of three degrees according to Balling with the addition of lactic acid (4 mL/L of medium) to inhibit the growth of bacteria. Pure cultures were incubated in a dark chamber at 22 ◦ C for 42 days with subculturing. Cultivated fungi were identified based on visual recognition of their morphological structures in sporulation. Finally, to evaluate the dispersion of fungi in the stems, two dead trees, the nonmonitored PPx (with visible boring dust and canopy yellowing signs) and the monitored PP6, were felled in June and October 2021, respectively. Stem discs were extracted at different heights from each felled tree. In total, five discs from PPx (i.e., 0, 1.3, 5.3, 9.3, 17.3 m; Figure S1) and twelve discs from PP6 (i.e., 0, 1.3, 3.3, 5.3, 7.3, 9.3, 11.3, 13.3, 15.3, 17.3, 19.3, 21.3 m; Figure S2). Discs from both trees were digitized in high image resolution. Measurements of stem wood area affected by fungi in each stem disc were performed on the corresponding pictures in the image editor GIMP 2.10 as the ratio of a blue stain area to a total area of a given disc with semiautomatic contouring (contrast-based threshold masking) of particular areas. 3. Results 3.1. Stem Sap Flow and Radial Size Changes During the in situ monitoring carried out in 2020 and 2021, the dieback of two trees (one each year) was documented by the abrupt cessation of upward stem sap flow (Q) occurring in the late spring (Figure 2). First, a temporal shift of 18 days in the vegetation season beginning was observed from 2020 to 2021. Seasonal sap flow in pine stems started on 12 April 2020 (day of the year (DOY) 103) and on 30 April 2021 (DOY 121), and the beginning of tree water transport in 2020 occurred earlier in comparison to the Qonset dates range observed in previous years (2015–2019). Thus, sap flow unalterably stopped on 8 May 2020, in tree PP1 and on 23 May 2021, in tree PP6, matching the timing of 26 and 23 days after the beginning of pine transpiration activity periods in each year (Figure 2a,c and Figure 3a,c), respectively. After these dates, the diurnal data pattern of Qreversed to the opposite (Figure 3a,c), corresponding to the data signature of Qmeasurements in dead wood obtained experimentally long before in cameral tests of the equipment. Finally, the sap flow cessation led to the canopy browning in the midsummer, complete defoliation, and total dieback of those trees by the end of the vegetation seasons. Interestingly, sap flow stopped in the tree PP10 on 21 August 2020, at three-quarters of the vegetation season (Figure 2a), a year with an average of 177 days of sap flow, the longest compared to the previously observed range (144–173 days) during the 2015–2019 period, where the rest of trees showed the stop of sap flow on 6 October. Nevertheless, the tree remained alive in 2021 without any visible signs of weakening, and Qmeasurements on the side part of the tree stem in the season of 2021 showed normal sap flow data patterns (Figure 2c). Diurnal cycles of stem circumference size variations (dRc) of the infected trees started to differ from the dRc of healthy trees 10–12 days before the sap flow termination, showing a continuous stem shrinkage in time (Figure 3b,d) and deviation in their regression ( Figure 4b,d ). A diurnal dRc data pattern change to the opposite of the normal water/growth-related cycle occurred on the first day of Qtermination. Healthy trees showed diurnal dRc dynamics corresponding to the day/night transpiration cycle, reaching the daily minimum at the peak sap flow with high atmospheric vapor pressure deficit and daily maximum at night [ 39 ], while dRc of the infested trees started to follow the air temperature variations. Later, a permanent decline of mean daily stem circumference size was observed in the trees PP1 and PP6 after the sap flow stop dates (Figure 2). Forests 2023,14, 1301 6 of 15 Forests 2023, 14, x FOR PEER REVIEW 6 of 15 Figure 2. Daily base-lined stem sap flow rates ((a,c)—Q, kg/cm/day) and daily stem circumference size changes ((b,d)—dRc, mm, adjusted) of the studied pine trees (PP1–PP13) at the POG site in the vegetation seasons of 2020 (a,b) and 2021 (c,d). Red lines represent the Q and dRc time series of the dead trees PP1 (a,b) and PP6 (c,d); bold black line represents Q in the tree PP10. Dark green lines represent the mean Q and dRc values of healthy trees each year. Diurnal cycles of stem circumference size variations (dRc) of the infected trees started to differ from the dRc of healthy trees 10–12 days before the sap flow termination, showing a continuous stem shrinkage in time (Figure 3b,d) and deviation in their regression (Figure 4b,d). A diurnal dRc data pattern change to the opposite of the normal water/growthrelated cycle occurred on the first day of Q termination. Healthy trees showed diurnal dRc dynamics corresponding to the day/night transpiration cycle, reaching the daily minimum at the peak sap flow with high atmospheric vapor pressure deficit and daily maximum at night [39], while dRc of the infested trees started to follow the air temperature variations. Later, a permanent decline of mean daily stem circumference size was observed in the trees PP1 and PP6 after the sap flow stop dates (Figure 2). Figure 2. Daily base-lined stem sap flow rates (( a , c )—Q, kg/cm/day) and daily stem circumference size changes (( b , d )—dRc, mm, adjusted) of the studied pine trees (PP1–PP13) at the POG site in the vegetation seasons of 2020 ( a , b ) and 2021 ( c , d ). Red lines represent the Qand dRc time series of the dead trees PP1 ( a , b ) and PP6 ( c , d ); bold black line represents Qin the tree PP10. Dark green lines represent the mean Qand dRc values of healthy trees each year. Forests 2023,14, 1301 7 of 15 Forests 2023, 14, x FOR PEER REVIEW 7 of 15 Figure 3. Hourly raw stem sap flow rate (Q, kg/cm/h, (a,c)) and stem radial size change (dRc, mm, (b,d)) data of the studied pine trees, representing the dieback of the trees PP1 in 2020 (a,b) and PP6 in 2021 (c,d). Figure 3. Hourly raw stem sap flow rate (Q, kg/cm/h, ( a , c )) and stem radial size change (dRc, mm, ( b , d )) data of the studied pine trees, representing the dieback of the trees PP1 in 2020 ( a , b ) and PP6 in 2021 (c,d). Forests 2023, 14, x FOR PEER REVIEW 7 of 15 Figure 3. Hourly raw stem sap flow rate (Q, kg/cm/h, (a,c)) and stem radial size change (dRc, mm, (b,d)) data of the studied pine trees, representing the dieback of the trees PP1 in 2020 (a,b) and PP6 in 2021 (c,d). Figure 4. Scatterplots of raw (not base-lined) hourly sap flow (Q, ( a , c )) and stem circumference change (dRc, ( b , d )) hourly values measured in the trees PP1 ( a , b ) died in 2020, PP6 ( c , d ) died in 2021 versus corresponding values measured in the healthy (control) tree PP2. Red dots represent data from Forests 2023,14, 1301 8 of 15 died/alive trees posterior to the start dates of irreversible dRc and Qchanges in each year related to the trees’ mortality, black dots show the ratios of the same values prior to it, and black lines show linear regression in these periods. 3.2. Development of Beetles and Fungi The quantitative estimation of the T. minor seasonal population, after the emergence of beetles from stems carried out in October 2021, showed a population density of 22.4 beetles per dm 2 of the parental generation and 37 pcs/dm 2 of the young generation in the lower part of the stems. At the same time, the estimation of T. minor population density, considering their intensity of maturation (secondary) feeding in pine shoots during the season of 2021 (data obtained in May 2022), resulted in an average number of seven shoots per 1 m 2 , characterizing the young generation of beetles population as high [ 40 ], capable of reaching 25–40 thousand beetles per hectare. The reconstruction of the beetle’s lifecycle stages using Taccu let to assign the timing of Qand dRc disturbance in the monitored colonized trees with certain phenological phases of T. minor (Figure 5). The beginning of T. minor seasonal activity—the dispersal phase, when some mature beetles start to fly and attack pine trees, occurred on the next day after the seasonal Qonset (SoS) in both years, on 13 April 2020 (DOY 104) and on 1 May 2021 (DOY 122) at Taccu = 94–96 ◦ C, respectively. The massive flight phase occurred two days later (Taccu = 111–114 ◦ C), and the diurnal fluctuations of dRc in the colonized trees started to deviate from healthy trees (Figure 4c,d) after 23 April 2020 (DOY 114) and 11 May 2021 (DOY 131) in the ending of this phase at Taccu = 176–184 ◦ C (i.e., 10–12 days after SoS). Further, the egg layout phase began simultaneously with the egg development phase on 25 April 2020 (DOY 116) and 14 May 2021 (DOY 135) at Taccu = 224–228 ◦ C. The sap flow diurnal patterns started to decline (Figure 4a,b) on 3 May 2020 (DOY 124) and 21 May 2021 (DOY 142) at Taccu = 275–332 ◦ C when the beetle’s eggs switched to the massive development phase. The upward sap flow termination in the PP1 and PP6 trees occurred on 8 May 2020 (DOY 128) and 23 May 2021 (DOY 144), 23–26 days since the SoS, and it corresponds to the massive egg development phase of T. minor at Taccu = 300–350 ◦C. Forests 2023, 14, x FOR PEER REVIEW 8 of 15 Figure 4. Scatterplots of raw (not base-lined) hourly sap flow (Q, (a,c)) and stem circumference change (dRc, (b,d)) hourly values measured in the trees PP1 (a,b) died in 2020, PP6 (c,d) died in 2021 versus corresponding values measured in the healthy (control) tree PP2. Red dots represent data from died/alive trees posterior to the start dates of irreversible dRc and Q changes in each year related to the trees’ mortality, black dots show the ratios of the same values prior to it, and black lines show linear regression in these periods. 3.2. Development of Beetles and Fungi The quantitative estimation of the T. minor seasonal population, after the emergence of beetles from stems carried out in October 2021, showed a population density of 22.4 beetles per dm 2 of the parental generation and 37 pcs/dm 2 of the young generation in the lower part of the stems. At the same time, the estimation of T. minor population density, considering their intensity of maturation (secondary) feeding in pine shoots during the season of 2021 (data obtained in May 2022), resulted in an average number of seven shoots per 1 m 2 , characterizing the young generation of beetles population as high [40], capable of reaching 25–40 thousand beetles per hectare. The reconstruction of the beetle’s lifecycle stages using Taccu let to assign the timing of Q and dRc disturbance in the monitored colonized trees with certain phenological phases of T. minor (Figure 5). The beginning of T. minor seasonal activity—the dispersal phase, when some mature beetles start to fly and attack pine trees, occurred on the next day after the seasonal Q onset (SoS) in both years, on 13 April 2020 (DOY 104) and on 1 May 2021 (DOY 122) at Taccu = 94–96 °C, respectively. The massive flight phase occurred two days later (Taccu = 111–114 °C), and the diurnal fluctuations of dRc in the colonized trees started to deviate from healthy trees (Figure 4c,d) after 23 April 2020 (DOY 114) and 11 May 2021 (DOY 131) in the ending of this phase at Taccu = 176–184 °C (i.e., 10–12 days after SoS). Further, the egg layout phase began simultaneously with the egg development phase on 25 April 2020 (DOY 116) and 14 May 2021 (DOY 135) at Taccu = 224–228 °C. The sap flow diurnal patterns started to decline (Figure 4a,b) on 3 May 2020 (DOY 124) and 21 May 2021 (DOY 142) at Taccu = 275–332 °C when the beetle’s eggs switched to the massive development phase. The upward sap flow termination in the PP1 and PP6 trees occurred on 8 May 2020 (DOY 128) and 23 May 2021 (DOY 144), 23–26 days since the SoS, and it corresponds to the massive egg development phase of T. minor at Taccu = 300–350 °C. Figure 5. Dynamics of daily stem sap flow (Q) of the infested PP1 (black solid line) and PP6 (red solid line) trees and accumulated daily mean positive air temperature (Taccu, dashed lines). Black lines—the data from 2020; red lines—the data from 2021. Columns represent the T.minor beetle Figure 5. Dynamics of daily stem sap flow (Q) of the infested PP1 (black solid line) and PP6 (red solid line) trees and accumulated daily mean positive air temperature (Taccu, dashed lines). Black lines—the data from 2020; red lines—the data from 2021. Columns represent the T.minor beetle phases (dimensionless): Fo—flight onset, Fm—massive flight, El—egg layout, Ed—egg development, Edm—massive egg layout/development, and Lfo—larvae feeding onset; grey columns—in 2020, purple—2021. Forests 2023,14, 1301 9 of 15 The fungus in the stem wood samples was identified as Pesotum piceae Crane & Schoknecht, Sporothrix Hektoen & Perkins, an anamorph of Ophiostoma piceae (Munch) H. et P. Sydow (Ophiostomatales: Ophiostomataceae). Conidial sporulation obtained during the cultivation formed a bunch of brown septate conidiophores united in coremia ( Figure S3 ). Conidia were located in a large mucous droplet at the apex of the coremia. Analysis for fungi detection in the sampled pine roots did not reveal the presence of any fungi. The analysis of stem disc cuts at several heights of the dead trees PP6 and PPx showed significant differences in the profile of the O. piceae fungi spread along and across the stem tissues in two-time intervals since the incursion of T. minor. About 60% of blue-stain lesions on wood tissues in the stem cross-area of the tree PPx at 1.3 and 5.3 m heights were observed at the end of June 2021 when the tree was felled. While only 4–8% are in the middle (9.3 m) and bottom (0 m) parts, but not at the upper part (0% at 17.3 m; Figure S1). Later by the end of the season, in October 2021, the tree PP6 showed blue-stain lesions in most of the stem, where fungi occupied 64–97% of the total cross-section areas. In contrast, only one part of the stem (at 13.3 m) showed to be much less (14%) affected by the fungi (Figure S2). Because T. minor beetles are the vector carriers of O. piceae fungi spores, the distribution of fungi in PPx provided an example of how the lower part of the stem was more accessible for T. minor beetles to penetrate through the bark, and it may also coincide with lower intensity of beetle attacks to this particular tree. The entire round spread of the fungi in the conductive tissues in the lower part of the stem led to the mortality of this tree. In contrast, the tree PP6 showed a higher effect of the fungal activity with the almost total detriment of sapwood along the stem length after ca. six months since the infestation and ca. five months since the sap flow termination. A less blue wood area at the height of 13.3 m in PP6 can only provide an assumption that the beetles did not inhabit this part of the trunk, but it is likely that the O. piceae later could eventually occupy this part of the trunk as well. 3.3. Wood Formation and Climate Response Wood formation monitoring revealed the onset of cell enlargement in 2021 between 10 June and 22 June (DOY 161–173). However, when comparing a healthy tree (e.g., PP2) with the infested tree PP6, no cambial activity was observed in the affected (died) tree (Figure 6). By this time, the beetle’s phenological cycle already passed to the larvae feeding phase at Taccu = 520–650 ◦C. Secondary growth from monitored trees was controlled by environmental factors during the growing season (Figure S4), favored by temperature during the mid-spring (April, r = 0.47; p< 0.001) and early autumn (September, r = 0.28; p< 0.05), whereas precipitation and water balance negatively affected tree secondary growth in the midspring (April, r = −0.38 and; p< 0.05 and r = −0.5 and; p< 0.001, respectively).