Abstract

Tree selection and inoculation

The following studies occurred over a 2-year period from 2018 to 2020. For the 2018 dehumidification kiln trials, 30 M. polymorpha trees (6.0 to 10.8 cm diameter at breast height [DBH]) were selected from the Hilo Forest Reserve (19°41.782′N, 155°13.309′W) and for the 2019 vacuum kiln study; 19 trees (20 to 32 cm DBH) were selected from the Waiākea Forest Reserve (19°37.361′N, 155°06.297′W), both on the east side of Hawai‘i Island near Hilo. Both sites had documented ROD mortality for several years (Vaughn et al. 2018). The trees were numbered with circular metal tags and a 3-m-long section along the main stem was demarcated with spray paint to indicate the future log to be harvested. For both trials, the trees were artificially inoculated by applying a fungal agar slurry to stem wounds as previously described by Hughes et al. (2020). Two wounds on the lower stem of trees were inoculated with the systemic wilt pathogen, C. lukuohia, whereas four wounds at increasing stem heights were inoculated with the canker pathogen, C. huliohia (Juzwik et al. 2019a). The C. lukuohia-inoculated wounds were 60 cm (for dehumidification trials) or 138 cm (for vacuum kiln trials) above the log bottom demarcation line, on opposite sides of the stem. In contrast, the four wounds receiving C. huliohia were made between 60 cm and 240 cm above the log bottom demarcation line with 60 cm vertical spacing between each wound placed in a spiral manner around the tree stem, thus allowing for formation of multiple cankers. After application of fungal inoculum to the sapwood, bark plugs were replaced over the inoculated wounds and sealed with duct tape.

For the 2018 dehumidification kiln study, a single fungal isolate was used for inoculum (C. lukuohia, P14-1-1; C. huliohia, P16-8). For the first experiment 12 trees were inoculated in April; an additional 18 trees were inoculated for the second experiment in June. In total 30 trees were inoculated, 15 trees per each Ceratocystis species. For the 2019 vacuum kiln trials a mixture of two fungal isolates was used per Ceratocystis species (C. lukuohia, P14-1-1 and P16-7; C. huliohia, P15-59 and P16-8). Inoculum production and methods were the same as mentioned above. To allow for more time for internal fungal colonization of C. huliohia, 13 trees were inoculated in August and C. lukuohia inoculations occurred in September (6 trees).

Tree felling and log preparation

Study trees were felled 70 and 100 days after inoculation for the first and second dehumidification kiln trials (2018), respectively, and 119 (C. lukuohia) and 147 (C. huliohia) days after inoculation for the 2020 vacuum kiln trials. For this study, the smaller-diameter main stems (≈8-cm large-end diameter) used in the dehumidification kiln trials are referred to as poles and the thicker main stems (≈20- to 25-cm large-end diameter) of the vacuum kiln trials are referred to as logs. After felling, side limbs were removed, poles were cut to a length of 3.0 m (dehumidification kiln trial) and logs to 2.45 m (vacuum kiln trial), and all wood was transported to the US Department of Agriculture (USDA) Agricultural Research Service (ARS) Pacific Basin Agricultural Research Center (PBARC) in Hilo for processing. The bark was removed by draw knife to the outer sapwood, and poles and logs were shortened to approximately 2.6 m (dehumidification kiln trial) and 2.0 m (vacuum kiln trial), respectively. A 4-cm-thick disk was removed from one of the logs' (vacuum kiln) or both poles' ends (dehumidification kiln) to assess pretreatment wood MC. To determine pretreatment C. lukuohia viability in ‘ōhi‘a wood, an additional 4-cm-thick disk of symptomatic (discolored) wood was removed from each pole/log end (two per wood length; Fig. 1A), labeled, and held in a cold room at 5°C until further processing. For C. huliohia-inoculated wood, sampling occurred in the areas adjacent to the inoculation points where staining associated with fungal colonization was most evident. Thus, four disks were removed per log for pretreatment fungus viability assays and held at 5°C (Fig. 1B). Final pole lengths for the dehumidification kiln trials were 2.5 m for C. lukuohia-inoculated wood and slightly shorter (2.1 m) for C. huliohia-inoculated wood as pretreatment viability samples were collected near the area with vascular staining. Vacuum kiln logs were at a final length of 1.7 m. The ends of the reduced-length wood sections were painted with liquid paraffin sealant (Clear Multi-Surface Waterproofer, Thompsons WaterSeal, Cleveland, OH or Anchorseal, Anchor-Seal Inc., Gloucester, MA) to minimize heat infiltration through the cut ends. Poles/logs were labeled with circular metal tags.

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Wood temperature, moisture monitoring, and drying schedules

Temperatures at the pole/log surface and within the wood were monitored in real time using thermocouple wires and data loggers to determine start of threshold temperature exposure timing. For each dehumidification kiln trial, three C. lukuohia- and three C. huliohia-infested ‘ōhi‘a poles (six total per kiln charge) were drilled in the base of the pole end and to the geometric center, thermocouple wires inserted, and each hole filled with plumbers' putty to prevent unwanted steam intrusion into the hole (Chen et al. 2017). A separate thermocouple wire was left on top of the pile of poles to monitor ambient air temperature within the kiln. All thermocouple wires in the kiln were fed outside via a small polyvinyl chloride conduit pipe (backfilled with foam) to two data loggers (Hobo, Onset Computer Corp., Bourne, MA). One C. lukuohia- and one C. huliohia-inoculated pole (two total) with thermocouple wires were held outside the kiln at ambient temperatures as unheated negative controls for the June trial and two poles per fungal species (four total) for the September trial.

For each kiln charge of the 2020 vacuum kiln study, three logs were selected for temperature monitoring. Per log, three holes were drilled into wood at radial depth of 70 percent toward the log's core at ⅓, center, and ⅔ of the log length. Another hole was drilled to the geometric center and one through the cut face of the log. Omega K-type thermocouple wires were inserted into the holes, the holes filled with plumbers' putty, and a single thermocouple placed on a log exterior to monitor ambient temperatures inside the kiln box. Thermocouple wires exited the kiln chamber via a sealed port to an outside computer data acquisition system (LabVIEW, National Instruments Corp., Austin, TX) for data collection and real-time thermal monitoring. A single log per fungal species with thermocouple wires was held outside the kiln at ambient temperatures per each vacuum kiln trial as a negative control. For the three vacuum kiln trials conducted, two drying schedules were used. For the first trial, the heating schedule was designed to kill Ceratocystis fungi by reaching the 56°C target temperature at a radial depth of 70 percent toward the core and then the vacuum drying process was initiated. For the second and third trials, the heating schedule was designed to increase the temperature more slowly to the targeted 56°C during the vacuum drying cycle (combining the heating and drying cycles).

For all trials with both kiln types, internal temperatures of logs were monitored to determine the time when the threshold temperature was reach for each kiln load. Wood MC was assessed from a subsample of each pole or log before and after heat treatments using an oven-drying method (Eckelman 1997; Fig. 1). To obtain wood MC, a 4-cm-thick disk (or a representative portion) was immediately weighed after cutting and placed into a drying oven (105°C) until a constant weight was obtained. Dry-basis wood MC was determined as (Eckelman 1997):

Kiln heating treatments

Log treatments were conducted using commercial equipment at local mills in Hilo and Waimea, Hawai‘i Island. For the 2018 trial, a dehumidification kiln (High Temperature DH System, Nyle Systems LLC, Brewer, ME) was used. ‘Ōhi‘a poles were placed on top of a commercial load of stickered hardwood flooring planks milled on site. The kiln operator made gradual temperature increases on the basis of his experience and knowledge. Once the target temperature of 60°C at the core for all thermocouple-monitored poles (n = 3 in each trial) was achieved, the temperature was held constant for a minimum of 5 hours. The kiln was then shut off and doors opened when the operator deemed it appropriate (to avoid wood warping or splitting). For the first trial (June 2018), poles from five C. lukuohia- and C. huliohia-inoculated trees each were placed in the kiln for heat treatment, with two ambient negative controls (12 poles total). For the second trial (September 2018), seven poles were placed in the kiln and two poles were held outside as ambient controls per fungal species (18 poles total). In total, 24 Ceratocystis-infested poles were kiln-treated along with six nonheated negative controls (30 poles for both trials combined).

A steam-heat-operated continuous vacuum kiln (iDry Standard, iDRY LLC, Barre, VT) with a solar-augmented (preconditioning) water-heating device was used to treat M. polymorpha logs (≈20- to 25-cm large-end diameter) in late January–early February 2020. Components of the system were a vacuum chamber, vacuum pump, water boiler, and control system. The chamber dimensions were 1.7 m wide by 1.7 m high by 4.3 m long. When all monitored logs reached 56°C at a radial depth of 70 percent to the core, the load was held for 30 minutes. Three trials (kiln charges) were conducted, but rate of heat increase and number of drying cycles differed between Trial 1 and Trials 2 + 3 to determine effects on total time required and resulting wood MC. Trial 1 used a test schedule to kill the Ceratocystis spp. in the logs and achieve a fast vacuum drying rate (referred to as kill + dry schedule); Trials 2 + 3 only focused on killing Ceratocystis and did not try to optimize log drying (referred to as kill-only schedule). In the first trial using the kill + dry schedule, five C. lukuohia- and C. huliohia-inoculated logs (10 total) were heated in the kiln with a single log per fungus species left outdoors (ambient temperature) under shade beside the kiln chamber to serve as negative controls (2 total). In the second (C. lukuohia logs only) and third (C. huliohia logs only) trials using the kill-only schedule, five logs were heated per kiln charge, with a single log serving as an ambient temperature control for each trial. In total, 20 Ceratocystis-infested logs were kiln treated and four nonheated Ceratocystis-infested logs served as negative controls (24 logs for both trials combined).

Posttreatment and Ceratocystis viability testing

After treatment, the thermocouple wires were removed and the logs were transported back to the USDA PBARC facility. For C. lukuohia-inoculated trees, log end segments (22 cm) were removed and discarded. Four 4-cm-thick disks were removed per log end with two used to assess posttreatment MC levels and two to assess Ceratocystis viability (Fig. 1A). For C. huliohia-inoculated trees, three disks were removed per log end, with two used for fungus viability assays and one for MC determination (Fig. 1B). Disks for pre- and posttreatment Ceratocystis viability testing were bagged and stored at 5°C.

Small sapwood chips were obtained with a chisel and mallet and used for carrot baiting (Moller and DeVay 1968). For the smaller-diameter poles of the dehumidification kiln study, two radial depths of up to 1.0 cm (outer zone) and 1.1 to 2.0 cm (inner zone) were sampled. For the larger logs from the vacuum kiln, the assayed zones were 0.5 to 2.5 cm (outer) and 3.0 to 5.0 cm (inner). On the basis of wood disk circumference, four (90° apart) and six (60° apart) regions around the disk were subsampled for the dehumidification and vacuum kiln studies, respectively. Control logs were treated in a similar manner. To bait Ceratocystis fungi, the excised wood chips were sandwiched between two carrot discs (approx. 0.5–1.0 cm thick), wrapped in laboratory film (Parafilm, Bemis Co., Neenah, WI), individually bagged, and incubated at ambient temperatures (≈24°C) for up to 30 days (Hughes et al. 2021). Baits were rated positive for viable Ceratocystis growth when grayish mycelia and fruiting bodies (perithecia) were visible on wood chips or carrot surfaces. For those baits in which perithecia did not fully mature, a qPCR detection assay was used to confirm the pathogen(s) from excised carrot bait pieces (Heller and Keith 2018). To validate the accuracy of our Ceratocystis visual identifications, a subsample of positive carrot baits was assayed using qPCR as described above (n = 56 total).

Wood temperature, MC monitoring, and drying schedules

For the 2018 dehumidification kiln, the first trial lasted a total of 22 days (526 total h), and logs were heated to 60°C at their core for 10 hours (Fig. 2A). In the second kiln trial, an unexpected loss of power occurred, and the kiln remained closed until the proper components were obtained and a service technician was able to restore function. This resulted in a longer than usual run time of 34 days (812 h in kiln). Poles were exposed to 60°C at their core for 5 hours (Fig. 2B). Core temperatures of the control poles oscillated from 21 to 29°C and 23 to 32°C for the first and second trials, respectively (Fig. 2). Once the target temperature was reached for each trial, the kiln was shut off and allowed to cool before wood was removed. Initial ‘ōhi‘a wood MC was 75 to 77 percent and 81 to 83 percent for the first and second trials, respectively. Dehumidification kiln treatment reduced the wood MC to approximately 14 to 16 percent, whereas natural drying of control poles stored under ambient conditions resulted in a MC of 30 percent (data not shown).

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For the first 2020 vacuum kiln trial using the kill + dry schedule, log temperature increased rapidly over 14 hours to 56°C at the target 70 percent radial core depth and the kiln was held at that log temperature for 30 minutes (Fig. 3A). The temperature was then lowered to approximately 47°C and held for additional vacuum cycles for 95 hours to provide some drying of the wood (4.5 days/110 h in kiln). For Trials 2 and 3 using the kill-only schedule, the temperature component of the treatment schedule was increased more slowly (over ∼48 h) until 56°C was reached at the targeted wood depth with minor emphasis on drying the wood (Fig. 3B). Wood for the vacuum kiln trials had initial MC values ranging from 73 to 89 percent. In the first trial, where drying was of interest, logs remained at 68 percent MC, whereas in the latter two trials, where drying was not a focus, logs retained a MC of 74 to 76 percent (data not shown).

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Dehumidification kiln trials of Ceratocystis viability

For both the first and second trials, viable C. lukuohia was recovered onto carrot baits in 94 to 100 percent of wood samples (n = 240) before kiln heating treatments (Table 1). Ceratocystis huliohia was recovered in 76 to 99 percent of carrot baits (n = 240). For both pathogens, the fungus was isolated more often from the outer sapwood zones than the inner (Table 1). In comparison, viable Ceratocystis species were never detected in carrot baits from kiln-heated wood samples (n = 384, data not shown). The negative control poles held outside the kiln under shade at ambient temperatures for a mean of 28 days commonly yielded the pathogens. Specifically, C. huliohia was isolated in 65 percent and C. lukuohia in 96 percent of carrot baits (n = 48 per fungal species) on the basis of three poles per species. All visually positive carrot-bait subsamples taken for qPCR validation were positive and congruent with the inoculated Ceratocystis species (data not shown). Thus, the dehumidification kiln treatments were successful in eradicating viable Ceratocystis propagules from the colonized poles. Like our previous work with a vacuum–steam unit, a threshold temperature of 60°C eradicated C. lukuohia, C. huliohia and the oak and walnut pathogens Bretziella fagacearum and Geosmithia morbida, respectively (Juzwik et al. 2019b, 2021, 2022). The current standard of 60°C for 60 minutes (heat treatment standard T314-a) was accepted for treating ash (Fraxinus spp.) firewood in quarantined areas of the United States against the heat-tolerant emerald ash borer (Agrilus planipennis; Myers et al. 2009, National Plant Board Firewood Guidelines 2020, USDA Treatment Manual 2022).

Table 1. Carrot-baiting recovery means for detection of rapid ōhia death-associated fungi Ceratocystis lukuohia and Ceratocytis huliohia from the outer and inner sapwood of debarked logs from artificially inoculated Metrosideros polymorpha trees before dehumidification kiln treatment.

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Vacuum kiln trials of Ceratocystis viability

In Trial 1 of the vacuum kiln, both C. huliohia- and C. lukuohia-colonized logs were used with the kill + dry treatment schedule. The pathogens were found from 25 to 78 percent of wood samples assayed (n = 288) and there was no pattern to fungal recovery on the basis of sapwood depth sampled (Table 2). Neither fungus was isolated from logs after the 4.5-day kill + dry kiln schedule. In contrast, fungal detection rates for control logs held outside the kiln were 63 percent (n = 24 subsamples) and 25 percent (n = 24 subsamples) for C. lukuohia and C. huliohia, respectively (data not shown).

Table 2. Carrot-baiting recovery means for detection of rapid ōhia death-associated fungi Ceratocystis lukuohia and Ceratocystis huliohia from the outer and inner sapwood of debarked logs from artificially inoculated Metrosideros polymorpha trees before vacuum kiln treatment.

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For the second and third trials, study logs received the more gradual kill-only schedule over 40 and 48 hours (Table 2). For the second trial, C. huliohia-infested logs were used, and the fungus was isolated from 31 percent of 144 subsamples (combined depths) before kiln treatment. Ceratocystis lukuohia-infested logs were used in the third trial and the pathogen was isolated from approximately 55% subsamples (n = 144, combined depth) before treatment. In these trials, more fungi were recovered from the inner sapwood zones than outer (Table 2). Ceratocystis huliohia was not isolated from disk subsamples in Trial 2 (n = 120) and C. lukuohia was not isolated in Trial 3 (n = 120) after their kiln treatments. In contrast, rates for fungal detection in the control logs held outside the kiln were 17 percent (n = 24 subsamples) and 29 percent (n = 24 subsamples) for C. huliohia and C. lukuohia, respectively. Thus, the vacuum kiln treatments were successful in eradicating viable pathogen propagules from the colonized logs. This threshold temperature eradicated Ceratocystis and several tree pathogens, including Phytophthora cinnamomi and Lasiodiplodia theobromae (Ramsfield et al. 2010; Juzwik et al. 2019b, 2021, 2022). The heating schedule of 56°C/30 min is the accepted wood treatment standard to eradicate gypsy moth (Lymantria dispar) and Asian longhorn beetles (Anoplophora glabripennis) in the United States (Myers and Bailey 2011, Heat Treatment Standard T314-b, National Plant Board Firewood Guidelines 2020, USDA Treatment Manual 2022) and is the international standard for treating solid wood packing material to prevent the unintentional spread of wood inhabiting pests and pathogens (ISPM 15; International Plant Protection Convention 2006).

Within this trial, log-core temperatures were monitored at a radial depth of 70 percent to the core since neither ROD Ceratocystis species colonizes the core heartwood (Hughes et al. 2020, Juzwik et al. 2019a). Although USDA log and firewood treatment schedules (T314) measure temperature to the log's core (USDA Treatment Manual 2022), in our study, wood temperatures at a 70 percent radial depth were similar to that of the core (Fig. 3) and thus increasing the target depth would likely not require extensive additional heating times.