Field burning experiments
The effect of exposure to flames and heat in a bonfire on the survival of Xtp in naturally infected pistachio wood was assessed in two field experiments. Both were conducted at the Department of Primary Industries Victoria, Irymple, in winter, to comply with fire bans imposed in other seasons in bushfire-prone areas. One was done in 2008 (34°15′14″S, 142°12′41″E, 63 m a.s.l.) and the second in 2009 (34°13′06″S, 142°11′16″E, 58 m a.s.l.). To minimize the likelihood of false negative results arising from the discontinuous distribution of Xtp, small pieces of wood confirmed to be infected were enclosed in mesh bags and attached with wire to metal poles, then retrieved for assessment after the bonfire.
Branches and twigs with stained xylem typical of dieback were taken in 2008 from a pistachio tree known to be infected in the Waite Campus orchard, University of Adelaide, South Australia (34°58′18″S, 138°38′00″, 127 m a.s.l.) (E. Facelli, The University of Adelaide, Australia, personal communication) and in 2009 from infected trees in a commercial orchard in Robinvale, Victoria (34°34′46″S, 142°46′47″E, 61 m a.s.l.) (Facelli et al., 2005). The presence of Xtp was confirmed by culturing as described by Facelli et al. (2005) with slight modifications and by PCR as described by Marefat et al. (2006). Briefly, small segments of branches or twigs were surface-disinfested by dipping in 95% ethanol and flamed. After cutting the ends and removing the bark with a sterile scalpel, fragments 0·5–1 mm thick and 5–10 mm in length were excised and soaked in 9 mL sterile distilled water (SDW) overnight at room temperature (approximately 22ºC). Aliquots (100 μL) of the resulting suspension were spread onto Petri plates of sucrose peptone agar (SPA) (Moffett & Croft, 1983) amended with 150 mg L−1 Benlate® (BSPA) (Facelli et al., 2005) in 2008 or onto nutrient agar (NA, Oxoid) supplemented with 10 mg L−1 cephalexin, 1 mg L−1 ampicillin and 0·7 mg L−1 gentamycin (NA+A) in 2009. NA+A was modified from XTS, a medium semiselective for Xanthomonas campestris pv. translucens (Schaad & Forster, 1985); the plating efficiency of Xtp on NA+A was 90–100%, in two replicates over time. Aliquots (1·5 mL) of the suspensions were also centrifuged, the pellets resuspended in 15 μL SDW and 1 μL used as template in PCR with primers specific for the pathogen (Marefat et al., 2006). Branches and twigs that were PCR-positive and resulted in isolation of Xtp-like colonies were cut into pieces 1 cm long (for branches 3–5 cm in diameter) or 5–7 cm long (for twigs 1·5–2 cm in diameter). Five to 12 such pieces of woody material (total c. 50 g), cut the day before the experiments, were enclosed in stainless steel mesh bags (0·9 mm aperture, 0·37 mm wire diameter, 50% open area) (Sefar Metal Mesh Pty Ltd) folded to form bags, to investigate the effect of direct exposure to the flames on survival of Xtp. At the same time, another set of pieces of wood was placed in glass Petri dishes (100 mm in diameter) before enclosing in wire mesh, to examine the effect of the heat of the fire on pathogen survival.
In 2008, six steel poles were placed upright at random within a 5- (length) × 3·5- (width) × 0·5- (depth) m pit, dug into soil (sandy clay loam) approximately 10 m from a grape vineyard, 35 days before igniting the bonfire. The soil was dry to the touch. One mesh bag containing exposed infected wood was attached to each of four poles at 20 cm and three poles at 50 cm above the pit floor and another bag was buried 5 cm below the pit floor at pole 6. One mesh bag containing infected wood in a Petri dish was attached to each of two poles at 20 cm and two poles at 50 cm above the pit floor. The pit was then filled with 32-day-old dried grapevine canes as fuel (Sosnowski et al., 2012). Another mesh bag containing infected wood was kept in the laboratory as a control.
In 2009, three replicate pits, 5 m apart, were dug at 5–15 m from a small grove of pistachio trees. Mesh bags containing infected wood exposed or placed inside Petri dishes (c. 20 of each) were attached to steel poles with wire at four positions in relation to the pit floor (0, 20 and 50 cm above and 5 cm below) and the poles were placed at five positions within each pit. The pits were then filled with branches, without leaves, cut from pistachio trees. Half of these branches had been pruned from trees in winter 2008 and left in the open, and the remainder had been cut and dried for about 7 weeks before the experiment. These materials were distributed evenly among the three pits. Five mesh bags, each containing 110–120 g infected wood, were kept in the laboratory as controls.
The temperature of the fire above and below the pit floor in which the infected wood was burned was estimated using methods modified from Lanoiselet et al. (2005). In brief, Tempilstik crayons (Tinco Ltd) with melting points of 50, 60, 70, 80, 90, 110, 130, 150, 170, 190, 210, 220, 230, 240 and 250°C and 40, 50, 60, 70, 80, 200, 400, 600, 788, 982 and 1200°C were used in 2008 and 2009, respectively. Pieces of crayon (one specific temperature per tube) were inserted into glass test tubes (60 × 5 mm), which were sealed with aluminium foil. The tubes were then placed into 100-mm-diameter glass Petri dishes, enclosed in mesh bags and attached to the poles at 0, 20 and 50 cm above and 5 cm below the pit floor. Additionally, one bag was buried 10 cm below the pit floor at the base of each pole. After the bonfire, the maximum temperature, as indicated by the Tempilstik crayons, was recorded for each position.
Bonfires were lit using a flame-thrower on 12 August 2008, a dry sunny day, and in 2009 in the afternoon of 16 July for pits 1 and 2 and the morning of 17 July for pit 3, when conditions were damp (see Table 1).
Table 1. Weather conditions and characteristics of the fires in three pits in which pistachio wood was burned in 2009. Fires in pits 1 and 2 were lit in the afternoon of 16 July and in pit 3 in the morning of 17 July. Weather data were based on data provided by http://www.eldersweather.com.au
| Air temperature (°C)||12·6||13·5||4·3|
| Relative humidity (%)||70||48||97|
| Wind speed (km h−1)||7 (gusts 11)||13 (gusts 20)||2 (gusts 5)|
| Prevailing wind direction||East–southeast||South||East–northeast|
|Characteristics of fire|
| Starting time||12:10 h||16:15 h||08:55 h|
| Accelerant time (min)||4||4·5||9·5|
| Intense burn duration (min)||20||20||20|
| Total burn time (min)||40||40–105||20–105|
Immediately after cooling, all mesh bags were retrieved and the contents weighed. The weight of the contents in mesh bags or Petri dishes before and after the bonfire was compared using a paired t-test with 95% confidence intervals. The contents of each mesh bag or Petri dish were assessed for colour, dryness and cracking compared with the control. Remains were then categorized as non-burned, partially burned, charcoal and ash, according to degree of charring. Non-burned material was defined as comprising pieces of wood that remained natural in colour with slightly moist bark. Partially burned material comprised pieces of wood that changed colour from light to dark brown or black, dried out, cracked on the surface. Charcoal comprised wood that had turned black, brittle and porous, and ash was mainly fine, grey powder. The proportions of non-burned and/or partially burned, or charcoal and/or ash in mesh bags or Petri dishes were expressed as percentages of the total number. Following assessment, three samples were taken at random from each mesh bag or Petri dish from each of the positions to assess survival of Xtp by culturing and PCR as described above. Where the remains were charcoal, they were first ground using a sterile mortar and pestle.
Survival of Xtp
In 2008, charcoal or ash was mixed thoroughly with a sterile teaspoon and samples of 0·1 and 0·5 g from each batch of charcoal or ash were incubated separately in 9 mL SDW overnight at room temperature (c. 22°C). Where sufficient remains were available, an additional portion of 1 g charcoal or ash was also assessed. Also, small amounts of charcoal or ash were placed directly on BSPA amended with 10 mg L−1 cephalexin, 1 mg L−1 ampicillin and 1·4 mg L−1 gentamycin (ABSPA; E. Facelli, The University of Adelaide, Australia, personal communication). Tissues (0·5–0·7 g) from non-burned and control wood were soaked in 9 mL SDW overnight at room temperature. Aliquots (100 μL) from all resulting suspensions were spread onto each of two plates of ABSPA. The suspensions were also used for pathogen detection by PCR (Marefat et al., 2006). In 2009, samples were prepared for assessment of survival and PCR in the same manner, except that each sample was standardized to 0·5–0·6 g. As the plating efficiency of ABSPA proved variable in 2008, NA+A was used to isolate Xtp from the remains of the bonfires in 2009.
In vitro temperature experiments
The effect of temperature on survival of Xtp in liquid culture media and in artificially and naturally infected wood was examined to establish the critical time–temperature relationship lethal to the pathogen. Xtp isolate DAR 75532, obtained from a diseased pistachio tree in a commercial orchard at Kyalite, New South Wales (Facelli et al., 2005) was used where a pure culture was required.
Thermal death time
The method of Brown (2009) was used. Cultures of Xtp were grown in sucrose peptone broth (SPB) and nutrient broth (NB) overnight at 28°C. From each suspension, 10 mL, with 109 colony forming units (CFU) mL−1, were transferred into a McCartney bottle then immersed in a water bath adjusted sequentially to 40, 45 then 50°C. These temperatures were chosen based on previous observations that the pathogen appeared to grow slowly at 40 and 50°C (data not shown). Mercury-filled thermometers were inserted into bottles of sterile SPB and NB to monitor the temperature of the broth cultures. Aliquots from each temperature treatment were removed aseptically every 10 min for up to 60 min, serially diluted 10-fold to 10−6, then 10 μL of each dilution were pipetted in triplicate onto SPA and NA. Controls comprised aliquots removed before the McCartney bottles were placed in the water bath. The plates were incubated inverted at 28°C in the dark for up to 14 days and CFU were enumerated. The experiment consisted of two replications over time. Data were subjected to analysis of variance (anova) using the statistical software genstat version 11.1 (Lawes Agricultural Trust). Treatment means were compared by the least significant difference (LSD) procedure at the 5% significance level.
Thermal death point
The above procedure was used to determine thermal death point except that the overnight suspensions of Xtp in SPB and NB were incubated at 50, 55, 60, 65 and 70°C for only 10 min (Brown, 2009). Aliquots from each temperature treatment were diluted 10-fold to 10−6 and three replicate drops plated onto SPA and NA to determine the temperature at which Xtp was killed in 10 min. The plates were incubated inverted at 28°C and CFU enumerated as above. The experiment was conducted once with three replicate bottles for each temperature treatment. CFU counts before and after the temperature treatment were compared using a paired t-test with 95% confidence intervals.
Survival in infected twigs and wood
Based on the results of experiments to examine the effect of burning and high temperature in culture, the effect of incubation at 40–60°C on survival of Xtp in inoculated twigs and naturally infected branch segments was assessed. Twigs were inoculated by a vacuum infiltration method modified from Salowi (2010). Twigs (0·7–1·2 cm in diameter and 7 cm long) were collected from confirmed pathogen-free 4-year-old P. vera cv. Sirora that had been maintained in pots in a shadehouse located at the Waite Campus or from confirmed disease-free trees (cv. Sirora, 33 years old) in a dieback-free commercial orchard at Saddleworth, South Australia (34°05′02″S, 138°46′70″E, 324 m a.s.l.). The twigs were vacuum-infiltrated with suspensions of Xtp (250 μL, 108 CFU mL−1) then placed in Petri dishes and sealed with Parafilm. After 10 days at 28°C, one-third of each twig was processed to confirm the presence of the pathogen and determine the population prior to heat treatment. The remaining two-thirds of those twigs that yielded an initial population of 104 CFU mL−1 or greater were used for the experiments.
For naturally infected wood, twigs from branches with excessive resinous exudate were collected from the aforementioned pistachio tree in the Waite Campus orchard to confirm infection status. Branches bearing twigs confirmed to contain viable pathogen were collected and cut into segments 2–3 cm long for small branches (1–2 cm diameter) and 1–3 cm long for larger branches (>2 cm diameter). These segments were then bisected longitudinally through visibly stained xylem so that each half was likely to contain bacteria. One section was processed immediately to determine the initial population of the bacteria and the other section was exposed to the designated temperature prior to counting bacteria.
Before exposing the twigs or branch segments to each temperature treatment, they were enclosed in Petri dishes, with a moist Whatman filter paper (grade 41, 90 mm diameter) wedged into the lid to prevent drying out. The Petri dishes were then sealed with plastic film (GLAD® Products Australia). Exposure time was recorded from the time that the incubator reached the designated temperature, generally 15–20 min after the plates of twigs or wood had been placed inside the incubator. All experiments were set up as a completely randomized design. The time required for internal tissues of a twig (c. 1 cm diameter) or wood piece (c. 1 cm thick) to reach 50, 55 and 60°C was determined using a Hastings Data Logger (Gemini Data Loggers (UK) Ltd). The pith temperature was measured independently three times by inserting the probe of the logger into the pith of three additional twigs or pieces of wood in a Petri dish. The area surrounding the point of entry of the probe was covered with heat-resistant plasticine. Data were recorded every 10 min for 4 h.
In a preliminary experiment with artificially infected twigs (experiment 1), a single plate containing five twigs was exposed to 50°C for 60, 120, 150 or 180 min or to 55 and 60°C for 60, 120 or 180 min. In a second experiment (experiment 2) with a new batch of inoculated twigs, three replicate plates, each comprising two twigs per plate, were exposed to 50, 55 or 60°C for the same durations as in experiment 1. Subsequently, new batches of twigs were exposed to 40°C for 30 or 60 min (experiment 3), or to 55 (experiment 4) and 60°C (experiment 5) for 15, 30, 45 or 60 min. Each temperature treatment, comprising three replicate plates with two twigs, was conducted twice over time.
In the experiment with naturally infected wood, there were five replicate plates, each containing three branch segments. The plates were placed in an incubator at 50°C for 60, 120 or 150 min.
After treatment, the twigs or branch segments were surfaced-sterilized, cut into pieces (0·5–1 mm) with secateurs that had been dipped in ethanol and flamed, then soaked in SDW overnight at room temperature. The viability of Xtp was assessed by transferring, in triplicate, 10-μL aliquots of serial dilutions to 10−5 of the resulting suspensions onto NA for the twigs or spreading of 100-μL aliquots of serial dilutions to 10−2 on NA+A for the branch segments. The plates were incubated at 28°C and CFU enumerated as described previously. Where the temperature treatment did not kill all the pathogen in the exposed twigs or branch segments, the bacterial counts after heating were compared with the initial population of the same twig or branch and the reduction in the number of viable bacteria was expressed as the geometric mean (Keck et al., 1995). The geometric mean is defined as ‘the nth root of the product of the data’ (Crawley, 2007). It is used to measure the central tendency of processes that change multiplicatively rather than additively (Crawley, 2007) and for data that have a logarithmic pattern (Lanley, 1979).