Effect of burning and high temperature on survival of Xanthomonas translucens pv. pistaciae in infected pistachio branches and twigs

Authors


E-mail: eileen.scott@adelaide.edu.au

Abstract

This paper reports the efficacy of burning and heat-treating pistachio branches and twigs as a means of disposing of prunings from trees infected with Xanthomonas translucens pv. pistaciae (Xtp). Burning of pistachio wood, naturally infected with Xtp, was conducted twice under field conditions. Viable Xtp was detected in some non-burned wood, but not in charcoal, ash or partially burned wood. Controlled laboratory experiments were conducted with pure cultures of Xtp and naturally and artificially infected pistachio wood. In liquid culture, 65°C was lethal to Xtp, whereas survival at 60°C or less varied with culture medium and duration of exposure. Xtp survived in infected wood exposed to 40–55°C for at least 60 min but was killed by exposure to 60°C for 15 min or more. Overall, the results of burning and heat treatment were consistent, and confirmed that burning was a reliable eradication technique to dispose of infected wood, such as prunings, providing the pathogen was exposed to a temperature of 60°C or greater for at least 15 min.

Introduction

Eradication of plant pathogens following an incursion is essential to safeguard the profitability and sustainability of plant industries in particular, and the socioeconomy and environment in general. Standard methods for eradication of plant pathogens include burning and burial of infected materials. Burning is preferred as it is considered to eliminate the affected materials, killing any pathogen they may contain (Ebbels, 2003). Although there have been reports where burning only reduced the incidence of, or contained, pathogens (Hardison, 1976; Pereira et al., 1996), there are numerous examples of successful eradication of plant pathogens by burning alone or where burning was a part of an integrated management programme. For example, fire blight was successfully eradicated from Australia (Rodoni et al., 2002) and Sweden (Gråberg, 1993), and citrus canker was eradicated from Thursday Island (Jones, 1991), Lambell’s Lagoon near Darwin (Broadbent et al., 1995) and the Emerald district of Queensland (Gambley et al., 2009). Despite the apparent success of burning infected or exposed trees as an eradication strategy for bacterial pathogens, it is possible that pathogens may survive in ash or non-burned plant debris, particularly if material is partially embedded in soil. The temperature and duration of burning are also important factors in pathogen mortality, which determine the success of the burning. However, such information has rarely been provided (Sosnowski et al., 2009, 2012).

Pistachio dieback is a bacterial disease of woody tissue that provides a local model with which to assess the effectiveness of existing strategies to eradicate exotic, systemic, bacterial pathogens of woody perennials that threaten Australian agriculture. The disease was first observed in Australian pistachio orchards in 1989 (Facelli et al., 2001) and now occurs in the main pistachio growing regions of Australia. It has caused the death of more than 10% of trees in some areas (Edwards & Taylor, 1998) and impedes the expansion of the Australian pistachio industry. The causal agent, Xanthomonas translucens pv. pistaciae (Xtp) (Giblot-Ducray et al., 2009), occurs mainly in woody tissue of the scion, Pistacia vera, where it usually induces staining of the xylem. The bacterium has been isolated less frequently from the rootstock, generally Pistacia integerrima (Facelli et al., 2009). The distribution of Xtp in pistachio wood is often discontinuous, while the bacterium has been isolated from leaves only rarely and not from roots or associated soil samples (Facelli et al., 2009). Although the natural means of survival and dissemination are not known, transmission of Xtp via pruning has been demonstrated (Taylor et al., 2005).

The aim of this study was to evaluate the efficacy of burning as a means of disposal of pruned branches and woody tissues infected with Xtp and, subsequently, the response of the pathogen to heat treatment. The experiments were intended to provide data on which to base future eradication strategies for controlling newly emerging bacterial pathogens of woody tissues.

Materials and methods

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
CharacteristicsPit 1Pit 2Pit 3
Weather conditions
 Air temperature (°C)12·613·54·3
 Relative humidity (%)704897
 Wind speed (km h−1)7 (gusts 11)13 (gusts 20)2 (gusts 5)
 Prevailing wind directionEast–southeastSouthEast–northeast
Characteristics of fire
 Starting time12:10 h16:15 h08:55 h
 Accelerant time (min)44·59·5
 Intense burn duration (min)202020
 Total burn time (min)4040–10520–105

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 10colony 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).

Results

Burning experiments

The fire in 2008 lasted for 30 min. Remains in mesh bags after the bonfire were mainly ash and a small amount of charcoal, while Petri dishes contained mainly charcoal and a few pieces of partially burned wood. The wood buried below the pit floor was not burned. The temperature crayons indicated that the fire exceeded 250°C at all locations where the samples were attached to poles above the pit floor, while temperatures 5 cm below the pit floor reached between 50 and 60°C. No viable Xtp was isolated from the remains, even from the non-burned wood, nor was the pathogen detected by PCR. Viable Xtp, c. 102 CFU mL−1, was isolated from all control wood pieces.

Total combustion time in 2009 varied between pits (Table 1). In general, after the fire was ignited, intense flames lasted for 20 min in all three pits. Small flames lingered for another 20 min in pit 1. Two corners (poles 4 and 5) in this pit furthest from the flame-thrower were exposed to little or no fire, so that the fuelwood and samples in mesh bags were not burned. In pit 2, after the intense flames subsided, small flames remained at two corners (poles 1 and 2) of the pit and the last flame died at pole 2, 105 min after ignition. The samples under the pit floor at pole 1 were partially burned and those at pole 2 were blackened on the surface. Dew was observed on the woody material in pit 3, delaying the start of the fire. The flames stayed mainly in the central part of this pit, but died off quickly after ignition. As considerable fuelwood at the four corners was not burned, the branches were piled up again at each corner close to the lingering flames until most were burned. Isolated flames continued for another 70 min in three corners (poles 1, 2 and 5) and 85 min in the other (pole 4).

Material retrieved from the 2009 burning experiment comprised non-burned and partially burned wood, charcoal and ash. Thirty-five, 40% and 45% of the mesh bags contained wood pieces burned to charcoal and/or ash in pits 1, 2 and 3, respectively. More mesh bags attached to poles 1, 2 and 3 (58%, 42% and 42%, respectively) contained wood pieces that were burned to charcoal and/or ash than those attached to poles 4 and 5 (33% and 25%, respectively). Approximately 87% of the mesh bags with wood pieces placed on the pit floor and 60% of those suspended 20 cm above the pit floor were burned to charcoal and/or ash, compared with 0% and 13% of the samples placed 5 cm below and suspended 50 cm above the pit floor, respectively. For wood pieces in Petri dishes, 25%, 30% and 35% of the dishes contained material that was burned to charcoal and/or ash in pits 1, 2 and 3, respectively. For all pits combined, 60%, 53%, 7% and 0% of Petri dishes contained wood burned to charcoal and/or ash on the pit floor, at 20 and 50 cm above the pit floor and 5 cm below the pit floor, respectively. Those wood pieces that were burned to charcoal and/or ash were generally in Petri dishes placed on the pit floor that had broken as a result of the heat. For wood in mesh bags and Petri dishes, weight after the bonfire was significantly less than prior to burning (Table 2).

Table 2.   Mean weight (g) of pistachio wood pieces, before and after burning, in mesh bags or Petri dishes attached to five poles at various positions above the floor in three pits in 2009. Weight of wood in each mesh bag or Petri dish before and after the bonfire was compared using a paired t-test with 95% confidence interval. Means were computed from weight data for 15 mesh bags or Petri dishes, from one position above the pit floor on each pole in each pit
 Sample position on pole above pit floorMean weight per bag or Petri dish (g)Paired t-test P value
Before bonfireAfter bonfire
Mesh bags−5 cm118·2386·05<0·0001
0 cm117·5719·84<0·0001
20 cm118·5237·57<0·0001
50 cm118·2673·73<0·0001
Petri dishes−5 cm55·5944·570·0019
0 cm55·7122·93<0·0001
20 cm56·2624·66<0·0001
50 cm55·5341·36<0·0001

Of 360 samples of wood, charcoal and ash from mesh bags and Petri dishes (180 each) processed in the survival assay, viable pathogen was isolated from 13 pieces of wood. Six of these wood pieces (two in two mesh bags and four in two Petri dishes) were buried 5 cm below the surface and three pieces (two in a Petri dish and one in a mesh bag) were suspended 20 cm above the pit floor on poles 4 and 5 in pit 1 (Fig. 1a,b). The other four were from three mesh bags, two suspended 50 cm above the pit floor on poles 2 and 4 in pit 1 and one on pole 4 in pit 3. All samples which yielded viable Xtp, except one from the west side of pit 1, were on the east side of pits 1 and 3 furthest from the flame-thrower. Viable pathogen was not isolated from any samples from pit 2. The population of viable pathogen recovered from samples suspended 20 and 50 cm above the pit floor ranged from 101 to 103 CFU mL−1, while that from samples buried 5 cm below the pit floor ranged from 102 to 104 CFU mL−1. The population of viable pathogen for wood pieces placed away from the fire as controls ranged from 102 to more than 104 CFU mL−1. Most wood that yielded viable Xtp was not burned and had slightly moist bark.

Figure 1.

 Isolation of viable Xanthomonas translucens pv. pistaciae (Xtp) on nutrient agar with antibiotics from the remains of (a) infected pistachio wood in mesh bags and (b) infected wood in Petri dishes along with (c) indicative temperature (°C) based on Tempilstik crayons, after bonfires in 2009. Each pit was 3 × 5 m and 0·5 m deep. Locations from which viable Xtp was isolated are denoted with (inline image) and those from which viable Xtp was not isolated with (inline image). A flame-thrower was applied to each pit following the direction of the prevailing wind: east–southeast for pit 1, south for pit 2 and east–northeast for pit 3.

Xtp was detected by PCR in all wood that yielded viable pathogen, including controls, mainly in non-burned and partially burned wood samples, except for one ash sample, for which only a faint amplicon was detected.

The maximum temperature recorded by melted crayons was equal to or >200°C but <400°C for most of the locations above the pit floor, and ranged from <40°C to >80°C at 5 cm below the pit floor (Fig. 1c). The heat penetrated 10 cm below the pit floor, and exceeded 80°C at some positions in pit 3.

In vitro temperature experiments

Thermal death time

The trend of pathogen response to 40, 45 and 50°C in both replications was similar. However, because of differences between replications in time required to kill the pathogen at 50°C, each data set was analysed separately and the mean populations (CFU mL−1) of suspensions exposed to the three temperatures for one replication are presented (Fig. 2). The population did not change (> 0·05) when the suspensions of Xtp cells in NB and SPB were exposed to 40°C for up to 60 min. The population decreased when suspensions in NB were exposed to 45°C for 10 min and this continued (< 0·001) as exposure time increased, whereas the population in SPB did not change over time. There was a significant reduction (< 0·001) of population when suspensions in both NB and SPB were exposed to 50°C for 10 min and viable pathogen was not recovered from NB suspensions after 60 min, whereas the pathogen continued to survive in SPB at 50°C for 60 min (Fig. 2). However, no viable pathogen was observed on NA and SPA in the other replicate of the suspensions exposed to 50°C for 30 and 40 min, respectively (data not shown).

Figure 2.

 Mean number of colony forming units (CFU; log transformation) of Xanthomonas translucens pv. pistaciae after exposure to 40 (diamond), 45 (square) or 50°C (triangle); (a) incubated in sucrose peptone broth and enumerated on sucrose peptone agar, (b) incubated in nutrient broth and enumerated on nutrient agar.

Thermal death point

Exposure to 50 or 55°C for 10 min significantly reduced (< 0·001) the population of Xtp in both NB and SPB suspensions but did not completely kill the pathogen (Table 3). Exposure to 60°C in NB completely killed the pathogen in 10 min, and significantly reduced the population in SPB. No viable pathogen was detected after exposure to 65 or 70°C for 10 min.

Table 3.   Thermal death point for Xanthomonas translucens pv. pistaciae in suspension in nutrient broth (NB) or sucrose peptone broth (SPB) exposed for 10 min to a range of temperatures, then enumerated (CFU mL−1) on nutrient agar (NA) or sucrose peptone agar (SPA), respectively. The experiment was conducted with three replicates
Temperature (°C)NB/NASPB/SPA
TreatmentControlaTreatmentControl
  1. aControls comprised aliquots from each of the three replicate liquid culture bottles, which were enumerated on SPA and NA before the bottles were immersed in water baths at 50–70°C.

  2. bPopulations of viable bacteria before and after treatment were estimated using the method of Miles & Misra (1938) and compared using a paired t-test with 95% confidence intervals.

504·2 × 105 b2·1 × 1093·4 × 1052·1 × 109
551·9 × 1031·7 × 1097·2 × 1052·0 × 109
6001·2 × 1098·8 × 1022·5 × 109
6501·9 × 10902·1 × 109
7002·1 × 10902·5 × 109

Survival in infected twigs and wood

Pith temperature increased rapidly in the first 10 and 20 min of incubation, from ambient conditions of about 22°C to approximately 4°C less than the designated temperatures, then slowly reached the designated temperatures in another 30–40 min (Fig. 3). Once the designated temperatures were reached, pith temperature stayed constant for the remaining incubation period. In the first experiment with artificially infected twigs, viable Xtp was isolated from one, four and three twigs of five that had been exposed to 50°C for 60, 120 and 150 min, respectively (Table 4). In the second experiment, viable Xtp was isolated from two of six twigs after exposure for 60 or 120 min, but not from twigs exposed for 150 min. However, in twigs that yielded viable pathogen, the number of viable bacteria after heat treatment decreased by at least 97% (from 9·3 to 3 × 103) compared with that before treatment. On average, only 1·702, 0·048–0·489 and 0·018% of the bacteria remained viable in the twigs exposed to 50°C for 60, 120 and 150 min, respectively (Table 4). Viable pathogen was not recovered from any twigs exposed to 50°C for 180 min, nor from twigs exposed to 55 or 60°C for 60 min or longer. In the third experiment, when artificially infected twigs were exposed to 40°C, the pathogen was isolated from the majority of twigs exposed for 30–60 min, although CFU varied (Table 5). The pathogen was isolated from artificially infected twigs that had been exposed to 55°C in experiment 4-1 (i.e. experiment 4, replicate 1), from a decreasing number of twigs with increasing time of exposure, but not in experiment 4-2 (experiment 4, replicate 2) with a new batch of twigs. The temperature recorded in the incubator for experiment 4-1 fluctuated between 51 and 52°C in the first 20 min. Nevertheless, the population of the pathogen after exposure to 55°C was 80 to almost 100% less than the initial population in the same twigs (Table 5). The pathogen was not isolated from twigs exposed to 60°C for 15 min or longer (experiment 5-1 and 5-2; Table 5).

Figure 3.

 Increase in pith temperature of pistachio twigs (c. 1 cm diameter) or wood pieces (c. 1 cm thick) incubated at 50, 55 or 60°C for 60 min. Temperature was recorded by inserting a probe connected to a data logger into the pith region of the twigs or wood pieces. The mean of three replicate measurements for each temperature is presented.

Table 4.   Recovery of viable Xanthomonas translucens pv. pistaciae from artificially infected pistachio twigs before (one-third of each twig) and after (remaining two-thirds of each twig) exposure to 50°C for various durations in two experiments. The percentage of viable bacteria remaining following heat treatment is given along with the geometric mean
Exposure duration (min)ExperimentNumber of twigs which yielded pathogen/number testedNumber of CFU mL−1Viable bacteria (%)Geometric meana (%)
Before treatmentAfter treatment
  1. CFU: colony forming units. N/A: not applicable

  2. aGeometric mean is a type of mean or average defined as ‘nth root of the product of the data’ (Crawley, 2007).

6011/53·0 × 1064·7 × 1020·016N/A
22/62·9 × 1052·4 × 1030·8281·702
1·2 × 1054·2 × 1033·500
12014/53·8 × 1072·0 × 1050·5260·048
2·4 × 1071·8 × 1030·008
2·1 × 1071·9 × 1030·009
8·0 × 1061·2 × 1040·150
22/63·2 × 1052·6 × 1030·8130·489
1·7 × 1055·0 × 1020·294
15013/52·3 × 1071·4 × 1040·0610·018
1·5 × 1075·3 × 1030·035
9·7 × 1062·7 × 1020·003
20/6N/A
18010/5N/A
20/6N/A
Table 5.   Recovery of viable Xanthomonas translucens pv. pistaciae from artificially infected pistachio twigs before (one-third of each twig) and after (remaining two-thirds of each twig) exposure to 40–60°C for various durations. Each temperature treatment comprised three replicate plates, each containing two twigs, and was repeated once. The percentage of viable bacteria remaining following heat treatment is given along with the geometric mean
Temperature (°C)Exposure duration (min)Experiment- ReplicationNumber of twigs, of 6, which yielded pathogenNumber of CFU mL−1Viable bacteria remaining (%)Geometric meana (%)
Before treatmentAfter treatment
  1. CFU: colony forming units. N/A: not applicable.

  2. aGeometric mean is a type of mean or average defined as ‘nth root of the product of the data’ (Crawley, 2007).

  3. bThe temperature recorded in the incubator for experiment 4-1 fluctuated between 51 and 52 in the first 20 min of incubation of twigs.

40303-134·4 × 1061·4 × 10631·81817·187
3·5 × 1073·3 × 10794·286
1·3 × 1082·2 × 1061·692
3-251·9 × 1081·9 × 10710·0003·344
4·0 × 1062·5 × 1040·625
6·3 × 1071·3 × 10720·635
1·1 × 107
3·3 × 10630·000
2·5 × 1072·7 × 1040·108
603-131·3 × 1062·5 × 10519·23125·357
2·3 × 1072·6 × 107113·043
2·0 × 1081·5 × 1077·500
3-261·9 × 1071·5 × 10778·94731·666
2·5 × 1072·2 × 10788·000
1·0 × 1071·5 × 1051·500
6·3 × 1061·6 × 1052·540
2·1 × 1073·0 × 107142·857
1·2 × 1073·2 × 107266·667
55154-1b62·0 × 1071·4 × 1057·0000·786
2·2 × 1065·5 × 1030·250
2·7 × 1075·3 × 10619·630
4·5 × 1073·4 × 1050·756
1·5 × 1072·5 × 1040·167
5·3 × 1062·9 × 1030·055
4-20N/A
304-131·6 × 1075·3 × 1020·0030·005
2·9 × 1063·3 × 100·001
1·1 × 1063·3 × 1020·030
4-20N/A
454-112·7 × 1051·3 × 1020·048N/A
4-20N/A
604-113·6 × 1068·7 × 1020·024N/A
4-20N/A
60155-1 & -20N/A
305-1 & -20N/A
455-1 & -20N/A
605-1 & -20N/A

Exposure of naturally infected pistachio wood to 50°C for 60–180 min did not completely kill the pathogen (Table 6). The number of viable bacteria recovered from branch segments heated for 60 or 180 min varied greatly, although the population of viable Xtp in the majority of branch segments decreased by more than 98% compared with that before exposure to this temperature. Although there was no clear trend in the number of viable bacteria in naturally infected wood with duration of exposure to 50°C, overall, pathogen survival was poor, as indicated by geometric means of 0·366–3·253 (Table 6).

Table 6.   Recovery of viable Xanthomonas translucens pv. pistaciae from naturally infected pistachio wood before (half of each branch segment) and after (other half) exposure to 50°C for various durations. Each temperature treatment comprised five replicate plates, each containing three pieces of wood. The percentage of viable bacteria remaining following heat treatment is given along with the geometric mean
Exposure duration (min)Number of branch segments which yielded bacteria/number of branch segments testedaNumber of CFU mL−1Viable bacteria remaining (%)Geometric mean (%)b
Before exposureAfter exposure
  1. CFU: colony forming units. N/A: not applicable.

  2. aOne branch segment from each of the 60 and 120 min durations was excluded as it yielded no colonies from either section before and after treatment.

  3. bGeometric mean is a type of mean or average defined as ‘nth root of the product of the data’ (Crawley, 2007).

608/147·3 × 1036·3 × 10386·3641·764
2·9 × 1044·0 × 1010·140
3·5 × 1043·0 × 1010·085
3·6 × 1042·0 × 1010·559
2·2 × 1048·0 × 1010·369
1·7 × 1045·7 × 10333·333
3·1 × 102·7 × 10385·761
6·4 × 1031·0 × 1010·156
1203/147·7 × 1036·0 × 1010·7830·366
4·2 × 1032·0 × 1010·482
7·7 × 1031·0 × 1010·130
1501/159·3 × 1033·0 × 10332·143N/A
1803/152·8 × 1031·0 × 1010·3533·253
4·0 × 1032·4 × 10360·401
6·2 × 1021·0 × 1011·613

Discussion

Burning in an open pit was an effective means of eradicating Xtp from pistachio wood providing that all the wood was burned completely or reached the lethal temperature for sufficient time to kill the pathogen. The lethal temperature for Xtp in vitro was 60–65°C, depending on the culture medium, and the pathogen was not recovered from twigs heated in an oven at 60°C for 15 min or more. Recovery of viable bacteria from wood that was incompletely burned or that penetrated the soil and escaped exposure to lethal temperature illustrated the importance of ensuring that all wood is burned or heated sufficiently.

In the field experiments, no viable pathogen was isolated from any of the samples after the bonfire when the infected wood was placed on the pit floor, where the temperature reached between 200 and 400°C at most locations, which suggested that eradication was achieved. Incomplete combustion at some locations furthest from the flame-thrower, for example poles 4 and 5 in pit 1, allowed the pathogen to survive. The majority of the wood pieces that yielded viable pathogen from these two locations were buried below the pit floor, where the temperature remained below 40°C, indicating that any pathogen in debris that penetrates into the soil might not be eradicated by burning, particularly if the pieces of wood are large and heat penetration is poor. Sosnowski et al. (2012) also reported that the fungus Elsinoe ampelina was eradicated from grapevine canes by burning, providing that infected canes did not penetrate the soil below the pit floor. Viable cells of the pathogen were also isolated from non-burned wood suspended above the pit floor; however, had these materials not been attached to poles, they are likely to have fallen into the flames and been incinerated. The relatively small number of bacteria isolated from the control pieces of wood in 2008 compared with 2009 may reflect the use of ABSPA rather than NA+A in the first year. Overall, the data suggest that buried or non-burned debris could act as a source of inoculum for subsequent disease if the pathogen surviving in buried wood was transported via human or other vectors to areas with susceptible hosts.

As the use of thermal crayons in the field experiments did not allow the establishment of time–temperature relationships nor the measurement of temperature reached inside woody tissues, in vitro experiments were conducted to elucidate the effect of exposure to heat over time. Xtp survived well in liquid SPB and NB at 40°C, and the pathogen was not eradicated when exposed to 45 or 50°C for 60 min or to 55 or 60°C for 10 min. Furthermore, heat tolerance was greater in SPB than in NB, which may be related to the production of extracellular polysaccharide in the presence of sucrose (Souw & Demain, 1979) that could protect the cells from heat. This phenomenon was reported by Leach et al. (1957), in that the thermal death point for Xanthomonas phaseoli was 2–4°C higher when grown on glucose–casein-hydrolysate agar, on which polysaccharide was produced, than in nutrient broth. Thermal death times reported for other xanthomonads have varied. For example, Keck et al. (1992) reported 50°C for 40 min to be lethal to Xanthomonas campestris pv. pelargonii, whereas cells of X. fragariae were killed at 56°C for 15 min or 52°C for 60 min (Turechek & Peres, 2009). Although 55°C was not lethal to Xtp, the population decreased significantly. The lethal threshold for the pathogen appeared to be 60°C in NB and 65°C in SPB, suggesting that the thermal death point lies between 60 and 65°C. In comparison, the thermal death point of Xanthomoas oryzae pv. oryzae was 53°C (Stall et al., 1993), although the influence of culture medium on survival was not specified.

Survival of Xtp was prolonged in pistachio wood, the pathogen being eradicated from inoculated twigs after exposure to 50°C for 180 min, but not from naturally infected branch segments. The time (50–60 min) required for internal tissues to reach designated temperatures in this study suggests that viable pathogen isolated from wood which had been incubated at 50°C for 60–180 min or more might have resided in the innermost tissues. Although the population was greatly reduced, the failure to kill all bacteria means that heating at 50°C would be unsuitable as a means of eradication. Xtp produces polysaccharide slime in planta (E. Facelli, The University of Adelaide, Australia, personal communication), which might protect the pathogen from damage by heat (Leach et al., 1957). Treating at 55ºC for 60 min failed in some cases to eradicate Xtp from artificially infected tissue, but it was always eradicated when the exposure was increased to 120 min, suggesting that any protective effect of polysaccharide slime was temporary.

The pathogen survived in liquid culture and artificially infected twigs at 40°C for 60 min, 50°C for 60–150 min, or in naturally infected wood at 50°C for more than 180 min. In contrast, in the field experiments it was not isolated from wood pieces at the few locations below the pit floor where crayons indicated a temperature of 40ºC or more. It may be that the pathogen in these wood pieces was reduced to below the detection limit of the method used for isolation. In addition, the observation that the wood was dry, cracked or turned brown to black indicates that it might have been exposed to the heat for a long duration. Desiccation as a result of heat might also affect the survival of the pathogen in the wood tissues, and may merit further research. The results of the burning experiment where viable pathogen was not isolated from any samples that reached 60°C were in agreement with the in vitro experiments, both in liquid medium culture and twigs, and once again, confirmed the lethal effect of this temperature on the pathogen’s survival.

In summary, Xtp was eradicated when infected wood was burned to charcoal or ash. This could be achieved by providing adequate penetration of flames and duration of heat. Failure to do this allowed the pathogen to survive in some cases. Incomplete eradication of this or a similar pathogen might result in reintroduction of the disease to areas where susceptible hosts are present and, consequently, cause eradication programmes to fail. Deep burial of the remains, which usually follows burning in eradication programmes, might help to prevent the escape of any pathogen surviving in incompletely burned wood.

Acknowledgements

The authors thank the staff of the Department of Sustainability and Environment, Mildura, Victoria for assistance with the fires, and CMV Farms, Robinvale and M. Wright, Saddleworth for providing plant materials. We also thank staff of the Department of Primary Industries Victoria and affiliates for assistance with preparing pits and materials. We thank E. Facelli for helpful discussions and A. Salowi and G. Kritzman for advice on their technique to infiltrate twigs with Xtp. This research was a part of the PhD project of the first author, supported by an Australian Endeavour Postgraduate Award.

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