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AUTHOR QUERY SHEET

Author(s): T. D. RAMSFIELD, R. D. BALL, J. F. GARDNER and M. A. DICKArticle Title: Temperature and time combinations required to cause mortality of a range of fungi colonizing woodArticle No.: TCJP 499269

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Can. J. Plant Pathol. (2010), 00(0): 1–8

ISSN 0706-0661 print/ISSN 1715-2992 online © 2010 The Canadian Phytopathological SocietyDOI: 10.1080/07060661.2010.499269

Forest pathology/Pathologie forestière

TCJP

Temperature and time combinations required to cause mortality of a range of fungi colonizing wood

Mortality of wood colonizing fungiT. D. RAMSFIELD, R. D. BALL, J. F. GARDNER AND M. A. DICK

Scion1, Private Bag 3020, Rotorua 3046, New Zealand

(Accepted 19 May 2010)

Abstract: The global movement of solid wood packaging material is an important pathway by which invasive organisms have increased their range. The International Standard for Phytosanitary Measures No. 15 (ISPM No. 15) was published by the Secretariat of the International Plant Protection Convention to provide guidelines for wood treatment to reduce the risk of accidental pest movement via solid wood packaging material. This study assessed the exposure temperature and time combinations necessary to kill a range of fungi and one species of Phytophthora colonizing wood. The ISPM No. 15 protocol (heat to 56 °C core temperature for 30 min) was included as one of the treatments in the experiment. The survival data collected in this experiment were utilized to develop a binomial generalized linear model that allowed statistical assessment of survival following treatment. The tolerance of the isolates to heat treatment was variable and it was found that the ISPM No. 15 protocol did not result in mortality of all species that were tested.

Keywords: heat treatment, invasive fungi, ISPM No. 15, phytosanitary measures, solid wood packaging material

Résumé: La circulation mondiale des matériaux d’emballage en bois massif constitue une des voies par laquelle les organismes invasifs ont étendu leur habitat. La Norme internationale pour les mesures phytosanitaires No 15 (NIMP No 15) a été publiée par le Secrétariat de la Convention internationale pour la protection des végétaux afin de fournir des directives quant au traitement du bois, visant ainsi à réduire le risque de dissémination accidentelle des organismes nuisibles que pourrait contenir le bois massif servant à la fabrication des emballages. Cette étude évalue la combinaison du temps et de la durée d’exposition au traitement nécessaire pour tuer une variété de champignons et une espèce de Phytophthora qui colonise le bois. Le protocole de la NIMP No 15 (chauffer le cœur du matériau à 56 °C pendant 30 min) a été inclus dans les traitements à évaluer au cours de l’expérience. Les données sur la survie collectées durant l’expérience ont été utilisées pour développer un modèle binomial linéaire généralisé qui permet l’évaluation statistique du taux de survie à la suite du traitement. La tolérance des isolats au traitement à la chaleur a varié et l’on a observé que le protocole de la NIMP No 15 n’a pas provoqué la mort de toutes les espèces testées.

Mots clés: Champignons envahissants, matériaux d’emballage en bois massif, mesures phytosanitaires, NIMP No 15, traitement à la chaleur

Introduction

International trade in wood and wood products, and theby-products of trade such as wooden pallets and dunnage,have resulted in the movement of pests around the world(Brockerhoff et al., 2006; Haack, 2006). The introductionof insects, such as the Asian longhorned beetle (Anoplo-phora glabripennis (Motchulsky)) to North America, via

this pathway has resulted in catastrophic invasions(Haack et al., 2010). Solid wood packaging materials areoften composed of relatively low-grade unprocessedwood of unknown origin and have been found to harboura variety of insects (Allen & Humble, 2002). In order toreduce the probability of accidental movement of pests inthis wood, the Secretariat of the International Plant Pro-tection Convention published the International Standard

Correspondence to: T. D. Ramsfield. E-mail: Tod.Ramsfield@scionresearch.com1Scion is the trading name of the New Zealand Forest Research Institute Ltd.

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for Phytosanitary Measures No. 15 (ISPM No. 15). Thisdocument describes phytosanitary measures to reduce therisk of the introduction and spread of quarantine pests viawood packaging material, one of which is exposure to atemperature of 56 °C to the core of the wood for 30 minutes(FAO, 2007). New Zealand, along with many other coun-tries, has adopted ISPM No. 15 in the Import HealthStandard for Wood Packaging Material from All Coun-tries (MAF, 2006). New Zealand requires a more extremeheat treatment protocol for imported sawn wood; theImport Health Standard for Sawn Wood requires that acore temperature of 70 °C be maintained for 4 hours(MAF, 2003).

The ISPM No. 15 temperature and time combinationof 56 °C for 30 minutes was experimentally developed inCanada to kill the nematode Bursaphelenchus xylophilus(Steiner & Buhrer) Nickle and its insect vectors (Smith,1992; Uzunovic et al., 2006). Since fungi may also bevectored on wood and have varying temperature thresh-olds (i.e. Chidester, 1937, 1939; Jones, 1973; Newbill &Morrell, 1991), an internationally standardized protocolwas developed by Uzunovic et al. (2006) to determinethe lethal temperature and time combinations for fungicolonizing wood and to determine if the heat treatmentprotocol of ISPM No. 15 is robust enough to kill fungicolonizing wood.

The objective of this research was to use the protocolof Uzunovic et al. (2006) to determine the lethal tempera-ture and time combinations for a range of different fungithat colonize wood.

Materials and methods

Fungi

Nine fungal genera and one species of Phytophthorawere selected for heat treatment. The fungi selected forthe study represent a range of fungal types associatedwith wood. The fungi include decay organisms, patho-gens and moulds that affect different parts of trees orwood and are considered to pose a biosecurity risk. Mem-bers of the Ascomycota included Cladosporium herbarum(Pers.) Link, Cladosporium tenuissimum Cooke, Fusariumcircinatum Nirenberg & O’Donnell, Lasiodiplodia theo-bromae (Pat.) Griffon & Maubl., Neonectria fuckeliana(C. Booth) Castl. & Rossman, Ophiostoma novo-ulmiBrasier, Sphaeropsis sapinea (Fr.) Dyko & B. Sutton.Members of the Basidiomycota included Armillarianovae-zelandiae (G. Stev.) Boesew., Phlebiopsisgigantea (Fr.) Jülich and Schizophyllum commune Fr.The one species from kingdom Chromista, phylumOomycota was Phytophthora cinnamomi Rands. Three

isolates of each species were selected for treatment, withthe exception of Cladosporium tenuissimum andCladosporium herbarum, for which there were one andtwo isolates, respectively. For each species, the isolateswere selected to represent as wide a geographic range aspossible to capture genetic variation within the species(Table 1).

Inoculation of wood blocks

Blocks of wood, 30 mm × 10 mm × 5 mm, were cut fromDouglas fir (Pseudotsuga menziesii (Mirb.) Franco),Monterey pine (Pinus radiata D. Don), and elm (Ulmus sp.).The blocks were cut from green timber from trees thatwere felled specifically for this experiment and the timebetween felling the trees and machining the blocks wasminimized by processing the wood within 2 days of fell-ing. Elm boards from a freshly felled tree were milledoffsite, couriered to the laboratory in plastic andmachined at the laboratory upon arrival within a week ofmilling. The blocks were mixed to randomly distributeblocks from the sapwood and heartwood and then auto-claved at 121 °C for 30 min. The blocks were inoculatedwith the test organisms by aseptically placing 20 woodblocks on 2% malt extract agar in a 95 mm square Petriplate, inoculating the plate with the selected isolate, fol-lowed by incubation at 20 °C until the wood blocks werecolonized. Ophiostoma novo-ulmi was inoculated ontoelm, N. fuckeliana was inoculated onto pine and the restof the fungi and P. cinnamomi were inoculated ontoDouglas fir.

Treatment

For each treatment, a total of six replicate blocks of woodthat were colonized by the same isolate were vacuumsealed inside a plastic bag using a Sammic model V421vacuum sealing machine (Sammic, Spain) and thenplaced in a water bath set at the temperature required toraise the core temperature of the wood block to the targettemperature. Removal of all the air from the plastic bagensured efficient heat transfer to the blocks by eliminat-ing the insulating effect provided by air trapped insidethe bag. Blocks were sealed into the bag immediatelyprior to treatment to reduce the effect of oxygen starva-tion. A thermocouple was inserted into the core of awood block and sealed within a plastic bag so that thetemperature of the water bath was set to achieve thedesired core temperature. The temperature of the waterbath was set with the same wood block and thermocouplefor every target temperature. When the water bath tem-perature was stabilized and the core target temperature

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achieved, the blocks were placed in the water bath andthen removed after exposure times of < 1 min, 30 min,60 min or 120 min. To ensure that the < 1 minute expo-sure was accurate, an experiment was conducted to timehow long it took for the wood block to reach the targettemperature from room temperature. Therefore, the woodblocks were allowed to reach the target temperaturebefore timing was started. Temperatures that were testedwere 25 °C (control), 41 °C, 46 °C, 51 °C, 56 °C, 61 °C,66 °C, 71 °C and 76 °C. All isolates were treated at eachtime and temperature combination from 25 °C to 71 °C,but S. commune was subjected to further testing at 76 °C.

All treatments involving F. circinatum, O. novo-ulmi,N. fuckeliana, L. theobromae, S. commune and one iso-late of P. gigantea were inoculated, treated and assessedunder strict quarantine conditions in the New Zealand Forest

Research Institute Ltd Quarantine and Containment Facil-ity (Ministry of Agriculture and Forestry laboratory ref-erence #2746).

Assessment

Immediately following treatment, the blocks of woodwere removed from the vacuum sealed bag and placedonto 2% malt extract agar. All blocks, including thosecolonized by P. cinnamomi, were incubated at 20 °C andvisually assessed for growth at regular intervals for up to21 days following treatment. The identity of the speciesemerging from the blocks of wood was confirmed bymorphology. If the isolate grew from the wood block fol-lowing treatment, the treatment was not consideredlethal. Of the 972 plates that were assessed, 13 became

Table 1. Fungi (and one species of Phytophthora) used in this study.

Fungus Isolate NZFS Collection location Host Collection date

Armillaria novae zealandiae 2340 Karamea, NZ Nothofagus sp. 11 May 2005Armillaria novae zealandiae 1853 Fiordland National Park, NZ Decayed wood 9 May 1994

Armillaria novae zealandiae 1026 Rotorua, NZ Pinus radiata 13 Feb 1995

Lasiodiplodia theobromae 2964 Federated States of Micronesia Cyrtosperma chamissonis 1 Jan 1987

Lasiodiplodia theobromae 2954 Western Samoa Psidium guajava 1 Mar 1982

Lasiodiplodia theobromae 2955 Mexico Mangifera sp. 1 Aug 1980

Cladosporium herbarum 89 New Zealand Pinus sp. 8 Apr 1964

Cladosporium herbarum 2963 Nelson, NZ Nicotiana tabacum 1 May 1978

Cladosporium tenuissimum 390 Tautara, NZ Eucalyptus nitens 10 Jun 2000

Fusarium circinatum 308A/1 Monterey, CA, USA Pinus radiata 1 Aug 1996

Fusarium circinatum 308C Ano Nuevo, CA, USA Pinus radiata 1 Aug 1996

Fusarium circinatum 1724 California Pinus radiata Unknown

Neonectria fuckeliana 1105 Waitane Forest, NZ Pinus radiata 4 Feb 2004

Neonectria fuckeliana 1068 Flagstaff Forest, NZ Pinus radiata 30 Oct 2003

Neonectria fuckeliana 2962 Flagstaff Forest, NZ Pinus radiata 23 Nov 2006

Ophiostoma novo-ulmi 184E New Hampshire, USA Ulmus sp. 1 Jan 1997

Ophiostoma novo-ulmi 1131 Auckland, NZ Ulmus sp. 19 Mar 1991

Ophiostoma novo-ulmi 184D Belgium Unknown 1 Jan 1980

Phlebiopsis gigantea 1015 Australia Pinus sp. 17 Nov 1995

Phlebiopsis gigantea 1958 Berwick Forest, NZ Pinus radiata 30 Oct 1995

Phlebiopsis gigantea 1530 Kaingaroa Forest, NZ Pinus radiata 18 Sept 2000

Phytophthora cinnamomi 102.16 Te Kao, NZ Pinus radiata 7 Jan 1999

Phytophthora cinnamomi 979 Whitford, NZ Quercus robur 3 Mar 2003

Phytophthora cinnamomi 1012 Auckland, NZ Quercus palustris 16 Apr 2003

Schizophyllum commune 1956 India Unknown 13 Sept 1995

Schizophyllum commune 2491 South Africa Unknown 26 Mar 1999

Schizophyllum commune 2490 Fiji Unknown 27 Jan 1999

Sphaeropsis sapinea 15.17 Kaingaroa Forest, NZ Pinus radiata 18 May 1999

Sphaeropsis sapinea 15.22 Cambridge, NZ Pseudotsuga menziesii 19 May 1999

Sphaeropsis sapinea 1033 Greymouth, NZ Pinus contorta 3 Jun 2003

NZFS is the culture collection of the New Zealand Forest Research Institute Ltd.

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contaminated and were not included in any of the analy-ses. These plates were A. novae-zelandiae 2340 at 46 °C< 1 min, and 46 °C for 30 min; A. novae-zelandiae 1026at 51 °C < 1 min; L. theobromae 2964 at 61 °C < 1 min;C. herbarum 89 at 61 °C for 30 min; C. herbarum 2963 at41 °C < 1 min; N. fuckeliana 1105 at 41 °C for 60 minand 46 °C for 120 min; P. gigantea 1530 at 46 °C for120 min and 56 °C < 1 min; P. cinnamomi 979 at 56 °C< 1 min; S. sapinea 15.17 at 46 °C for 120 min; S. sap-inea 15.22 at 61 °C < 1 min.

Statistical analysis

A binomial generalized linear model with logit link func-tion was fitted to the proportion (x/6) emerging using Rversion 2.8.1 (R Development Core Team 2009). Themodel fitted was:

y ∼ binomial(n,p)

where y is the (vector of) observed proportions emerging,n the binomial sample size (here 6 for each observation),and p is corresponding vector of fitted proportionsemerging. The coefficients a, b, c were fitted in separatemodels for each species or in a combined model withseparate coefficients for each species. Data for all isolatesof each species were combined and the model predictionswere calculated at the species level.

At most times and temperatures there was either 100%(6/6) or 0% (0/6) emergence. Often there would be warningsof fitted values being very close to 0 and 1 and very largestandard errors. This was a result of data from two species:Armillaria novae-zelandiae and Phlebiopsis gigantea. Thesetwo species were omitted to solve this problem (the other9 species fit). These two species had low incidence and verylow predicted values at temperature 56 °C/time 30 min, butthe coefficients for these species had high standard errors.

A ‘robust deviance’ estimate was calculated based onthe 9 species fit and used to adjust standard errors. Pre-dicted proportion, p, emerging and 95% confidence inter-vals (q2.5%, q97.5%) for the proportion emerging attemperature 56 °C after time 30 min was calculated. Themodel was also used to predict the temperature at which99% and 99.99% mortality occurred after 30 min expo-sure. The robust deviance estimate was 3.11 and standarderrors were adjusted by multiplying by the square root ofthis quantity, i.e. 1.76. The robust deviance statistic hasan asymptotic chi-squared distribution. The reason for

using the robust deviance, rather than the standard devi-ance, is because the latter is distorted by cells with 0% or100%. As a general rule, it is necessary to have anexpected value of 5 or more alive and 5 or more dead ineach cell (cf. count out of 6 in the raw data) for the devi-ance test statistic to be approximately chi-squared. Therobust deviance calculation overcomes the problem bypooling cells with similar predicted values until expectedvalues of dead and alive are sufficiently high in each cell.

Results

After treatment at a core temperature of 25 °C, as acontrol, with exposure for < 1 min, 30 min, 60 min and120 min, all isolates of every fungus and P. cinnamomiemerged from 100% (6/6 replicates) of the treated blocks.Thus, vacuum sealing the wood blocks and depriving thefungi of oxygen for the period of time that was requiredto complete treatment was not lethal to any of the isolatestested. This result also confirmed that the wood blockswere successfully colonized by the organism of interest.

Mean survival of every species at every temperature/time combination is shown graphically in Fig. 1A and B.Armillaria novae-zelandiae was found to be very suscep-tible to heat treatment, A. novae-zelandiae only survivedthe < 1 min and 30 min exposure times at 41 °C and< 1 min at 46 °C. The second most susceptible specieswas P. cinnamomi; isolate 102.16 was killed at alltemperature/time combinations greater than 41 °C for30 min. All temperature/time combinations greater than46 °C for 120 min were lethal to isolate 979 and greaterthan 51 °C for 30 min were lethal to isolate 1012. It wasobserved that S. commune was very heat tolerant. Threeof six replicates of isolate 1956 and 6 of 6 replicates ofisolate 2491 survived 66 °C for < 1 min, but the otherexposure times at 66 °C were lethal. When the tempera-ture was raised to 71 °C, 3 of 6 and 1 of 6 replicates ofisolates 1956 and 2491 of S. commune survived 30 minexposure but the rest of the exposure times were lethal.The temperature had to be raised to 76 °C before therewas 100% mortality at all exposure times for these twoisolates.

The predicted survival following treatment at 56 °C for30 min and the temperatures that resulted in predicted99% and 99.99% mortality following 30 min exposure(Table 2) demonstrate the variability of susceptibility toheat treatment. Schizophyllum commune was the mostheat tolerant species that was tested and the model pre-dicted 99.3% survival following treatment at 56 °C for30 min (Table 2). Of concern from a regulatory stand-point was 27.6% predicted survival of F. circinatum, thecausal agent of pitch canker disease, following treatment

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Mortality of wood colonizing fungi 5

Fig. 1. A and B. Survival following heat treatment. Each plot represents mean survival (out of 6 replicate blocks) ± standard error of the meanfor three isolates of each species at each time/temperature combination, with the exception of C. herbarum, for which there were two isolatesand C. tenuissimum, for which there was one isolate (and hence no S.E.).

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Fig. 1. (Continued).

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Mortality of wood colonizing fungi 7

at 56 °C for 30 min. It is predicted that exposure for30 min to a minimum temperature of 61.7 °C or 68.9 °Cis necessary to cause 99% or 99.99% mortality of F. circi-natum, respectively (Table 2). Other fungi that were rela-tively heat tolerant were L. theobromae, O. novo-ulmi andS. sapinea (Fig. 1A, Table 2).

Discussion

To reduce the probability of movement of invasive organ-isms, several countries have invoked regulations that statethat solid wood packaging material must be treated accord-ing to protocols outlined in ISPM No. 15, one of which isheat treatment to a core temperature of 56 °C for 30 min(FAO, 2007). Our study has demonstrated that fungi andP. cinnamomi infecting wood can be killed by heat treat-ment but that different species, as well as different isolatesof the same species, vary in their susceptibility to heat(Fig. 1A and B). The ISPM No. 15 protocol results in regu-lation at the pathway level, rather than individual species,but the results of our in vitro study demonstrated that expo-sure to 56 °C for 30 min is not lethal for all fungi.

The fungi that were selected represented a range offungal types. Basidiomycetes included A. novae-zelandiae,P. gigantea and S. commune. Armillaria novae-zelandiaeis a root disease agent, while P. gigantea and S. communeare white rot decay fungi. The remainder of the fungiselected were ascomycetes and these included L. theobro-mae, a coelomycete that causes twig dieback and leafblight, and C. herbarum and C. tenuissimum which are pig-mented moulds. Important ascomycete pathogens that wereselected included F. circinatum, the causal agent of pitchcanker of P. radiata, N. fuckeliana, which is associated

with flute canker of P. radiata, S. sapinea, the causalagent of diplodia whorl canker and shoot tip dieback andO. novo-ulmi, the causal agent of Dutch elm disease.Pathogens in the genus Phytophthora have been respons-ible for some of the worst plant disease problems thathave been recorded and P. cinnamomi was selected forthis study. These organisms were selected because theyrepresent a broad cross-section of fungal types as wellas organisms that colonize different regions of thehost plants. Additionally, C. herbarum, P. giganteaand S. commune are common saprophytic fungi thathave been recovered from imported and exported wood,while the rest are pathogens that infect wood.

It was observed that the lethal temperature/time com-binations for each organism were achieved through eitherof two treatment regimes: (i) Exposure to a critical hightemperature for less than one minute; or (ii) A prolongedexposure to a temperature 5 °C lower than the criticaltemperature that was lethal. For all species that weretested, aside from S. commune, and one isolate each ofC. herbarum, O. novo-ulmi, F. circinatum and S. sapinea,exposure to 61 °C for less than one minute was lethal.It was necessary to raise the temperature to 76 °C to killS. commune with a less than one-minute exposure, likelybecause S. commune is a chlamydospore-forming basidi-omycete (Stalpers, 1978). Chlamydospores are thick-walled, long-term, survival structures that are producedby many fungi and their presence increases the toleranceof the fungus to heat. The remainder of the fungi in thesample population are non-chlamydospore forming, andthey were killed by exposure to lower temperatures. Theother chlamydospore-forming organism in the study wasP. cinnamomi, yet Gallo et al. (2008) have shown that

Table 2. Regression parameters for the binomial generalized linear model developed in this study, estimated survival () following exposure to 56 °C for 30 minutes with 95% confidence interval and the predicted temperature (°C) that causes 99% and 99.99% mortality following 30 minutes exposure.

Species a b c p (95% C.I.) T 99% (°C) T 99.99% (°C)

Armillaria novae-zelandiae 919.2 −29.4 −19.9 0.000 (0.000, NaNa) 41.4 41.6Cladosporium herbarum 34.7 −0.85 −0.70 0.065 (1.0 × 10−5, 0.04) 52.0 58.6

Cladosporium tenuissimum 26.8 −1.27 −0.47 0.018 (0.0009, 0.25) 57.6 67.4

Fusarium circinatum 38.3 −1.05 −0.64 0.276 (0.19, 0.38) 61.7 68.9

Lasiodiplodia theobromae 41.5 −0.83 −0.73 0.097 (0.025, 0.31) 59.3 65.6

Neonectria fuckeliana 44.06 −1.511 −0.803 0.002 (1.1 × 10−4, 0.05) 54.2 59.9

Ophiostoma novo-ulmi 30.26 −1.017 −0.520 0.089 (0.030, 0.23) 60.4 69.3

Phlebiopsis gigantea 1342.5 −27.6 −26.3 0.000 (0.000, NaN) 47.6 47.8

Phytophthora cinnamomi 19.66 −0.281 −0.455 0.001 (3.9 × 10−5, 0.033) 51.2 61.4

Schizophyllum commune 46.74 −0.677 −0.704 0.993 (0.935, 0.999) 61.4 69.6

Sphaeropsis sapinea 31.68 −0.71 −0.588 0.025 (0.005, 0.118) 57.6 65.4

aNaN designates ‘not a number’. This occurred when the upper limit of the confidence interval could not be calculated. This is because on the logit scale, theupper limit may be infinite, then becoming NaN on back-transformation to a probability.

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exposure of P. cinnamomi chlamydospores to 40 °C for1–2 hours was lethal, which correlates well with theresults of this experiment.

Heat treatment of wood to kill colonizing fungi hasbeen researched in the past, both for wood export pur-poses (Jones, 1973) and to kill decay fungi in wood des-tined for service, such as poles, railway ties, or structuraltimbers (Chidester, 1937, 1939; Newbill & Morrell,1991). Jones (1973) found that treatment of oak woodinfected by the oak wilt pathogen Ceratocystisfagacearum (Bretz) Hunt for 48 h in 43 °C air or 24 h in54 °C air or 43 °C water for 48 h or 49 °C water for 12 hkilled the pathogen. Chidester (1937, 1939) studied theeffect of heat on the mortality of Gloeophyllum sepi-arium (Wulfen) P. Karst., Meruliporia incrassata (Berk.& M.A. Curtis) Murrill and Neolentinus lepideus (Fr.)Redhead & Ginns, Fomitopsis rosea (Alb. & Schwein.)P. Karst, Gloeophyllum trabeum (Pers.) Murrill andAntrodia serialis (Fr.) Donk infecting wood blocks. Itwas recommended that core time/temperature combina-tions of 66 °C for 75 min, 77 °C for 30 min, 82 °C for20 min, 93 °C for 10 min or 100 °C for 5 min were neces-sary to kill all of the fungi that were tested. Chidester(1939) concluded that 66 °C is the minimum core tem-perature necessary to sterilize wood, which was consist-ent with the results of this study as every fungus, with theexception of S. commune and one isolate of F. circina-tum, was killed by exposure to 66 °C or higher (Fig. 1Aand B).

From an import/export treatment perspective, a rapidexposure to a core temperature of 76 °C would result inmortality of all the organisms that were tested in thisexperiment. It is also likely that the regime of 70 °C atthe core for 4 h as specified by the Import Standard forSawn Wood (MAF, 2003) would also be lethal to allspecies that were tested as 70 °C was the lowest pre-dicted temperature that resulted in 99.99% mortality ofall species after 30 min exposure. Although the 56 °Ccore temperature for 30-min regime of ISPM No. 15killed many of the species that were treated in this exper-iment, not all species were killed. If the objective of aheat treatment is to kill all fungi infecting wood prod-ucts, the results of this study suggest that the ISPM No.15 protocol of 56 °C core temperature for 30 min is notsufficient to guarantee mortality of all fungi present inthe wood. Indeed, for 99.99% predicted mortality, all buttwo species that were tested (A. novae-zelandiae andP. gigantea), required exposure to a temperature higherthan 56 °C for 30 min.

Acknowledgements

The authors thank Jamie Agnew, Elizabeth Orton, RitaTetenburg, Rebecca Ganley and Anna Hopkins for technicalassistance. Suggestions from two anonymous refereesstrengthened the manuscript. This research was funded bythe New Zealand Ministry of Agriculture and Forestry.

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