• pinewood nematode;
  • quarantine;
  • pesticide;
  • control


  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Materials And Methods
  5. 3 Results
  6. 4 Discussion
  7. Acknowledgements
  8. References


The pinewood nematode (PWN) Bursaphelenchus xylophilus is an important conifer disease worldwide. It is the direct cause of the death of millions of pines in south-east Asia (mainly Japan, China and Korea) and has been established in Portugal since 1999. The phasing out of methyl bromide has created an urgent need for alternative treatment of wood packaging materials. The effect of sulfuryl fluoride (SF), a broad-spectrum fumigant used to control insects, was tested in Pinus pinaster boards naturally infested by PWN.


Boards were fumigated for 24 h at three different temperatures (15, 20 and 30 °C) with dosage ranges of 3169–4407, 1901–4051 and 1385–2141 gh m−3 respectively. Treated wood was sampled for nematode identification and counting, before treatment and after 24 h, 72 h and 21 days. No survival was found in the 15 °C and 30 °C treatments, while at 20 °C the mortality ranged from 94.06 to 100%. Some reasons for the survival at 20 °C are presented.


Results confirm SF to be an effective quarantine treatment for PWN at 15 and 30 °C. Further studies are needed to obtain the most effective dosage at 20 °C, and to determine the toxicity of SF fumigation on B. xylophilus at other temperatures, especially at 25 °C. © 2012 Society of Chemical Industry

1 Introduction

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Materials And Methods
  5. 3 Results
  6. 4 Discussion
  7. Acknowledgements
  8. References

The pinewood nematode (PWN), Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle, native to North America, is the causal agent of pine wilt disease, which has killed millions of pines in Japan[1] and China[2] since its introduction. It was first detected in Portugal in 1999 (this being the first record for Europe) in a dead maritime pine (Pinus pinaster Aiton) at Pegões, Setúbal Peninsula.[3] To infect a healthy pine tree, the nematode needs to be transported by an insect vector usually of the Monochamus genus.[4, 5] In Portugal the only known vector is the native Monochamus galloprovincialis.[6]

Since the detection of the PWN in Portugal, significant efforts have been made to control the disease by cutting and destroying infested pine trees before the exit of the insect vector or by luring insect vectors with traps containing semiochemicals to capture them during their flight period.[7] In situ and real-time methods for early detection of the pine trees killed by PWN are also being developed and are important management tools to assess the effective impact of the nematode in the mortality of the pines, sorting B. xylophilus-induced mortality from trees killed by scolitids and other causes.[8, 9]

Long-distance dispersal of the nematode and its vectors is related to the global commerce and transport of untreated round and sawn wood, and nematodes of the Bursaphelenchus genus are often detected in wood packaging materials worldwide.[10-16] To prevent such dispersal, International Standard for Phytosanitary Measures (ISPM) No. 15 for wood packaging material was first published in 2002 and later revised in 2009. In this last revision, emphasis was placed on ‘phytosanitary measures that reduce the risk of introduction and spread of quarantine pests associated with the movement in international trade of wood packaging material made from raw wood’. Wood packaging material covered by this standard includes dunnage but excludes wood packaging (plywood) made from wood processed in such a way that it is free from pests.

Two control measures are currently included in the standard, namely fumigation with methyl bromide and heat treatment. Nevertheless, methyl bromide is currently being phased out of use globally and is no longer registered for use in the European Union (EU). Therefore, new alternatives are needed to eliminate nematodes from wood packaging materials. Among these, sulfuryl fluoride (SF) is a broad-spectrum fumigant used to control insects and tested on the pinewood nematode, and is known to have better timber penetration qualities than methyl bromide.[17] This gas is commercialised under the commercial brands Vikane®, ProFume® and Zythor®, which are registered in various countries for the control of stored-product insects and wood-destroying pests. In the EU, SF has been granted Annex 1 listing under EU Directive 98/8/EC (Biocides) for Product Type 8 (Wood Preservative) and Product Type 18 (Insecticides). Recently, Annex 1 listing under EU Directive 91/414 (Pesticides) was granted with effect from 1 November 2010, and the pest types include insects and nematodes.

Sulfuryl fluoride has been submitted and evaluated for inclusion in ISPM-15. Following its evaluations, the Technical Panel on Phytosanitary Treatments (TPPT) considered that published papers and historical use of SF fumigations have demonstrated that this treatment eliminates efficiently insect pests of concern in wood packaging material used in international trade. The present study was carried out to provide additional information to previous studies by Soma et al.,[18] Dwinell et al.[19, 20] and Flack et al.[21] on the toxicity of sulfuryl fluoride against PWN, so that SF could be included in ISPM-15.

2 Materials And Methods

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Materials And Methods
  5. 3 Results
  6. 4 Discussion
  7. Acknowledgements
  8. References

2.1 Collection and preparation of wood material

A total of 29 dead maritime pine trees (P. pinaster) infested with the PWN were felled in February 2010 in Comporta, Portugal. The lower trunk was sawed into boards (5 cm thick × 10 cm wide × 45 cm long), while thinner wood sections and branches, with diameters between 10 and 20 cm, were cut into 40 cm logs to fill up the fumigation chambers. The wood was taken to the Instituto Nacional de Investigação Agrária e Veterinária (INIAV) laboratories in Oeiras and kept in climatic chambers at 25 °C and 70% relative humidity (RH) for 60 days to increase the number of propagative life stages of the nematodes to the target density of at least 100 000 nematodes per treatment, as required for Probit 9 statistical analysis. Subsequently, the temperature and RH were reduced to 12 °C and 50% for 7 days to promote the development of the dispersal juvenile stage (JIII), as this is considered to be the pinewood nematode's most resistant life stage.[22] This was done to obtain at least 60% of JIII nematodes, as required by the TPPT.

2.2 Fumigation chambers and fumigant

In the INIAV laboratories in Oeiras, Portugal, two 12.2 m long shipping containers were lined with 4 mm thick polystyrene insulation panels (WallmateTM; The Dow Chemical Company, Midland, MI) and placed at approximately 2.5 m above the floor to create an inner chamber. Thermostatically controlled air conditioners and coil-in-oil electric heaters were used to achieve the required temperatures.

Inside each shipping container, seven 1 m[3] polyvinyl chloride (PVC) chambers with galvanised steel frames were placed, representing six treatment chambers and one untreated (control) chamber. Each of the six chambers was connected to a 30 m long × 4 mm inner diameter SF introduction tube, a 20 m thermocouple line and two 30 m long × 2.5 mm monitoring tubes located in the top and bottom open air space of each chamber. The untreated chamber had the same set-up, except that no tube was introduced into the entry port, which was sealed. Temperature was measured using thermocouple lines and data loggers (TinytagTM; Gemini Data Loggers, Chichester, UK), which were placed inside the untreated chambers and set to record temperature and relative humidity at 15 min intervals.

Each fumigation chamber contained 36 boards, forming a stack of four piles, nine boards high, and another identical volume formed by at least nine logs. Nine replicate boards to be sampled were marked and placed in sets of three at the top, middle and bottom of the stack. The wood from each tree was dispersed among all treatments. The chambers, partially loaded with wood, were held within each shipping container set at the target temperature and allowed to acclimatise for approximately 48 h prior to introduction of SF.

A cylinder of commercial-grade ProFume® gas fumigant (99.9% sulfuryl fluoride; Dow AgroSciences, Indianapolis, IN) was placed on a Yellow Jacket® digital electronic weighing scale (Ritchie Engineering Company Inc., Bloomington, MN) and connected to the introduction tubes. The required amount of SF was dispensed via a needle valve to achieve target dosages into the polyvinyl chloride fumigation chambers.

Fumigations were conducted on 3, 8 and 13 May 2010 under three temperature regimes for the following target dosages: 3200 and 4000 gh m−3 at 15 °C; 2300, 2875 and 3450 gh m−3 at 20 °C; 1400 and 1750 gh m−3 at 30 °C. The concentration of SF was monitored using an infrared spectrometer (Spectros® Reporter IR; Spectros Instruments Inc., Hopedale, MA) at 0.5, 2, 4, 6, 12 and 24 h. There were three replicates for each dosage/temperature combination. Concentration readings were entered into Fumiguide® software (Dow AgroSciences, Indianapolis, IN) in order to calculate accumulated dosages.

Following 24 h exposure, SF was aspirated from the chambers via extraction tubes and fans for approximately 2 h. Before handling the treated wood, complete exhaustion of SF was confirmed with an SF-ExplorIR device (Spectros Instruments Inc., Hopedale, MA).

2.3 Sampling for nematode extraction and counting

Boards (treated and untreated) were sampled for nematode presence on four occasions: before fumigation, after treatment, at 72 h and at 21 days after treatment. Separate wood bioassays were prepared for each sampling interval and treatment.

Wood samples for the nematode extraction consisted of 8 cm segments of each end of the wood piece, sawed to obtain a mixed sample of 100 g of wood cubes of approximately 1 cm[3], using a mechanical timber saw. After the sampling of each treated wood piece, the equipment was carefully brushed, vacuumed and washed with alcohol, to avoid cross-contamination.

Live nematodes were extracted using the modified tray method,[23] in which the wood sample was wrapped in paper and ‘etamine’ tissue and then placed on a plastic mesh overlaying a plastic tray. The tray was filled with tap water until the wood sample was completely immersed. After 48 h, the sample was removed and the water in the tray was passed through a 400 mesh (38 µm) sieve. The remaining material was washed with distilled water in small plastic or glass containers, and stored at 4 °C until evaluation.

Samples obtained before fumigation were homogeneously mixed, and a 1 mL aliquot was placed inside a counting slide (Chalex Corporation, Grasonville, MD). The total number of PWN present (mixed stages) and the number of JIII were determined. If fewer than 100 individuals were present, a new aliquot was removed and the process was repeated until 100 individuals were counted or 5 × 1 mL accounted for. For the remaining sampling dates (day 1, day 3 and day 21 after fumigation), the entire sample volume was placed in a Doncaster petri dish (Doncaster, Kent, UK); the number of PWN present and the total number of JIII were determined. Each sample was evaluated separately by stereomicroscopy, and nematodes were identified by morphological characters and counted at the INIAV (Oeiras) and NemaLab/ICAAM (Évora).

2.4 Wood moisture content

After nematode extraction, each wood sample was oven dried (type U50; Memmert GmbH, Schwabach, Germany) for 48 h and weighed again. The percentage of initial moisture was calculated by subtracting the dry weight from the initial weight, and then dividing by the initial weight.

2.5 Statistical analysis

The data were subjected to statistical analysis to meet the requirements of a quarantine treatment.[24] A direct method of efficacy calculation (exposing more than 100 000 individuals to the treatment) followed that of Couey and Chew,[25] with the appropriate adjustment for control mortality.[26] This adjustment was necessary to account for natural mortality in the untreated controls. Other basic statistics such as ANOVA were performed using the Statistica® software (StatSoft, Inc., Tulsa, OK) package.[27]

3 Results

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Materials And Methods
  5. 3 Results
  6. 4 Discussion
  7. Acknowledgements
  8. References

3.1 Fumigation parameters

The fumigations were conducted using a chamber wood-loading factor of 16.2% (one pile of boards and one pile of logs with dimensions of 45 × 40 × 45 cm). The measured SF dosage ranges achieved were 3169–4407 gh m−3 at 15 °C, 1901–4051 gh m−3 at 20 °C and 1360–2141 gh m−3 at 30 °C. Because an unexpectedly low sorption of wood during the first set of fumigations at 20 °C resulted in a higher dosage than expected, during subsequent assays a correction to the amount of SF introduced was necessary. Fumigant leakage was measured from one of the fumigation chambers at 20 °C, and additional SF was immediately injected. However, the final obtained dosage of 1901 gh m−3 was below the minimum desired and therefore was not considered during subsequent analysis.

ANOVA adjusted to SF dosages measured in chambers revealed highly significant statistical differences for all tested temperatures and made it possible to determine three homogeneous groups for consideration in further analysis: F(15 °C) = 25.717, df = 5, 30, P < 0.0001; F(20 °C) = 112.34, df = 7, 40; P < 0.0001; F(30 °C) = 34.766, df = 5, 30, P < 0.0001.

The system was effective in achieving consistent temperatures following SF introduction in all fumigations. The temperatures recorded in the chambers were close to the target temperatures of 15, 20 and 30 °C in all assessment periods (Table 1).

Table 1. Measured experimental parameters (temperature, wood moisture content and dosage; mean ± SEM) in sulfuryl fluoride (SF) fumigationsa of Pinus pinaster boards infested with Bursaphelenchus xylophilus for 24 h in 1 m3 chambers. Letters after sulfuryl fluoride achieved dosages are LSD homogeneous groups. Rows in bold represent mean values of replicates for each treatment (temperature/dosage). WMC: wood moisture content
Fumigation parameterSF concentration (g m−3) at time (h) after introductionMean dosage (gh m−3)
Temp. (°C)WMC (%)Dose (g m−3)n0.5 h2 h4 h12 h24 h
  1. a

    Fumigations were conducted during April 2010 in Oeiras, Portugal. The CT (concentration × time) values were calculated from the concentrations below. The loading factor of the fumigation chambers was 16.2% (vol:vol).

14.6 ± 1.3425.3 ± 2.2109000000
14.6 ± 0.8926.7 ± 1.5314091451231371331323169 a
14.8 ± 1.1029.6 ± 1.4112091511281361361353217 a
14.7 ± 0.6728.2 ± 1.07130.0 ± 10.0018148.0 ± 3.00125.5 ± 2.50136.5 ± 0.50134.5 ± 1.50133.5 ± 1.503193
14.4 ± 0.5530.0 ± 0.5713091551521591561553691 b
15.4 ± 1.9528.4 ± 1.2314091691461641601583774 b
14.9 ± 1.0229.2 ± 0.69135.0 ± 5.0018162.0 ± 7.00149.0 ± 3.00161.5 ± 2.50158.0 ± 2.00156.5 ± 1.503733
15.2 ± 1.6428.3 ± 1.1217092051841911831844400 c
14.2 ± 0.8430.6 ± 0.5316092001771871861834407 c
14.7 ± 0.9529.5 ± 0.66165.0 ± 5.0018202.5 ± 2.50180.5 ± 3.50189.0 ± 2.00184.5 ± 1.50183.5 ± 0.504404
19.4 ± 1.5227.4 ± 0.6709000000
19.4 ± 1.1429.4 ± 1.07909106949490842145 a
19.2 ± 0.8430.5 ± 0.489091141019999962352 a
19.5 ± 0.5828.6 ± 0.709091021041061051032488 a
19.4 ± 0.4929.5 ± 0.4690.0 ± 0.0027107.3 ± 3.5399.7 ± 2.9699.7 ± 3.4898.0 ± 4.3694.3 ± 5.552328
18.8 ± 1.3030.4 ± 1.0909000000
18.4 ± 0.5531.5 ± 1.4014091691661591601533768 b
18.3 ± 1.8634.3 ± 3.2014091701671641631583852 b
18.4 ± 0.9132.9 ± 1.73140.0 ± 0.0018169.5 ± 0.50166.5 ± 0.50161.5 ± 2.50161.5 ± 1.50155.5 ± 2.503810
20.0 ± 0.4136.0 ± 2.7017091811781781761724036 c
20.5 ± 0.7129.8 ± 0.4416091861821801771694045 c
19.2 ± 1.3033.2 ± 0.9915091761721721711664051 c
19.7 ± 0.7133.0 ± 1.06160.0 ± 5.7727181.0 ± 2.89177.3 ± 2.91176.7 ± 2.40174.7 ± 1.86169.0 ± 1.734044
31.4 ± 1.1427.7 ± 0.1009000000
30.8 ± 0.8426.9 ± 0.4860954595958571360 a
31.6 ± 1.1426.5 ± 0.3750961586159571385 a
30.4 ± 0.5528.5 ± 0.4060940686464631482 a
30.9 ± 0.5527.3 ± 0.2956.7 ± 3.332751.7 ± 6.1761.7 ± 3.1861.3 ± 1.4560.3 ± 1.8659.0 ± 2.001409
31.2 ± 0.8430.1 ± 0.9270986787676751793 b
31.2 ± 0.8429.0 ± 0.2180973787877761804 b
31.2 ± 0.5629.5 ± 0.4775.0 ± 5.001879.5 ± 6.5078.0 ± 0.0077.0 ± 1.0076.5 ± 0.5075.5 ± 0.501799
30.4 ± 0.5527.0 ± 1.0680998949291892141 c

The tested wood moisture content (WMC) varied from just 17% to 47% (average values per treatment in Table 1). Differences were found in WMC between boards where PWN was eliminated and boards tested at 20 °C where survival was recorded (F(1,178) = 30.29, P < 0.0001). The relatively flat sorption curves of SF by P. pinaster wood in each fumigation chamber (Fig. 1) indicate that pinewood has poor SF sorption.


Figure 1. Parameters of the fumigation with sulfuryl fluoride (SF) of Pinus pinaster boards naturally infected with pinewood nematode Bursaphelenchus xylophilus at three temperatures. Sorption curves registered in fumigation chambers at 15 °C (A), 20 °C (B) and 30 °C (C). Relation of final CT product (concentration × time) in fumigation chambers at 15 °C (D), 20 °C (E) and 30 °C (F).

Download figure to PowerPoint

3.2 Fumigation effect on PWN populations

The PWN counts 48 h before fumigation of boards at 15 °C ranged from 166 000 to 883 000 (JIII) and from 21 000 to 253 000 (adults and all juveniles excluding the JIII stage) per dosage. At 30 °C, pretreatment counts ranged from 170 000 to 1 052 000 (JIII) and from 47 000 to 207 000 (adults and all juveniles excluding the JIII life stage) per dosage. The results of SF fumigations at the nine temperature–dose combinations are presented in Table 2. In boards fumigated at 15 and 30 °C, 100% mortality of PWN was achieved in all post-fumigation assessment periods (24 h, 72 h and 21 days) and for every dosage tested (3169–4407 gh m−3). In boards at 20 °C, PWN survival was measured at all dosages, although for the 2328 gh m−3 SF dosage the nematodes were recovered from only one of the 27 analysed boards on all sampling occasions. The 20 °C PWN counts 48 h before fumigation revealed very high PWN populations, ranging from 279 000 to 1 011 000 (JIII life stage) and from 170 000 to 268 000 (adults and all juveniles excluding the JIII life stage). Nematode populations in the boards persisted during the 21 days of the assay.

Table 2. Nematode numbers before and after sulfuryl fluoride fumigation of Pinus pinaster boards infested with Bursaphelenchus xylophilus (PWN) at three temperatures (15, 20 and 30 °C) for 24 h in 1 m3 chambers. The numbers of B. xylophilus (total nematodes and percentage at life stage JIII) in boards were counted before fumigation and in samples taken after 24 h, 72 h and 21 days
Fumigation parametersTotal number of nematodes (mean/g wood × weight of infected boarda)
48 h before fumigationAfter fumigation
24 h72 h21 days
  1. a

    Mean weight of infested boards = 1500 g.

  2. b

    N: number of infested boards.

  3. c

    NR: not recorded.

Temp. (°C)Dose (gh m−3)NbTotal PWNJIII life stage (%)NbTotal PWNJIII life stage (%)NbTotal PWNJIII life stage (%)NbTotal PWNJIII life stage (%)
1509424 00072.819546 00077.279395 00083.659177 00070.18
15319318405 00085.96000000000
153733181 630 00081.82000000000
154404181 190 00074.74NRcNRNR000000
20091 067 00081.389461 00087.029200 00072.18993 00084.99
202328272 780 00075.10118010013001150
2009551 00074.239402 00082.639301 00079.469426 00092.70
20381018814 00056.35110528.575283020.10524 76019.11
204044273 055 00063.10432972.6910452013.591036 98019.94
30091 182 00088.999684 00080.789609 00093.949112 00087.45
301409271 952 00082.94000000000
301799181 454 00087.10NRNRNR000000
3021419800 00075.39NRNRNR000000

Table 3 contains the corrected values for observed nematode mortality according to ISPM-15 requirements and Probit 9 calculations. At both 15 and 30 °C, the results provide Probit 9 efficacy and validate the SF dosage in the schedule to be proposed to ISPM-15 (Table 4). Values presented for the range between 20 and 30 °C should be considered as a basis for future studies.

Table 3. Percentage mortality of Bursaphelenchus xylophilus in boards of Pinus pinaster after sulfuryl fluoride fumigation. Percentages are relative to nematode populations in untreated control boards at each sampling date
Fumigation parameters24 h after fumigation72 h after fumigation21 days after fumigation
Temp.(°C)Dose (gh m−3)% Mortalitya% Mortalityb% Mortalitya% Mortalityb% Mortalitya% Mortalityb
  1. a

    Not adjusted for untreated mortality.

  2. b

    Adjusted by the Henderson–Hilton formula, accounting for natural mortality observed in the respective control.

150 6.7456 58.3097 
20056.7905 81.2446 88.2027 
20026.9409 45.3743 22.6292 
30042.1557 48.4793 89.3823 
Table 4. Proposed concentration–time dosages and effective doses of sulfuryl fluoride (SF) to eliminate Bursaphelenchus xylophilus in Pinus pinaster boards
Mean temperature (°C)Minimum target CT dosage (gh m−3)SF dose (g m−3)Minimum concentration (g m−3)
at 0.5 hat 2 hat 4 hat 12 hat 24 h
30 and above1400828778735841

4 Discussion

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Materials And Methods
  5. 3 Results
  6. 4 Discussion
  7. Acknowledgements
  8. References

Previous studies on sulfuryl fluoride toxicity mainly focused on various insects such as termites, wood-boring beetles and fruit flies.[28-32] The present study proves that SF is effective in eliminating high populations of B. xylophilus in infested wood boards. In fact, the nematode load in the fumigated boards was higher than the standard Probit 9 requirements (minimum of 100 000 individuals),[33, 34] and, even with the highest survival treatment (a total of 3810 nematodes at 20 °C), almost 800 000 nematodes were eliminated with fumigation, confirming the toxicity and efficacy of the gas in eliminating the PWN.

The survival of the nematode in the 20 °C treatment without a dose–response relationship is difficult to explain, although it may be possible that nematodes survived fumigation at the egg stage, whereas under more unfavourable temperatures of 15 and 30 °C the eggs would have been much less abundant or even absent. It is known that insect eggs are less susceptible to SF fumigation than other life stages because the shell limits the passage of the gas; therefore, control of eggs requires an increased exposure time or increased concentrations of SF.[28, 35-41] The same phenomenon applied to nematodes would explain the survival of some populations at 20 °C and the absence of B. xylophilus in almost all samples taken 24 h after fumigation (absent in 66 of 72 samples), as the egg is the only life stage not detected in the wood samples immersed in water, and after 21 days at favourable temperatures these same boards contained adult nematodes.

Another possible explanation for survival at 20 °C is that variations in board moisture content (with higher content where survival occurred) could have influenced SF penetration, as water-saturated pinewood is known to reduce the penetration of SF considerably.[42] However, the level of moisture in all boards was generally low, with the highest mean value of just 33%, which is distant from water saturation and would hardly be a barrier to SF entry. Indeed, pre-tests at Fera UK using freshly cut pine logs confirmed that effective penetration of SF at WMC above 60% occurred in a range of pine and hardwood species.[43]

The results at 15 and 30 °C assured Probit 9 efficacy and validated the toxicity of the SF dosage in the proposed ISPM-15 schedule (Table 4), significantly reducing the risk of introduction and/or spread of quarantine pests, as stated by ISPM-15 norms. On the other hand, the Probit 9 standard for quarantine treatment efficacy (99.9968% mortality), which was originally recommended for tropical fruit flies, may be too stringent for quarantine pests of insects on poor hosts.[44] Similarly, living pine trees should be considered as the host plant to the PWN and its insect vector, rather than debarked wood packaging material, which is exposed to changing environmental conditions and is a very poor host that poorly supports nematode development. Furthermore logs with bark would be expected to absorb less SF than processed boards, as SF penetration in logs occurs mainly at the cut ends, whereas in boards all sides are permeable.

Mortality rates resulting from quarantine fumigations show widely varying risks and do not necessarily give foolproof levels of security.[45] The stated goal of ISPM-15 is to ‘reduce the risk of introduction and/or spread of quarantine pests’ and to describe measures that ‘significantly reduce the risk of pest spread’. Such a reduction was achieved in the present study, given the size of the eradicated nematode populations.

The infested wood had been obtained from dead pines in locations where various species of bark and wood-boring beetles were present, such as scolitids (Ips sexdentatus and Orthotomicus erosus) and the cerambycids Arhopalus syriacus and M. galloprovincialis, normal insect fauna associated with dead P. pinaster in Portugal.[7, 46] Although not quantified, all larvae, pupae and adult insects present in the wood were killed with SF fumigation at the three tested temperatures, thereby confirming previous results on the efficacy of SF as an insecticide.[29, 32, 47, 48] The limited survival of the pinewood nematode in low-moisture-content wood[49] and the combined eradication of both the nematode and its insect vector from SF-fumigated boards effectively remove the risk of dispersing pine wilt disease, even if low levels of B. xylophilus survive, because the nematode depends on a living vector in the wood for dissemination to a healthy host tree.

Overall, the present results confirm that fumigation with SF is a suitable quarantine treatment for wood and wood packaging materials infested with B. xylophilus, and that SF may be an alternative chemical fumigant needed for commodity quarantine treatments for pests in the absence of methyl bromide. Additional studies are needed, however, to determine the effective dosage and/or exposure time at 20 °C to obtain 100% mortality, and to determine the toxicity of SF fumigation on B. xylophilus at other temperatures, especially at 25 °C.


  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Materials And Methods
  5. 3 Results
  6. 4 Discussion
  7. Acknowledgements
  8. References

The authors thank Adérito Matos, Vitor Gonçalves and Francisco Martins for processing thousands of wood samples to extract nematodes, while Sofia Basto and Ana Bela Carvalho provided laboratory assistance, from INIAV. Margarida Fontes and Maria Antónia Lampreia assisted in the identification and counting of nematodes. The authors are grateful to Pedro Serrasqueiro and António Moreira of Herdade da Comporta for allowing the cutting of PWN-infected pine trees, as well as to Paulo Verdasca from Madeca Saw Mills for making the boards from infested pine logs. Thanks also go to Cecília Rego from Instituto Superior de Agronomia (Technical University of Lisbon) for her statistical advice. José Roca Michavila (Roca Defisan S.L.) and Luis Múrias (Fumitech) gave valuable technical assistance for the fumigation operations, while Duncan Rix and Christopher Guy (Dow AgroSciences) gave logistical support for fumigation equipment. Thanks are also extended to Gaynor Cleisham (Dow AgroSciences) for administrative support and to Steve Nolting (Dow AgroSciences) for statistical advice. Roddie Burgess and Hugh Evans (UK Forestry Commission) assisted in the supply of wood logs for the preliminary trials at Fera, UK.


  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Materials And Methods
  5. 3 Results
  6. 4 Discussion
  7. Acknowledgements
  8. References
  • 1
    Mamiya Y, The pinewood nematode, in Plant and Insect Nematodes, ed. by Nickle WR. Marcel Dekker, New York, NY, pp. 589626 (1984).
  • 2
    Yang BJ and Wang QL, Distribution of the pinewood nematode in China and susceptibility of some Chinese and exotic pines to the nematode. Can J For Res 19:15271530 (1989).
  • 3
    Mota MM, Braasch H, Bravo MA, Penas AC, Burgermeister W, Metge K et al., First report of Bursaphelenchus xylophilus in Portugal and in Europe. Nematology 1:727734 (1999).
  • 4
    Linit MJ, The insect component of the pine wilt disease in the United States, in Pathogenicity of the Pine Wood Nematode, ed. by Wingfield MJ. APS Press, St Paul, MN, pp. 2639 (1987).
  • 5
    Kishi Y, Pine Wood Nematode and the Japanese Pine Sawyer. Thomas Comp Limited, Tokyo, Japan, 302 pp. (1995).
  • 6
    Sousa E, Bravo MA, Pires J, Naves P, Penas AC, Bonifácio L et al., Bursaphelenchus xylophilus (Nematoda; Aphelenchoididae) associated with Monochamus galloprovincialis (Coleoptera; Cerambycidae) in Portugal. Nematology 3:8991 (2001).
  • 7
    Bonifácio L, Impact and evolution of pine wilt disease in the affected area south of the Tagus River, Portugal. PhD Thesis, Science Faculty of Lisbon University, Lisbon, Portugal, 209 pp. (2009).
  • 8
    Bonifácio L and Sousa E, Early detection methods for the detection of pine wood nematode infections in standing maritime pines in Portugal. Silva Lusitana 19:4960 (2011).
  • 9
    Cardoso JMS, Fonseca L and Abrantes I, Direct molecular detection of the pinewood nematode, Bursaphelenchus xylophilus, from pine wood, bark and insect vector. Eur J Plant Pathol 133:419425 (2012).
  • 10
    Braasch H, Gu J, Burgermeister W, Brandstetter M and Metge M, Bursaphelenchus africanus sp. n. (Nematoda: Parasitaphelenchidae) found in packaging wood exported from South Africa to Ningbo/China. J Nematode Morphol Syst 9:7181 (2006).
  • 11
    Braasch H, Gu J and Brandstetter M, Bursaphelenchus burgermeisteri sp. n. (Nematoda: Parasitaphelenchidae) in packaging wood from Japan – a second species of the ‘africanus’ group. J Nematode Morphol Syst 10:3948 (2007).
  • 12
    Gu J, Braasch H, Burgermeister W and Zhang J, Records of Bursaphelenchus spp. intercepted in imported packaging wood at Ningbo, China. For Pathol 36:323333 (2006).
  • 13
    Li H, Trinh PQ, Waeyenberge L and Moens M, Bursaphelenchus chengi sp. n. (Nematoda: Parasitaphelenchidae) isolated at Nanjing, China, in packaging wood from Taiwan. Nematology 10:335346 (2008).
  • 14
    Li H, Trinh PQ, Waeyenberge L and Moens M, Characterization of Bursaphelenchus spp. isolated from packaging wood imported at Nanjing, China. Nematology 11:375408 (2009).
  • 15
    Tomiczek C, Braasch H, Burgermeister W, Metge K, Hoyer U and Brandstetter M, Identification of Bursaphelenchus spp. isolated from Chinese packaging wood imported to Austria. Nematology 5:573581 (2003).
  • 16
    Tomminen J, Pinewood nematode, Bursaphelenchus xylophilus, found in packing case wood. Silva Fennica 25:109111 (1991).
  • 17
    Scheffrahn RH, Su N-Y and Osbrink WLA, Precise dosage delivery method for sulfuryl fluoride in small chamber fumigations. J Econ Entomol 80:705707 (1987).
  • 18
    Soma Y, Naito H, Misumi T, Mizobuchi M, Tsuchiya Y, Matsuoka I et al., Effects of some fumigants on pine wood nematode, Bursaphelenchus xylophilus, infecting wooden packages. 1. Susceptibility of pine wood nematode to methyl bromide, sulfuryl fluoride and methyl isothiocyanate. Res Bull Plant Prot Jap 37:1926 (2001).
  • 19
    Dwinell LD, Thoms E and Prabhakaran S, Effect of sulfuryl fluoride on the pinewood nematode in pine wood. Proc Annu Int Res Conf on Methyl Bromide Alternatives and Emissions, San Diego, CA, pp. 94104 (2003).
  • 20
    Dwinell LD, Thoms E and Prabhakaran S, Sulfuryl fluoride as a quarantine treatment for the pinewood nematode in unseasoned pine. Proc Annu Int Res Conf on Methyl Bromide Alternatives and Emissions, San Diego, CA, p. 68 (2005).
  • 21
    Flack E, Barak A, Thoms E and Messenger M, Confirmation of proposed sulfuryl fluoride quarantine dosages for pinewood nematode control. Proc Annu Int Res Conf on Methyl Bromide Alternatives and Emissions, San Diego, CA, 2 pp. (2008).
  • 22
    Tomminen J and Nuorteva M, Pinewood nematode, Bursaphelenchus xylophilus, in commercial sawn wood and its control by kiln-heating. Scand J For Res 7:113120 (1992).
  • 23
    Penas AC, Dias LS and Mota MM, Precision and selection of extraction methods of aphelenchid nematodes from maritime pine wood, Pinus pinaster L. J Nematol 34:6265 (2002).
  • 24
    Follet PA and Nevan LG, Current trends in quarantine entomology. Annu Rev Entomol 51:539585 (2006).
  • 25
    Couey HM and Chew V, Confidence limits and sample size in quarantine research. J Econ Entomol 79:887890 (1986).
  • 26
    Henderson CF and Tilton EW, Tests with acaracides against brown wheat mite. J Econ Entomol 48:157161 (1955).
  • 27
    Statistica, Version 6.1 . StatSoft, Inc., Tulsa, OK (2003).
  • 28
    Thoms EM and Scheffrahn RH, Control of pests by fumigation with Vikane® gas fumigant (sulfuryl fluoride). Down to Earth 49:2330 (1994).
  • 29
    Bell CH and Savvidou N, The toxicity of Vikane (sulfuryl fluoride) to age groups of eggs of the Mediterranean flour moth (Ephestia kuehniella). J Stored Prod Res 35:233247 (1999).
  • 30
    Reichmuth Ch, Scholler M, Dugast J-F and Drinkall MJ, On the efficacy of sulfuryl fluoride against stored-product pest moths and beetles, in Proc Int Conf on Controlled Atmosphere and Fumigation in Stored Products, 21–26 April 1996, ed. by Donahaye EJ, Navarro S and Varnava A. Printco Ltd, Nicosia, Cyprus, pp. 1723 (1997).
  • 31
    Schneider BM and Hartsell PL, Control of stored product pests with Vikane gas fumigant (sulfuryl fluoride), in Proc 7th Int Working Conf on Stored-product Protection. Vol. 1, ed. by Zuxun J, Quan L, Yongsheng L, Xianchang T and Lianghua G. Sichuan Publishing House of Science and Technology, Beijing, China, pp. 406408 (1999).
  • 32
    Zettler JL, Leesch JG, Gill RF and Tebbets JC, Chemical alternatives for methyl bromide and phosphine treatments for dried fruits and nuts, in Proc 7th Int Working Conf on Stored-product Protection. Vol. 1, ed. by Zuxun J, Quan L, Yongsheng L, Xianchang T and Lianghua G. Sichuan Publishing House of Science and Technology, Beijing, China, pp. 554561 (1999).
  • 33
    Magnusson C and Schröder T, Technical protocol for testing nematodes during treatment development. International Forestry Quarantine Research Group Meeting, September 2008, Rome, Italy (2009).
  • 34
    Schröder T, McNamara DG and Gaar V, Guidance on sampling to detect pine wood nematode Bursaphelenchus xylophilus in trees, wood and insects. EPPO Bull 39:179188 (2009).
  • 35
    Kenaga EE, Biological, chemical, and physical properties of sulfuryl fluoride as an insecticidal fumigant. J Econ Entomol 50:16 (1957).
  • 36
    Outram I, Effects of the fumigant sulfuryl fluoride on the gross metabolism of insect eggs. Fluoride Q Rep 3:8591 (1970).
  • 37
    Su N and Scheffrahn RH, Efficacy of sulfuryl fluoride against four beetle pests of museums (Coleoptera: Dermestidae, Anobiidae). J Econ Entomol 83:879882 (1990).
  • 38
    Drinkall MJ, Dugast JF, Reichmuth Ch and Scholler M, The activity of the fumigant sulfuryl fluoride on stored product insects, in Proc 2nd Int Conf on Insect Pests in the Urban Environment, ed. by Wildey KB. Heriot-Watt University, Edinburgh, UK, pp. 525528 (1996).
  • 39
    Zettler JL, Gill R, Armstrong JW and Vail PV, Toxicity of sulfuryl fluoride (Vikane) to fruit flies in laboratory tests, in Proc Int Conf on Controlled Atmosphere and Fumigation in Stored Products, Fresno, CA, 29 October–3 November 2000, ed. by Donahaye EJ, Navarro S and Leesch JG. Executive Printing Services, Clovis, CA, pp. 155166 (2001).
  • 40
    Soma Y, Yabuta S, Mizoguti M, Kishino H, Matsuoka I, Goto M et al., Susceptibility of forest insect pests to sulfuryl fluoride. 1. Wood borers and bark beetles. Res Bull Plant Prot Jap 32:6976 (1996).
  • 41
    Soma Y, Naito H, Misumi T and Kawakami F, Effects of gas mixtures of sulfuryl fluoride and methyl bromide on forest insect pests. Res Bull Plant Prot Jap 35:1519 (1999).
  • 42
    Scheffrahn RH, Su NY and Hsu RC, Diffusion of methyl bromide and sulfuryl fluoride through selected structural wood matrices during fumigation. Mater Organisms 27:147155 (1992).
  • 43
    Scheffrahn RH and Thoms EM, Penetration of sulfuryl fluoride and methyl bromide through selected substrates during fumigation. Down to Earth 48:1519 (1993).
  • 44
    Follet PA and McQuate GT, Accelerated development of quarantine treatments for insects on poor hosts. J Econ Entomol 94:10051011 (2001).
  • 45
    Landolt PJ, Chambers DL and Chew V, Alternative to the use of probit 9 mortality as a criterion for quarantine treatments of fruit fly (Diptera: Tephritidae) infested fruit. J Econ Entomol 77:285287 (1984).
  • 46
    Sousa EM, Naves PM and Bonifácio L, Distribution of bark and wood-boring insects in maritime pine trees infected with Bursaphelenchus xylophilus in Portugal. Proc XXI Int Congr of Entomology, Iguassu, Brazil, p. 498 (2000).
  • 47
    Barak AV, Wang Y, Zhan G, Wu Y, Xu L and Huang Q, Sulfuryl fluoride as a quarantine treatment for Anoplophora glabripennis (Coleoptera: Cerambycidae) in regulated wood packing material. J Econ Entomol 99:16281635 (2006).
  • 48
    Barak AV, Messenger M, Neese P, Thoms EM and Fraser I, Sulfuryl fluoride as a quarantine treatment for Emerald Ash Borer (Coleoptera: Buprestidae) in ash logs. J Econ Entomol 79:603611 (2010).
  • 49
    Sousa E, Naves P, Bonifácio L, Henriques J, Inácio ML and Evans H, Survival of Bursaphelenchus xylophilus and Monochamus galloprovincialis in pine branches and wood packaging material. EPPO Bull 41:203207 (2011).