Technical protocol for testing nematodes
2009-TPFQ-39 Rev. 1
Agenda Item 6.4
Technical Protocol for Testing Nematodes during Treatment Development.
2008.07.13
Christer Magnusson
Bioforsk
Høgskoleveien 7
N-1432 Aas
Norway
Background:
The Technical Panel of Phytosanitary Treatments (TPPT) was established by the Standards
Committee of the Commission for Phytosanitary Measures (CPM) in 2004 to “Evaluate treatments
and recommend which ones should be included in standards adopted be the CPM”.
Pine wood nematode (PWN) Bursaphelenchus xylophilus, the causal agent of pine wilt disease, is an
important forest pest organism causing considerable damage in East-Asia and restricted areas of
Europe mainly Portugal. PWN is considered to pose a threat to coniferous forests world-wide, and it
has been intercepted repeatedly in timber, lumber and wood packaging material (WPM) moving in
international trade. Most NPPO:s have imposed strict regulation against PWN in traded wood
products, and request documentation to demonstrate freedom from PWN in imported wood products
and WPM associated with all commodities to be free from PWN. For WPM freedom of PWN is
demonstrated by the marking stating that the material has been exposed to a phytosanitary
treatment, i.e. Methyl Bromide fumigation of heat treatment in accordance with the ISPM 15 in
order to, kill PWN and its vectors. At present a new standard: “Management of phytosanitary risks in
the international movement of wood, with or without bark” is being developed as a complement to
ISPM 15.
For environmental reasons there is a need to find alternative treatments to Methyl Bromide
fumigation. Also alternatives to heat treatment would be of value for certain wood products.
Therefore new and efficient treatments against PWN are urgently needed. Several proposals for
such treatments against nematodes (i.e. PWN) in wood gave been proposed, but so far all have
failed to be accepted by the TPPT mostly because of a weak experimental designs.
Objective:
To document the effect of a treatment on the survival of pinewood nematode (PWN)
Bursaphelenchus xylophilus in wood of commercial types and dimensions.
Important biological aspects of PWN:
The life stages of PWN include both propagative and dispersal stages (Fig. 1). In conditions
favourable for reproduction the propagative life stages (Fig. 1 A) dominate in wood, and include
developing eggs and eggs containing the first juvenile stage J 1. Inside the egg the J1 moults to the
next juvenile stage J2, which hatches from the egg. In wood the nematodes feed on living plant cells
and on fungal mycelia. The second juvenile stage (J2) is followed by the J3 and J4 and the latter
moults into adult females and males, which, mate and lay eggs. The generation time is 3 days or
longer depending on temperature, which means that the population will reach high densities in a
short time if conditions remain favourable.
However, falling moisture levels, temperatures and shortness of nutritional resources trigger the
development of dispersal juvenile life stages (Ishibashi & Kondo 1977). This development starts by
the propagative J2 moulting into the third dispersal juvenile stage JIII (Fig. 1 B). This stage is
considered as a resting stage in the life cycle of PWN (Mamiya 1984) and has the thickest cuticle of
all life stages (Kondo & Ishibashi 1978). It has large energy (lipid) reserves and probably a high
capacity of migration. This is the stage, which in nature accumulates in the wood surrounding the
pupal chamber of Monochamus and moults (Fig.1 D) into the fourth dispersal stage J IV, which then
invades the tracheal system of the vector beetle to be carried to new trees where it moults to adult
in the feeding scars of the beetle (Fig. 1 E). In the absence of the insect the J III will accumulate in
drying wood, and if conditions improve it may moult to normal propagative J 4.
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Technical protocol for testing nematodes
2009-TPFQ-39 Rev. 1
Agenda Item 6.4
Figure 1. Life stages of the pine wood nematode (PWN),
Bursaphelenchus xylophilus. Propagative stages (blue) and
dispersal stages (red). A. Propagative phase. B. Propagative J2
moults into the dispersal third stage JIII. C. Dispersal JIII moults into
propagative J4. D. Dispersal JIII moults into dispersal JIV. Dispersal
JIV moults into adult in feeding scars of Monochamus beetle.
Requirements to the treatment procedure:
There should be detailed descriptions of the experimental facilities, and the environmental
conditions during the treatment. The treatments should be made with the nematodes in situ. In
order for the treatment to be well documented and reproducible the process parameters should be
registered throughout the treatment using calibrated instruments and following standard
procedures. The position of sensors should be chosen to account for possible gradients in vital
treatment parameters. The treated material and controls must be protected against nematode
colonisation during storage and incubation.
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Description of experimental facilities and equipment used
Environmental conditions
Treatment carried out with nematodes in situ
Treatment parameters well documented
Target the most challenging situation
Reproducible procedures
Full protection against post-treatment colonisation
Requirements to nematode populations:
Wood is naturally inhabited by several species of nematodes, so the density of PWN needs to be
established initially. As stated in ISPM 28 “Phytosanitary treatments for regulated pests” (FAO
2009), the life stages of the pest associated with the article moving in trade is the stage for which a
treatment should be proposed and established, and when several life stages are present (like PWN
in wood), the frequency and density of the most resistant life stage (J III) should be determined and
demonstrated to be the dominant stage in the material used for testing a treatment.
The importance of the physiological state and the thickness of the J III cuticle in relation to high
temperature and fumigants is not known, but this stage is widely recognized as the resting stage of
PWN. It should not be a problem to obtain material with a JIII frequency of more than 60 %, since
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Technical protocol for testing nematodes
2009-TPFQ-39 Rev. 1
Agenda Item 6.4
this stage has been reported to form 90 % of the PWN population in naturally infested wood of Pinus
densiflora and reach densities of more than 90 ind. per gram of wood (Soma et al. 2002).
How to identify the third dispersal stage JIII
This life-stage is easily spotted in suspensions by its dark intestine,
rounded tail and vigorous movements. Good pictures showing the
habitus of JIII can be found in Soma et al. (2002). The development of
the genital primordia, i.e. the cells that will form the gonad is
commonly used in separating life-stages of nematodes. Penas et al.
(2008) made a detailed study on the morphology of PWN from Portugal,
and found that determination of the JIII stage can be supported by
studying the development of the genital primordium. Staining
nematodes with 1 % acid orcein for 48 hours facilitated the observations
of this structure. Although the length of the genital primordia are quite
similar in J3 and JIII (22-51 um and 25-42 um respectively), the body
length of J3 is 327-544 um compared to 620-788 um in JIII. From what is
known for other nematodes the genital promordium of J III would
contain three cells. The structure of the genital primodium in J4 is not
shown in Penas et al. (2008), but can be expected to contain four cells
compared to three in JIII.
In regions of the world where PWN is not known to occur, full-scale experiments with PWN may not
be possible. A surrogate species in this situation could be the close relative Bursaphelenchus
mucronatus, which in most regions is considered to be non-pathogenic. Apart from the aspect of
pathogenicity this species has the same bionomics as PWN. It is not know if the J III-stage of
B.mucronatus has the same tolerance to heat and chemicals as the JIII-stage of PWN. However, B.
mucronatus could serve as a good model for developing treatments, which then could be verified in
experiments with PWN.
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Provide an analysis of the nematodes occurring in the test material
Provide a density estimate of PWN
Provide a frequency of the third dispersal stage (JIII)
The species B. mucronatus could probably serve as a surrogate species in the absence of
PWN.
Level of control:
Since considerable time the Probit 9 statistical standard has been required as a level of efficacy for
the treatment of commodities carrying high-risk pests like the PWN. To meet this standard the
treatments must kill 99.9968 % of the pest in a test of at least 100 000 pest individuals.
Requirements to test material:
Test material should be wood of normal commercial types like logs, poles, sawn lumber and particle
forms, which are made from tree species susceptible to PWN. Since PWN J III needs to be kept in its
natural physiological state during testing all tests must be made with nematodes in situ in wood.
The nematode densities should reflect a normal outbreak situation, i.e. nematode densities varying
between 100 and 20 000 ind. per gram of wood (Tab. 1).
Naturally infested pinewood from areas where the pine wood nematode occurs may be used. Normal
densities in material so far used in treatment tests have been 100-200 ind. per gram wood (Soma et
al. 2002, 2003, 2005, 2006). If naturally infested wood is not available, nematodes from cultures of
Botrytis cinerea may easily be inoculated and multiplied in fresh stems (Hisai et. al. 2004).
Nematodes will increase rapidly in numbers (Schroeder pers.comm.), and as the wood dries the
frequency of JIII will increase.
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Technical protocol for testing nematodes
2009-TPFQ-39 Rev. 1
Agenda Item 6.4
To be able to meet the probit 9 criteria it is important to select material with high enough initial
densities of nematodes, so experimental units should contain >100 000 nematodes before
treatment. Considering the densities recorded in outbreak situations or reached in log inoculations
this requirement can easily be fulfilled. For the dimensions used by Soma et al. (2005) the boards
(3x15x45 cm) and the lumber (15x15x30-40 cm) can be calculated that the boards would have
contained pre-treatment nematode densities of at least 168 000 and 560 000 – 750 000 nematodes
per unit.
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The wood for testing should be of normal commercial types and dimensions
Nematodes kept in their natural physiological state by “in situ in wood”-experiments
Nematode densities should be 100-200 ind. per gram wood (dw)
Each test unit should contain > 100 000 nematodes
Statistical considerations:
The number and size of samples, taken from the experimental units need to consider the variation
in nematode numbers caused by their clumped distribution in wood. In some instances whole units
may need to be investigated. Such samples may be used in an experiment to compare the survival of
nematodes in treated and untreated samples.
Another possibility may be to obtain representative samples to fit a reasonable statistical model
describing the evolution of (the number of) nematodes in wood. Based on the fitted model we must
be able to estimate the survival rate of nematodes, both with and without the actual treatments.
The properties of the model must be described and evaluated, and the uncertainty in all the
estimates must be stated.
Verification of nematode survival:
Samples (wood shavings) can be obtained by using a drilling machine with a spiral bit (Ø=17 mm)
operated at low speed to avoid frictional heat, which could kill the nematodes. Since small
population levels may be below the level of detection, samples need to be incubated for 6 and 21
days at +25oC before extraction. Nematode survival is investigated by the Baermann funnel
technique. Ideally funnels should be suitable for extracting larger than normal wood samples
(Fig.2). The outlet of the funnel is fitted by a plastic tubing closed by a metal clip. BY opening the
metal clip the water containing the nematodes can be collected in a beaker. In one hour the
nematodes have settled at the bottom of the beaker. After removing the supernatant, the sediment
can be adjusted to a known volume, which can be stirred and subsampled. Nematodes in the
subsample can be counted in a counting dish under the stereo-microscope by personnel with
competence in distinguishing PWN from other nematodes.
Expert competence:
Although, the Diagnostic protocol of Bursaphelenchus xylophilus is helpful in the diagnostic work,
expert nematological competence and adequate microscopic equipment is needed for the correct
assessment of the frequency of PWN and the JIII stage in the test material. It is a great advantage to
consult statistical expertise early in planning the experiment.
Acknowledgements:
Dr. Torfinn Torp of Bioforsk was helpful in discussions on statistics.
References:
FAO 2009. International standards for phytosanitary measures (ISPM) No. 28. Phytosanitary
treatments for regulated pests (2009): 1-10.
Hisai, J., Hirata, K. & Suzuki, K. 2004. Investigation of multiplication of inoculated pine wood
nematode (Bursaphelenchus xylophilus) inside red pine log (Pinus densiflora). RES. BULL. PL.
PROT. JAPAN 40: 79-82.
Ishibashi, N. & Kondo, E. 1977. Occurrence and survival of the dispersal forms of pine wood
nematode, Bursaphelenchus linicolus Mamiya and Kiyohara. Appl. Ent. Zool. 12: 293-302.
Kiyohara, T. & Suzuki, K. 1978. Nematode population growth and disease development in the
pine wilting disease. Eur. J. For. Path. 8: 285-292.
Kondo, E. & Ishibashi N. 1978. Ultrastructural differences between the propagative and dispersal
forms in the pine wood nematode Bursaphelenchus lignicolus, with reference to the
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Technical protocol for testing nematodes
2009-TPFQ-39 Rev. 1
Agenda Item 6.4
survival. Appl. Ent. Zool. 13:1-11.
Magnusson, C., Thunes, K.H., Nyeggen, H., Overgaard, H., Rafoss, T., Haukeland, S., Brurberg,
M.B., Rasmussen, I., Strandenæs, K-A., Økland, B. & Hammeraas, B. (2007). Surveillance of Pine
Wood Nematode (PWN) Bursaphelenchus xylophilus – Norwegian Surveys 2000-2006. Bioforsk
Report 2 (104): 22 pp + V.
Mamiya, Y. 1976. Pine Wilting Disease Caused by the Pine Wood Nematode, Bursaphelenchus
Lignicolus, In Japan. JARQ 10: 208-211.
Mamiya, Y. 1984. The pine wood nematode. In: Nickle, W.R. (ed.). Plant and Insect Nematodes.
Marcel Dekker Inc.: 589-626.
Penas A.C, Bravo M.A, Valadas V. & Mota M. 2008. Detailed morphobiometric studies of
Bursaphelenchus xylophilus and characterisation of other Bursaphelenchus species (Nematoda:
Parasitaphelenchidae) associated with Pinus pinaster in Portugal. J. Nematode Morphol. Syst.
10, 137-163.
Soma, Y., Goto, M., Ogawa, N., Naito, H. & Hirata, K. 2005. Effects of Some Fumigants on
Mortality of Pine Wood Nematode, Bursaphelenchus xylophilus Infecting Wooden Packages. 5.
Mortality of Pine Wood Nematode and Fumigation Standards by Methyl Iodide. RES. BULL. PL.
PROT. JAPAN 41: 1-7.
Soma,Y., Komatsu, H, Abe, Y., Itabashi, T., Matsumoto, Y. & Kawakami, F. 2006. Effects of
Some Fumigants on Pine Wood Nematode, Bursaphelenchus xylophilus Infecting Wooden
Packages. 6. Mortality of Pine Wood Nematode and Longhorn Beetles by Methyl Iodide Tarpaulin
Fumigation. RES. BULL. PL. PROT. JAPAN 42: 7-13.
Soma, Y., Goto, M., Naito, H., Ogawa, N., Kawakami, F., Hirata, K., Komatsu, H. & Matsumoto,
Y. 2003. Effects of Some Fumigants on Pine Wood Nematode, Bursaphelenchus
xylophilus Infecting Wooden Packages. Mortality and Fumigation Standards for Pine wood
Nematode by Methyl Bromide. RES. BULL. PL. PROT. JAPAN 39: 7-14.
Soma,Y., Naito, H., Misumi, T., Tsuchiya, Y., Mitsobuchi, M., Matsuoka, I., Kawakami, F., Hirata,
K. & Komatsu, H. 2002. Effects of Some Fumigants on Pine Wood Nematode, Bursaphelenchus
xylophilus Infecting Wooden Packages. 2. Mortality of Pine Wood Nematode by
Methyl Bromide Tent Fumigation. RES. BULL. PL. PROT. JAPAN 38: 13-19.
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Technical protocol for testing nematodes
2009-TPFQ-39 Rev. 1
Agenda Item 6.4
Table 1. Some field densities in wood of the pinewood nematode (PWN) Bursaphelenchus xylophilus
reported in the literature
TREE
Pinus
Pinus
Pinus
Pinus
Pinus
Pinus
Pinus
sylvestris
densiflora
densiflora
densiflora
densiflora
thunbergii
thunbergii
PART
branches
lumber
lumber
lumber
lumber
7 yrs
18 yrs
NOS NEM
20 0000
>100
204-1682
130-237
149-231
< 10 000
2000
BASIS
gr.dw
gr.(?)
gr.(?)
gr.(?)
gr.(?)
g.dw
g.ww
REGION
U.S.A/Illinois
Japan/Ibaraki
Japan/Ibaraki
Japan/Ibaraki
Japan/Ibaraki
Japan
Japan
REFERENCE
Malek & Appleby (1984)
Soma et al. (2005)
Soma et al. (2006)
Soma et al. (2003)
Soma et al. (2002)
Mamiya (1976)
Kiyohara & Suzuki (1978)
Figure 2. Baermann funnel technique for the extraction of pine
wood nematode (PWN) Bursaphelenchus xylophilus. Wood shavings
for extraction are placed on a paper filter supported by a metal
net in a plastic funnel (Ø=28,5 cm). Each funnel is fitted with
plastic tubing closed by a metal clip. Funnels are filled with water
and left for 48 hrs at +25 oC. A transparent plastic lid to avoid
cross contamination of samples covers each funnel.
Magnusson et al.(2007).
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