Harmey, M.A.

Lawler, C.

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Fundam. appl. NemalOl., 1993, 16 (6), 521-523
Immunological detection of nematode antigens
on the surface of a wood section
Colm LAWLER and Matthew A. HARMEY
Department of Botan)', University College Dublin, Beljield, Dublin 4, Ireland.
Accepted for publication 8 March 1993.
Summary - Antigens of Bursaphelenchus xylophilus and B. mUCTonalllS were detected on a debarked and kiln-dried Pinus sylvestris
section. The antigens were examined using a polyclonal antibody developed against B. xylophilus. The antibody could detect the
nematode proreins on the surface of the wood at a lower limit of 0.4 f..lg but could not discriminate between the two species
examined.
Résumé - Détection immunologique d'antigènes provenant de nématodes à la surface du bois - Des antlgenes
provenant de Bursaphelenchus xylophilus et B. mucronatus ont été détectés à la surface d'une tranche de Pinus sylvesl11s écorcée et
séchée au four. Ces antigénes ont été examinés à l'aide d'un anticorps polyclonal dirigé contre B. xylophilus. L'anticorps peut
détecter les protéines du nématode à la surface qu bois jusqu'à un taux inférieur à 0,4 f..lg, mais ne peut différencier les deux espèces
en cause.
Key-words : Immunodiagnosis, wood section, Bursaphelenchus xylophilus, Bursaphelenchus mUCTonatus.
Table 1. Nemarode popuJations used in this srudy.
nemarodes Bursaphelenchus xylophilus (see: Steiner &
Buhrer, 1934; Mamiya & Kiyohara; 1972; Nickie el al.,
1981) and Bursaphelenchus mucronalus Mamiya & Enda,
1979.
Materials and Inethods
EXTRACTION OF NEMATODE PROTEIN
B. xylophilus and B. mucronaLUs populations were
raised on 2 week-old cultures of a sterile strain of Bolry-
lis einerea. The nematodes, containing mixed life stages,
were harvested using sterile distilled water (SDW) and
were stored at - 20 oc. Nematode homogenates were
prepared by grinding the samples in liquid nitrogen,
followed by suspension in SDW. The protein concen-
tration was determined, using the Bradford assay (Brad-
ford, 1976).
Bursaphelenchus mUCTonatus
J 13 Japan-Yachiyo
Host
Pinus el/ioui
P. halapensis
P. sylvesms
P. denszflora
P. densiflora
P. thunbergii
CountryCode
Bursaphelenchus xylophilus
US4 USA-Florida
US9 USA-Arizona
US 15 USA-ruinois
J3 Japan-Nishiaizu
Jl0 Japan-Ueki
A variery of matrices are available for the immunolog-
ical detection of plant pathogens. These include nitro-
cellulose or other similar membranes used in immu-
noblotting (Towbin el al., 1979) and polysryrene or
other suitable plastics in microtitre plates used in the
Enzyme Linked Immunoabsorbent Assay or ELISA
(Harlow & Lane, 1988). These synthetic matrices have
been used in the immunodiagnosis of plant pathogenic
nematodes such as Globodera roslOchiensis and Globodera
paUida (Schots el al., 1990).
Immunological assays on natural plant surfaces such
as wood may have the potential to detect certain plant
pathogens. Tests performed on plant surfaces may how-
ever suffer interference from the presence of endoge-
nous compounds such as peroxidases which may cause
similar enzyme-linked colorimetrie reactions. The sur-
face itself may have a contrasting or unsuitable back-
ground colour which would hinder the use of colori-
metrie detection methods. If an immunological assay is
to be performed on a plant surface then the conditions
that apply ta synthetic matrices must equally apply to
this natural matrix. These include the capaciry ta absorb
or retain antigen, minimal background reaction and the
use of a subsrrate which can facilitate quantitative or
qualitative analysis.
In tlùs paper we describe the immunological detection
of nematade antigens applied on a debarked Pinus syl-
veslris wood section. The tests combine aspects of EL-
ISA and immunobloning and involved the use of micro-
waving ta fix the antigen on the wood surface. The
antigens examined were present in homogenates of
mixed life cycle stages of various populations of the
155N 1164-5571/93/06 S 4.00/ Gauthier-Vil/ars - OR5TOM 521
C. Lawler & M. A. Ha/mey
c0.20.42
B
A
F
E
o
}Jg
c
Fig. 1. Immunological test on Bursaphelenchus antigens ap-
plied omo a Pimls syives/n's wood section. The antigens were
applied in quantities of 2, 0.4 and 0.2 jJ.g and the control (C)
consisted of ID jJ.I of IX PBS. The B. xylophiius populations
examined were US4, US9, US 15, J3 and Jl0 (rows A-E, re-
spectively). The B. mucronalus population examined was J13
(row F.).
PREPARATION OF POLYCLONAL SERUM
One mg/one ml homogenates of B. xylophilus QI0) of
mixed life cycle stages were prepared using the methods
already described. These suspensions were mixed with
equal volumes of Freund's complete adjuvant (Harlow
& Lane, 1988) and injected subcutaneously into two
New Zealand White rabbits. This was repeated twice
more over a 4 week-period at 2 week-intervals. The an-
tigen concentration remained the same, but an equal
volume of Freunds adjuvant minus the Mycobaeteriurn
luberculosis bacteria or also known as Freund's incom-
piete adjuvant was used in this case. After 6 weeks the
rabbits were bled and the antibody titre was determined
using an indirect ELISA system.
lMMUNOLOGICAL TEST ON WOOD SECTION
The wood section used was 10 x 5 x 1 cm in dimen-
sion. The section was obtained from a debarked and
kiln-dried P. sylvestris log collected at Dublin port. Two
fJ-g of each nematode protein were added to the wood
surface and five and ten fold dilutions of the respective
suspensions were added to the surface. The negative
control consisted of 2 fJ-I of 1X phosphate buffered sa-
line (PBS), (lOX PBS; 1.38 M NaCI, 0.038 M KCI,
0.021 M KH2PO., 0.092 M Na2HPO.12H20, pH 7.3-
7.5). The suspensions were allowed to dry onto the
surface after which the dry wood section was then mi-
crowaved at 650 watts for 1.5 min to fix the antigen onto
the wood surface. This was followed by blocking in
PBS/0.3 %. Tween 20 for 1 h at room temperature. The
section was then incubated in 1: 1000 dilution in
PBS/0.05 %. Tween 20 (Wash Buffer) of the polyclonal
antibody raised against B. xylophilus antigen for 2 h at
room temperature. This was followed by five one min-
ute washes with wash buffer to remove any unbound
antibody. This was fo11owed by incubation in al: 1000
dilution of porcine anti-rabbit antibody conjugated to
horse radish peroxidase or HRPO (Dako-Paks), in wash
buffer for 2 h at room temperature. The section was
then washed as before and the added substrate contain-
ing 3'3'-diaminobenzidine (DAB), H 20 2, 4-chloro-
naphtol, ail in PBS, as described by Young (1989), was
used to detect the reaction. The wood section was
washed in disti11ed water to stop the reaction and irnme-
diately photographed.
For nematode populations used in this study, see Ta-
ble 1.
Results
Nematode proreins at a range of concentrations were
spotted onto the wood surface and were irnmobilised
using microwaves. These proteins were subsequently
examined using a polyclonal antibody raised against B.
xylophilus (Fig. 1). Positive areas appeared as black dots
on the pale brown surface of the wood as seen in the
reactions involving 2 fJ-g of nematode protein. The black
colour changed to a dark brown after 24 h but the colour
persisted for up to a number of weeks. The use of micro-
waving to fix the antigen onto the wood surface should
inactivate any peroxidase activity. However the activity
of unknown interfering compounds present on the wood
surface resulted in a similar background reaction to that
involving the nematode protein. The background had
varying degrees of intensity as seen in Fig. 1. The reac-
tion involving 2 fJ-g of nematode protein corresponds to
approximately 130 nematodes (unpubl.). The lower
522 Fundam. appl. NemaLOl.
limit of detection was 0.4 f.lg which represents approxi-
mately 25 nematodes.
There is no difference in the reaction involving the B.
xylophilus populations (Fig. 1, rows A, B, C, D, E) and
the B. mucronalus population (Fig. 1, row F) examined
using the polyclonal antibody. This is seen in the similar
intensity of the reaction in Fig. 1. In this case it was not
possible to differentiate the two Bursaphelenchus spp.
using the polyclonal antibody.
Discussion
As part of an attempt ro develop an immunological
test to detect B. xylophilus in timber sections the possi-
bility of performing the test directly on the timber sur-
face was investigated. Homogenised nemarodes were
used as antigen because the use of microwaving causes
intact nematodes to be Iysed and therefore internai anti-
gens as weil as cuticular antigens will be exposed to
subsequent antibody detection. However any nema-
todes which may be detected must be on or near the
wood surface. Therefore the test would not have the
capacity to detect sub-surface nematodes present in the
wood section. The application of such a test for the
detection of B. xylophilus directly on the timber would
be limited for a number of reasons. These include the
fact that the nematode is often present beneath the sur-
face and is also dispersed within the timber section in
aggregates of one ro ten, depending on the size and type
of section examined (Yik & Birchfield, 1981). Therefore
the lower limit of detection of approximately 25 nema-
rodes on the wood surface using the polyclonal antibody
would not detect the numbers which are representative
of a natural infestation.
However the use of natural plant material as the anti-
gen absorbing matrix in an inununological test, may be
exploited in the detection of certain surface pathogens.
The use of the antibodies directly on the wood surface
has a number of positive aspects and include the ability
ro detect the presence of surface pathogens without the
requirement of antigen extraction. Applications of such
a technique would include a decrease in the time and a
reduction in costs needed ro perform such a test. There-
fore after the surface pathogen has been identified by
immunological tests on synthetic matrices it could be
further confLrmed by similar tests on the plant surface
which could yield information on pathogen distribution.
In the case of the detection of B. xylophilus the nema-
rode must be present on the surface and exist in reason-
able quantities to allow detection. The positive identifi-
cation of B. xylophilus will depend on the specificity of
the antibodies involved. In the case of B. xylophilus the
use of monoclonal antibodies offers the potential to de-
Vol. 16, n° 6 - 1993
Immunological deleclion on wood surface
tect certain life stages of the various populations exam-
ined. Finally the choice of substrate is important and
must yield a colour reaction of sufficient strength and/or
contrast to be used in an enzyme linked assay. In this
case the combination of 4-clùoronaphtol and 3'3-diami-
nobenzidine along with HzOz in IX PBS yields such a
result.
Acknowledgement
The authors would like ro thank Dr. G. de Guiran, INRA,
Antibes, France, for donating the various Bursaphelenchus xy-
lophilus and B. mUC1'Onacus populations. This work was sup-
ported by E.C. Grant no. 89/3990002.
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