Plant Pathology Bulletin 17: 111-118, 2008
Effects of cranberry juice and pineapple
juice on Pratylenchus coffeae
Bie Yun Tsai
Department of Plant Pathology and Microbiology, National Taiwan University, Taipei, Taiwan
E-mail: firstname.lastname@example.org; Fax: +886-2-2363-6490
Accepted for publication: February 25, 2008
Tsai, B. Y. 2008. Effect of cranberry juice and pineapple juice on Pratylenchus coffeae. Plant Pathol.
Bull. 17: 111-118.
The nematodes killed by cranberry juice often had puffed cuticle similar to the blisters on human
skin after been scalded by boiling water. There were 75.7 % P. coffeae with puffed cuticles after 96 h
exposure to cranberry juice. None of the nematodes in the fructose or pineapple juice treatment had
puffed cuticle. Cranberry juice was very effective in killing all the developmental stages of P. coffeae.
The mortality rates were 100% for J2 and male, 97 % for female and 99 % for J3+J4 after 96 h
exposure. Dilution of cranberry juice to 1:5 rendered it ineffective in killing P. coffeae but 71.5 % of
nematodes were paralyzed after 96 h exposure. Cranberry juice remained very effective in killing P.
coffeae after boiling for 2 minutes. The canned pineapple juice was completely ineffective in killing P.
coffeae. However, hand-squeezed fresh pineapple juice caused high mortality of the nematodes.
Boiling for 2 min rendered it completely ineffective. The cranberry juice and pineapple juice have
different nematicidal principles as indicated by heat stability test and the difference in their ability to
cause puffed cuticle. The infection of mung bean roots by P. coffeae was significantly reduced by
cranberry juice and fresh pineapple juice in the growth chamber tests.
Key words: control, cranberry, juice, pineapple, Pratylenchus
Marigolds (Tagetes spp.) are well known to produce
Chemical nematicides have been the major means of
nematicidal compounds (3) and Tagetes patula has been
control for plant-parasitic nematodes, an important
demonstrated to be an effective catch crop for long-term
category of plant pests, to ensure good quality and
control of Pratylenchus penetrans in strawberry
abundance of crop production. However, pesticide residues
cultivation and the effect lasted three years, longer than the
in the vegetables, fruits, and cereals are a potential threat
effect of soil fumigation (10). Moreover, Pudasaini et al. (17)
to our health. The pollution of ground water and the
reported that culturing marigold for 105 days reduced P.
environment by pesticide residues is also of great concern.
penetrans populations at greater depths than soil
Therefore, alternative methods for the control of
fumigation. The extract of marigold was also effective in
nematodes, such as biological control, crop rotation, green
controlling Meloidogyne incognita (26). Tsay, et al. (23)
manure and biofumigation have gained increasing interests
studied Asteraceae plants and found that Gaillardia
in recent years. Moreover, the search for natural
pulchella was effective in the control of M. incognita and
nematicides has also been conducted by numerous
Rotylenchulus reniformis. Brassica residues were used to
control the citrus nematode Tylenchulus semipenetrans (24).
Zasada and Ferris (27) found that B. hirta and B. juncea meal
juice, Ocean Spray Australia Pty., Ltd.), canned pineapple
was effective against M. javanica and T. semipenetrans.
juice (100% juice, Typhone, Taiwan), fresh pineapple fruit,
Other vegetables, such as cabbage, onion, and radish, and
and Fong Leng fructose (Fonen and Fonher Enterprise Co.
spices, such as chili pepper and wasabi, have been reported
Ltd., Taiwan, 75% Sugar (90% Fructose, 5% Glucose, and
to be effective against M. javanica (21, 22). The leaves of
5% other sugar)) were purchased from the local
Magnolia grandiflora L. was reported to be effective
supermarket. Fructose (Sigma) was also used for
against the pine wood nematode Bursaphelenchus
comparison. Slices of the fresh pineapple fruit were put in
xylophilus (11). A medicinal herb, wild feverfew, Parthenium
a polyethylene bag and squeezed by hand to obtain fresh
hysterophorus, was reported to exhibit nematicidal activity
pineapple juice and used immediately for the experiments.
against M. incognita (9). The essential oil of
Chrysanthemum coronarium flowerheads showed strong
Effects of cranberry juice and pineapple juice
nematicidal activity against M. artiellia (16). Moreover, C.
on P. coffeae
coronarium was applied to the soil as a green manure to
Parallel lines, approximately 0.4 cm apart, were
control M. incognita and M. javanica on tomato roots (4).
drawn with a fine blue marking pen at the bottom of Petri
While extracts or amendments of various plants,
dishes to facilitate the counting of nematodes under
including medicinal plants (21, 9), cover crops (10), vegetables
dissecting microscope. The aliquots of 3 ml of cranberry
(24, 21, 27), trees (11), and flowers (16) have been tested for their
juice and 2 ml of distilled water were added to small Petri
nematicidal activities, there has been no report on fruits
dishes (5.7 diam.) and mixed well. Then 1 ml of nematode
and their activity against plant-parasitic nematodes, other
suspension was added to the Petri dish to make 1:1
than tomato fruits (21). However, the effects of fruits on
dilution of the Cranberry Classic and mixed well (final
animal parasites have been documented. Papaya,
concentration = 13.5% pure cranberry juice). The Petri
pineapple, and fig all have been found to have
dish was then sealed with Parafilm to avoid changing the
anthelmintic activities (2, 14, 18, 20). This paper reports the
concentration of the juice due to evaporation. Distilled
effects of cranberry juice and pineapple juice on the lesion
water was used in place of the juice for the control. To
nematode, Pratylenchus coffeae (Zimmermann) Filipjev &
make 1:5and 1:10 dilution of the Cranberry Classic (final
concentration = 4.50 and 2.45 % pure cranberry juice,
respectively), 1 ml and 0.5 ml of juice, respectively, 2 ml
MATERIALS AND METHODS
of nematode suspension and 3 ml of distilled water were
loaded to the Petri dish. Canned pineapple juice (1:2
dilution) and hand-squeezed fresh pineapple juice (1:2 and
Preparation of nematodes
1:5 dilutions) were tested the same way as the cranberry
The lesion nematode P. coffeae was originally
juice. The sealed Petri dishes were then incubated at 28
collected from the experimental station of the National
for 24 h, 48 h, and 96 h. At each sampling time, the
Taiwan University and cultured on excised tomato roots in
nematodes were counted under a dissecting microscope
Gamborg's B5 medium (GIBCOBRL, Life Technologies,
(Olympus SZ61). Those nematodes that were not moving
Inc. Grand Island, NY, U.S.A.) following surface
after touching with a nematode pick were considered dead.
sterilization with H O for 2 min. The nematodes from one-
Since there were many nematodes immobile but responded
month old culture were extracted with modified Baermann
to touch in the dilution test of cranberry juice, the
funnels, collected daily and kept at 15
. They were used
immobile nematodes were transferred to distilled water
within three days. The concentration of nematodes was
and the live and dead nematodes were counted after 24 h
adjusted to 200 nematodes/ml for the in vitro tests and
to determine % paralyzation. To examine the response of
1000 nematodes/ml for the in vivo tests.
the different developmental stages of P. coffeae, male,
female, and J2 were counted separately. The third- and
Source of juices
fourth- stage juveniles were difficult to separate under a
Cranberry juice (Cranberry Classic, 27% cranberry
dissecting microscope and were combined in the counting.
Pratylenchus controlled by cranberry and pineapple juice
In the preliminary test, the cuticle of some nematodes
number of nematodes penetrated the roots were counted
killed by cranberry juice was found to puff up at certain
under a dissecting microscope. The % infection was
places. The nematodes with and without puffed cuticle
calculated as the number of nematodes penetrating the
were counted separately for comparison. Because fructose
roots/ the number of nematodes inoculated per cup
was added in the commercially made cranberry juice, Fong
100%. There were four replicates for each treatment, and
Leng Fructose and fructose (Sigma) were also tested to
experiment was repeated twice.
determine if fructose could cause puffed cuticle. All the
processes were carried out in a lamella flow hood to avoid
fungal contamination from the air. There were four
replicates for each treatment. The experiment was repeated
The nematodes killed by cranberry juice often had
puffed cuticle (Fig. 1) similar to the blisters on human skin
scalded by boiling water. The location of puffed cuticle
Heat stability test
was variable. The head, the middle of the body and the tail
all have been observed to exhibit puffed cuticle. Usually
Cranberry juice and hand-squeezed pineapple juice
the puff was on one side of the nematode body (Fig. 1A),
were heated in a microwave oven to boil for 2 min in a
however, some puff occurred at both sides of the body
beaker covered with saran wrap. Distilled water was added
(Fig. 1B). The length of the puffed cuticle was also
to the boiled juices to maintain the original volume to keep
variable, some was short and some was longer than half of
the juices from been concentrated by evaporation. After
the length of the body. The body contents at the puffed
cooling to room temperature, the boiled juices were tested
location appeared to stick together and had a narrower
as described above. Distilled water was used in place of
appearance than the other places of the nematode body.
the juices for the control. Boiled water was used as a
The cuticles of nematodes treated with fructose (Sigma),
second control to determine if boiling could cause
Fong Leng fructose, and hand-squeezed fresh pineapple
suffocation of the nematodes due to loss of oxygen in the
juice appeared normal (Fig. 1C and 1D). Different
process of boiling. There were four replicates for each
developmental stages of P. coffeae all had puffed cuticles
treatment. The experiment was repeated twice.
(Table 1) but the male had the lowest percentages of
nematodes with puffed cuticles. There were 75.7 % P.
Protection of plant roots by the juices
coffeae with puffed cuticles after 96 h exposure to
Mung bean seeds were surface sterilized with 70%
cranberry juice. None of the nematodes in the fructose or
ethanol for 4 minutes and rinsed three times with tap
pineapple juice treatment had puffed cuticle.
water. The seeds were then sown in sterile sands in plastic
Cranberry juice was very effective in killing all stages
cups and kept at 28
in a growth chamber with 16 h
of P. coffeae (Fig. 2). There were 88 % mortality for J2,
photoperiod. The seedlings were used five days after
J3+J4, and female nematodes, and 94% mortality for male
germination. The aliquot of 1 ml of nematode suspension
nematodes after 24 h exposure. The mortality was 100%
was added to 150 g of sterile sands in a plastic cup (7 cm
for J2 and male, 97 % for female and 99 % for J3+J4 after
diam. at top, 4 cm diam. at bottom), along with 30 ml of
96 h exposure. Since the sensitivity of different stages of P.
cranberry juice (1:1, 1:2, and 1: 5 dilution, vol/vol) or
coffeae to cranberry juice was very similar, data of
fresh pineapple juice made in a blender at 1:2 or 1:5
different stages were pooled in the other experiments.
dilution (w/v). Tap water was used in place of the juice for
Dilution of cranberry juice to 1:5 rendered it
the control. The cups were then sealed with saran wrap and
ineffective in killing P. coffeae but 71.5 % of nematodes
rubber band to prevent evaporation. They were incubated
were paralyzed after 96 h exposure (Fig. 3). There were
in a growth chamber for four days. After the
30.7 % nematodes paralyzed at the dilution of 1:10 after
incubation, saran wrap was removed and the mung bean
96 h exposure.
seedlings were each transplanted to a cup. They were
Cranberry juice remained very effective in killing P.
returned to the growth chamber and kept for three days.
coffeae (Fig. 4) after boiling for 2 minutes. Boiled water
Then the roots were stained with acid-fuchsin (6). The
control showed that boiling did not cause suffocation of
possessed nematicidal activities against P. coffeae. It was
The canned pineapple juice was completely
very effective against all the developmental stages of P.
ineffective in killing P. coffeae (Table 2). However, hand-
coffeae (Fig. 2). Since different stages of P. coffeae exist in
squeezed fresh pineapple juice caused high mortality of
soil, it is important to have an understanding on the
the nematodes. There were 89.5 % of the nematodes killed
response of different stages to a control strategy. Although
by hand-squeezed fresh pineapple juice after 96 h
the mortality of males was significantly higher than the
exposure. Dilution to 1:5 significantly lowered its
other stages after 24 h exposure, there was no significant
effectiveness and boiling for 2 min rendered it completely
difference between the mortality of different stages at 96 h
The infection of mung bean roots by P. coffeae was
The killing of P. coffeae by cranberry juice was often
significantly reduced by cranberry juice and fresh
accompanied by puffed cuticle (Fig. 1). Although fructose
pineapple juice (Fig. 5). There was 100% reduction of
was added in the commercial cranberry juice, the results
infection in the cranberry juice (1:1 dilution) and fresh
showed that cranberry juice and not fructose was
pineapple juice (1:2 dilution) treatment. The effectiveness
responsible for causing the symptom (Table 1). Puffed
was reduced by dilution of the juices.
cuticle appeared in all developmental stages of P. coffeae.
The percentages of nematodes with puffed cuticles were
lowest in male and highest in J3+J4. It is possible that the
cuticles of different developmental stages had certain
The cranberry juice has been found to have medical
difference which affected their response to cranberry juice.
value in preventing urinary tract infection and tooth decay
The appearance of dead nematodes was considered to be
(19). The present findings showed that cranberry juice also
related to the nematicidal ingredients (13, 8). Kong, et al. (13)
Fig. 1. Cuticle of Pratylenchus coffeae. (A) Puffed cuticle on one side of the body of adult female Pratylenchus coffeae
treated with cranberry juice; (B) Puffed cuticle on both sides of the body of adult female P. coffeae treated with cranberry
juice; (C) Cuticle of P. coffeae treated with fructose; (D) Cuticle of P. coffeae treated with fresh pineapple juice.
Pratylenchus controlled by cranberry and pineapple juice
Table1. Percentages of Pratylenchus coffeae with puffed cuticle
% Nematodes with puffed cuticle 1
Cranberry 24 h
Cranberry 96 h
Fructose (Fong Leng)
Means within each row followed by different letters are significantly different at P
0.05 according to Duncan's multiple range
test after arcsine square root transformation.
Fig. 2. Mortality of different developmental stages of Pratylenchus coffeae after exposure to cranberry juice (1:1 dilution)
for 24, 48, and 96 hours. Bars with different letters within each exposure time are significantly different at P
according to Duncan's multiple range test after arcsine square root transformation.
Fig. 3. Effect of cranberry juice diluted to 1:5 and 1:10 on mixed stages of Pratylenchus coffeae. Bars with different letters
within each category are significantly different at P
0.05 according to Duncan's multiple range test after arcsine square
reported that B. xylophilus killed in the treatment with
muscle activity blocker levamisole hydrochloride appeared
to be semicircular shape and those treated with morantal
tatrate appeared to be coiled shape. Choi, et al. (8) reported
that B. xylophilus killed by onion oil exhibited an
extended, straight form. None of the above mentioned
literatures reported puffed cuticles of nematodes. A high-
molecular-weight constituent of cranberry juice was
reported to inhibit the Helicobacter pylori adhesion to
human gastric mucus (5). It was also found to have
inhibitory effect on influenza virus adhesion and
infectivity (25). The high-molecular-weight constituent
Fig. 4. Mortality of Pratylenchus coffeae treated with
boiled cranberry juice. Bars with different letters are
responsible for the inhibition of cranberry juice to bacteria
significantly different at P
0.05 according to Duncan's
and virus has not been identified. It was only reported as
multiple range test after arcsine square root
NDM (Nondialysable material). Whether the nematicidal
principle in cranberry juice responsible for causing the
puffed cuticles was the same as NDM needs further
The hand-squeezed fresh pineapple juice was also
found to be highly effective in killing P. coffeae in this
study (Table 2). Canned pineapple juice was not effective
against P. coffeae. On the contrary, the nematodes were
more active in canned pineapple juice than in the control.
The reason for it is unknown. The nematicidal effects of
pineapple juice on animal parasites was attributed to
cysteine proteinases (20). Whether the nematicidal effect of
fresh pineapple juice on plant-parasitic nematodes
discovered in the present studies is also due to cysteine
proteinases needs further research. If it is the case, then it
Fig. 5. Effect of cranberry juice (CJ 1:1, 1:2, 1:5 dilution)
is understandable that pineapple juice lost its nematicidal
and fresh pineapple juice (FPAJ 1:2, 1:5 dilution) on the
infection of Pratylenchus coffeae on mung bean roots.
effects after boiling (Table 2), since enzymes are often heat
Bars with different letters are significantly different at P
labile. The canned pineapple juice may have lost its
0.05 according to Duncan's multiple range test after
effective ingredient in the canning process which involved
arcsine square root transformation.
heating of the juice.
Dilution of cranberry juice to 1:5 (vol/vol) rendered it
ineffective in killing P. coffeae but the nematodes were
Table2. Effect of pineapple juice on mixed stages of
paralyzed by the treatment (Fig. 3). The paralyzing effect
% Mortality 1
remained at 1:10 dilution to a lesser degree. Paralyzation
of nematodes has been reported to be useful in plant
Canned pineapple juice
protection. Extract of Crotalaria leaves was found to cause
Fresh pineapple juice (1:2 dilution)
77.6 a 89.5 a
paralyzation of the root-knot nematodes and protected
Fresh pineapple juice (1:5 dilution)
tomato roots from infection by the nematodes (12).
Boiled fresh pineapple juice (1:2 dilution)
In the heat stability test, boiled water control showed
Means within each column followed by different letters are
that boiling did not cause suffocation of the nematodes. It
significantly different at P
0.05 according to Duncan's
is possible that the oxygen carried in the nematode
multiple range test after arcsine square root transformation.
Pratylenchus controlled by cranberry and pineapple juice
suspension was sufficient for the nematodes as the
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from the air into the water. Boiling did not affect the
Nematicidal activity of onion (Allium cepa) oil and its
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components against the pine wood nematode
(Fig. 4), indicating that the nematicidal principle in
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