Download INFLUENCE OF BACTERIA AND TEMPERATURE ON THE

INFLUENCE
OF BACTERIA
AND TEMPERATURE
ON THE
REPRODUCTION
OF CAENORHABDITIS
ELEGANS
INFESTING
MUSHROOMS
(NEMATODA:
RHABDITIDAE)
(AGARICUS
BISPOR US)
BY
P. S. GREWAL
Entomology and Insect Pathology Section,
Institute of Horticultural Research, Worthing Road, Littlehampton,
West Sussex BN17 6LP, UK
Ten species of bacteria associated with Caenorhabditiselegans,a saprobic rhabditid nematode
infesting cultivated mushroom (Agaricusbisporus),were isolated and identified. In monogenic
var. anitratus, A. calcoaceticus
var. lwoffi,
calcoaceticus
cultures, five species of bacteria (Acinetobacter
Enterobactercloacae, Pseudomonasmaltophilia and Serratia liquefaciens)sustained the growth and
reproduction of C. elegan.vfor several generations. Bacillus cereusand Pseudomonassp. supported
growth and reproduction of the nematode, but resulted in smaller populations. E. amnigenusand
P. aeruginosacould support nematode growth and reproduction for the first 2-3 generations;
Bacillus sp. could support growth but not reproduction. The reproductive capacity of parthenogenetic female C. elegansvaried with temperature and bacterial food source. Cubic equations were fitted to the data on nematode fecundity. Temperature optima for reproduction were
estimated. Temperature significantly affected generation time of the nematode but bacterial
species had little effect. The significance of interactions between C. elegansand its associated
bacteria in mushroom culture is discussed.
Keywords:Caenorhabditiselegans,Agaricusbisporus, rhabditid nematodes, mushroom saprobes,
bacteria, temperature, reproduction.
Saprobic rhabditid nematodes are frequently found in the compost and casof the mushroom,
ing material (peat and chalk mixture) used for cultivation
with
mushroom
are
associated
Agaricus bisporus. They
poor
growing
generally
conditions and have been held responsible for low yields (Klingler & Tschierpe,
1980; Ross & Burden, 1981; Kaufman et al., 1984). However, the processes of
are poorly understood
and attempts to elucidate the role of these
pathogenesis
nematodes
as
bacteria-feeding
pests in the mushroom
industry have been
&
1966; Ingratta
Olthof, 1978; Gerrits,
equivocal (Hesling,
1980).
The pathogenicity
of such nematodes
to mushroom
is difficult to demonand
strate because
eelworms
need live bacteria
as food for maturation
reproduction
(Nicholas,
1984) and because A. bisporus requires the activity of
bacteria for its sporophore
induction
(Eger, 1972). Escherichia coli has been
a
into the biology and food
used
as
food
bacterium
for
research
widely
&
of
nematodes
1976; Anderson &
Nicholas,
dependence
free-living
(Andrew
of the bacteria
Coleman,
1981, 1982; Schiemer, 1982, 1987) but identification
73
culture
associated with the saprobic rhabditid nematodes
infesting mushroom
and of the interactions
between such nematodes
and bacteria have not been
made.
A recent survey (Grewal & Richardson,
in press) showed Caenorhabditis elegans
be
the
common
nematode
to
most
saprobic rhabditid
Dougherty
(Maupas)
associated
with mushroom
culture in the UK. It is a bacteria-feeding
herwith a rapid life cycle and high reproductive
maphrodite
capacity (Dusenbery,
1980; Wood, 1988). The present paper reports a study of the bacterial flora
associated with C. elegans and of the interactions
between C. elegans and bacteria
in laboratory
culture.
MATERIALS AND METHODS
Nematode culture. C. elegans was isolated from a sample of compost collected from
a mushroom
farm near Taunton,
extracted
.i
Somerset, UK and the nematodes
Baermann
funnel
The
the
nematodes
technique (Hooper,
using
(mostly
1986).
;l
were cultured
with associated
bacteria
at 22°C on 3 % (W/V)
juveniles)
nutrient
agar (Oxoid Ltd.) in Petri plates.
:4
Isolation and characterisation of bacteria. All the bacteria used in this study were
isolate of C. elegans. Bacterial flora associated with
isolated from the Taunton
the nematodes
were isolated by inoculating
nematodes
onto
freshly-extracted
five plates (about 20 nematodes/plate)
of nutrient agar. Bacteria from the gut
of surface-sterilized
nematodes (see below) were isolated by tissue homogenisa- ??
was diluted to 1:10,000 in a saline solution (Akhurst,
1980). The homogenate
tion (0.85 % w/v sodium chloride) and the suspension
s
plated on nutrient agar
48
h
of
After
incubation
at
26
isolates
bacteria
25°C,
plates (0.1 ml/plate).
(13
from each group) were selected on the basis of colony morphology
for further
at 25°C for 24
study. The chosen isolates were purified by 2 or 3 sub-cultures
h on nutrient agar plates, transferred
to nutrient agar slopes and stored at 4 ° C
for further testing.
The form of the colonies of each isolate was then examined.
Colony
motility, spore formation and morphology
appearance,
(Doetsch,
1981) were
noted. Gram reactions (Gregersen,
1978) and tests for both catalase (Lelliott
& Stead, 1987) and oxidase reactions (Kovacs, 1956) were made. The oxidative
and fermentative
two
ability of each isolate was determined
by inoculating
media (Hugh & Leifson, 1953). The isolates
tubes of oxidation-fermentation
were further characterised
using the appropriate
Analytical Profile Index (API
tests including
LOPAT
Products
Ltd) and a range of other confirmatory
at
4
and
41
°C
and
al.,
gelatin
liquefication
1966),
growth
(Izard et
(Lelliott et
and
citrate
utilisation
lecithinase
al.,
al., 1981),
production
(Parry et
1983)
were done.
Axenization of nematodes. For all the experiments,
gravid females were collected from 7 to 8-day-old agar cultures and placed in saline solution (0.95 %
74
W/V sodium chloride) in cavity-blocks for five minutes. The female nematodes
were washed three times with 0.1 % 'Thimerosal'
(W/V sodium ethyl mercurithiosalicylate,
Sigma Ltd) and left in sterile distilled water over-night
during which time egg-laying and hatching occurred. Newly-hatched
juveniles
were washed four times each with 'Thimerosal'
and with antibiotics
(Strep100?tl/ml and Chloramphenicol
tomycin
50jj).l/ml, Sigma Ltd) together with
alternate washes of sterile distilled water every five minutes. Sintered glass funnels (pore size 50[Lm, Sigma Ltd) were used to wash the nematodes
with the
sterilants.
Sterile juveniles were collected in a drop of sterile water and placed
on plates of 3 9lo nutrient
at 25°C for 48h and
agar; they were incubated
checked for bacterial contaminants.
Nematodes
from bacteria-free
plates were
washed with sterile distilled water and used immediately.
Reproduction of C. elegans on associated bacteria. All the species of bacteria
isolated from C. elegans were compared as food substrates supporting
nematode
Each
was
streak
inoculated
and
on
nutrient
3
9lo
reproduction.
species
grown
in
mm
100
Petri
mm
with
each
25
agar
square
equal compartplates (18
deep),
ments (Sterilin,
for 24h at 25°C.
nematode
Sterile,
UK),
second-stage
inoculated (one juvenile contained in 10{jd of sterile
juveniles were individually
distilled water/compartment)
onto the bacterial lawns, using a Pasteur pipette,
and were incubated
at 20°C (temperature
19 to 21°C in
ranges between
mushroom
&
Wood, 1985). Fifteen
compost/casing
during cropping,
Flegg
were
for
each
bacterium.
After
four, eight and twelve
compartments
prepared
the
size
of
the
nematode population
days,
(eggs, juveniles and adults) in five
of each of the ten treatments
was determined
compartments
by washing, with
distilled water, the contents of each compartment
into a separate
beaker.
were
in
and
were
fixed
formalin
solution.
nematodes
2
%
Specimens
Eggs
counted.
The data were tested for independence
of variance,
square-root
transformed
and subjected to analysis of variance (Snedecor,
1956).
Nematode fecundity. The reproductive
female C.
capacity of parthenogenetic
10,
22, 25
15,
20,
elegans was studied at a range of constant temperatures
(5,
and 28°C) in monoxenic
cultures of the three species of bacteria (species that
Bacterial
supported
vigorous reproduction
during the previous experiment).
lawns were grown at 25 ° C for 24h on 3% nutrient agar in square Petri plates
each with 25 equal compartments.
were
Sterile,
second-stage
juveniles
inoculated
onto the lawns in l0pLl sterile distilled water using a sterile Pasteur
and five replicates
pipette. One juvenile was inoculated into each compartment
of each bacterium
at each temperature
were prepared. Juveniles were allowed
to develop to adults and to lay eggs. As soon as the eggs started to hatch, the
adults were transferred,
until death, to similarly-prepared
fresh bacterial lawns
for further egg-laying.
and
hatched
Eggs
juveniles (from both batches) were
washed with distilled water into 100 ml beakers, fixed in 2% formalin solution
and counted.
75
and
Data were tested for independence
of variance, square-root
transformed
to
of
Based
on
the
of
cubic
variance.
fit,
equations
subjected
analysis
goodness
were fitted using a third degree polynomial
regression model (Draper & Smith,
1966). The cubic equation was:
Y=a+bt+ct2+dt3
and a, b, c and d are regression
Where Y = no. of eggs laid, t = temperature
as
The precision of the estimated
coefficients.
regression (R 2) was calculated
R2 = (sum of squares due to regression).(sum
of squares about mean)-1. Diffor
of cubic equations
enabled
the optimum
ferentiation
temperature
with
each
bacterium
to
be
calculated.
nematode
reproduction
Nematode generation time. The duration of a single generation
of C. elegans was
and bacterial inoculum.
studied in similar conditions
of temperature
Square
in each compartment
were used. Generation
Petri plates with one juvenile
to the time of hatching of the
time was recorded from the time of inoculation
on
As soon as egg-laying started, observations
first egg in each compartment.
five compartments
in each treatment
were recorded at 30 minute intervals
and the time recorded.
Data were tested for
until the first egg hatched
to logarithms and subjected to analysis
of variance, transformed
independence
of variance.
RESULTS
Isolation and characterisation of bacteria. Ten species of bacteria were isolated
from C. elegans immediately
after its extraction
from compost. Bacteria were
as: Acinetobacter calcoaceticus var. anitratus, A. calcoaceticus var. lwoffi,
identified
Bacillus cereus, Bacillus sp., Enterobacter amnigenus, E. cloacae, Pseudomonas
aeruginosa, P. maltophilia, Pseudomonas sp. and Serratia liquefaciens.
Reproduction of C. elegans on associated bacteria. Nematode
growth was supported by all the bacteria and adult female C. elegans were observed 3 days after
inoculation
(Fig. 1). However, reproductive
capacity varied greatly. Four days
after inoculation
most offspring (mean = 230-358) were in cultures containing
A. calcoaceticus var. anitratus, A. calcoaceticus var. lwoffi, E. amnigenus and S.
B.
liquefaciens; but only 14-75 eggs were found in compartments
containing
cereus, Bacillus sp., Pseudomonas sp. and P. aeruginosa.
Five species of bacteria (A. calcoaceticus var. anitratus, A. calcoaceticus var.
lwoffi, E. cloacae, P. maltophilia, and S. liquefaciens) supported nematode
growth
and reproduction
for several generations.
During 12 days, monoxenic cultures
of A. calcoaceticus var. anitratus, and A. calcoaceticus var. lwoffi yielded most (Fig.
1A,B).
E. amnigenus and P. aeruginosa supported nematode growth and reproduction
but multiplication
then declined or stopped (Fig. lE, G).
for 2-3 generations
C. elegans populations
increased
with B. cereus and
slightly but steadily
Pseudomonas sp. With Bacillus sp., inoculated
juveniles
grew to adults and
76
Fig. 1. Reproduction of C. elegansat 20°C on ten species of associated bacteria. Data are squareroot transformations of the mean numbers of progeny (eggs, juveniles and adults) on each
bacterium after 4, 8 and 12 days post-inoculation. A = A. calcoaceticusvar. anitratus, B = A.
= E. amnigenus, F = E. c/oaca?
ca/?oac?cuy var. lzc?offi,C = B. cereus, D = Bacillus sp., E
calcoaceticus
E=E.
G = P.
cloacae,G=P.
aeruginosa, H = P. maltophilia, I = Pseudomonassp. and J = S. liquefaciens.Bars represent L.S.D.
(p < 0.05) at 36 df for comparing bacteria.
started to lay eggs but most of them either did not hatch or the juveniles died
soon after hatching.
Nematode fecundity. When the mean yields of nematodes
from the three
bacteria were compared at a range of temperatures
(Table I), it was found that
female C. elegans laid maximum
of eggs at 15°C and minimum
numbers
numbers at 10 ° C . C. elegans did not reproduce at 5 or at 28 ° C with any of the
three bacteria and the juveniles
died 3-4 days after inoculation.
Nematode
was
fecundity
highest when A. calcoaceticus var. anitratus was present. Interaction
between temperatures
and bacterial species was significant
that the effects of temperature
on reproduction
of C.
(p < 0.05),
suggesting
differ
in
monoxenic
the
Females
laid
cultures
of
three
bacteria.
most
elegans
at
15°C
when
cultured
with
A.
calcoaceticus
var.
and
S.
eggs
lwoffi
liquefaciens,
and at 20°C when cultured with A. calcoaceticus var. anitratus (Table I).
Cubic equations were used (goodness of fit of the quadratic
model was not
of
for C. elegans
for
the
estimation
significant,
optimum temperature
p > 0.05)
77
TABLE I
Effects of temperature and bacterial food source on fecundity of C. elegans.
square-root transformations of the mean total numbers of eggs laid.
Data are
var. lwoffi, Sl = S. liquefactens.
*Aa = A. calcoaceticus
var. anitratus, Al = A. calcvaceticus
LSD (p < 0.05) at 60 d. f. :
(a) for comparing grand means = 0.211
(b) for comparing grand means with the same level of temperature = 0.48
TABLE II
Effects of temperature and bacterial food source on mean generation time (hours) of C.
elegans. Data are log. transformations of mean generation time (hours).
*Aa = A. calcoaceticusvar. anitratus, Al = A. calcoaceticusvar. lwoffi, Sl = S. ligue, faciens;
- = no growth (nematodes died 3-4 days after inoculation)
LSD (p < 0.05) at 60 d. f. :
(a) for comparing grand means = 0.014
(b) for comparing grand means with the same level of temperature = 0.032
at which maximum
fecundity (i.e. the temperature
eggs were laid)
The
estimated
bacterium
(Fig. 2).
temperature
optima were: 16.5,
for
A.
var.
A.
15.5°C
calcoaceticus
calcoaceticus var. lwoffi and
anitratus,
lines were fitted to the estimated
ciens respectively.
Regression
of the estimated
fecundity and the precision
regression
(R2) was
(Fig.
2).
with each
16.1 and
S. liquefanematode
calculated
78
Nematode generation time. Mean generation
time of C. elegans in monoxenic
cultures of three bacteria was significantly
decreased (p < 0.05) by increased
in 54.2h at
temperature
(Table II). C. elegans completed one mean generation
at
25°C, 59.6h at 22°C, 73.6h
20°C, 106.2h at 15°C and 233.3h at 10°C. The
three species of bacteria did not differ significantly (p < 0.05) in their effects on
time.
generation
Fig. 2. Fitted cubic equations and regression lines showing the effects of temperature and
bacterial food source on fecundity of C. elegans.R2 represents the precision of the estimated
regression.
DISCUSSION
Differential
nematodes
to bacterial species
responses of various rhabditid
have been observed in the past (Sohlenius,
et
1968; Tietjen
al., 1970; Andrew
& Nicholas, 1976; Anderson & Coleman,
1981). The present study showed that
all ten species of bacteria tested supported
of C.
growth and development
a
selective
bacterial
feeder.
and
nematode
the
is
not
However,
elegans
suggests
in their
when the nematodes
continued
variation
feeding monoxenically,
reproductive
capacity was observed. For instance, A. calcoaceticus var. anitratus
79
and A. calcoaceticus var. lwoffi sustained vigorous reproduction
of the nematode
for several generations,
E. amnigenus and P. aeruginosa supported
reproduction
not
for
and
C.
could
at
all
on
Bacillus sp.
2-3
only
generations
elegans
reproduce
studies of Andrew and Nicholas
The present results confirm the comparative
excellent
(1976). They found that Escherichia coli and P. fluorescens supported
and resulted in
of C. elegans for several generations
growth and reproduction
P.
adults.
and
but provery large
aeruginosa supported
growth
reproduction
than P. fluorescens. Reproduction
was less vigorous
duced smaller populations
on Bacillus subtilis. B. mycoides supported
of the
growth, but not reproduction,
nematode.
in the reproduction
of C. elegans on various species of
The differences
bacteria may be due to differential nutritive value and/or biomass of bacteria.
Apart from the inherent differences in the nutritive value of bacterial species,
the growth conditions also affect the quality of bacterial cells (Schiemer,
1982).
The choice of static populations
of bacteria rather than replenishing
cultures
with a food source may have directly affected both the quality and quantity of
and ultimately
the size of nematode
bacterial populations
populations.
after the first few
The inhibition/retardation
of C. elegans reproduction
by bacteria such as E. amnigenus and P. aeruginosa could be due to
generations
a limited food supply (Schiemer,
of
1982, 1987) and/or to accumulation
bacterial by-products
toxic to the nematodes.
Bacteria are known to produce
metabolic
& Van Duuren,
nematicidal
1957; Bergmann
products (Johnson,
inhibition
of
C.
Bacillus
elegans reproduction
by
sp. suggests
1959). Complete
the involvement
of a toxin. Ignoffo and Dropkin (1977) reported that a therwas active against
mostable
toxin from Bacillus thuringiensis (beta exotoxin)
and
redivivus.
avenae,
Aphelenchus
Meloidogyne incognita
Panagrellus
Bottjer et al.
that
B.
kurstaki
and
B.
found
(1985)
thuringiensis subsp.
thuringiensis subsp.
israelensis were toxic to eggs of the nematode,
Trichostrongylus colubriformis.
The response of C. elegans (in monoxenic
cultures of E. coli) to temperature
has been studied by Byerly et al. (1976) who reported
that egg-laying
was
The
affected
of
C.
to
by temperature.
response
elegans
temperature
significantly
is affected by the bacterial food source and the temperature
optima for maxivaries with the species of bacteria used as food. This finmum egg-production
ding has a direct relevance to mushroom culture as (i) the bacterial flora in casing material changes during cropping (Eger, 1972; Doores et al., 1987) and (ii)
temperatures
vary between 15 and 28°C during different phases of mushroom
of C. elegans populations
in
growth (Flegg & Wood, 1985). So the development
material is not only governed by temperature,
or
mushroom
compost/casing
total bacterial biomass, but also by the quality of food (i.e. the relative frequency of bacterial species).
and Coleman (1982) found that Caenorhabditis sp. isolated from a
Anderson
in monoxenic
short grass prairie in Colorado had a niche breadth of 20-30°C
isolate of C. elegans
cultures with Pseudomonas cepacia. By contrast, the Taunton
80
could not reproduce at 28 ° C. Sudhaus (1980) found that species collected from
the tropics always had higher lethal temperature
tolerance than sibling species
from temperate
The
also corroborates
the finpresent investigation
regions.
al. (1975) who reported
that C. elegans did not tolerate
dings of Lyons et
below 10°C.
temperatures
The size of C. elegans populations
by the nature of the
may be determined
bacterial flora. There are two broad sources of bacteria in mushroom
culture
which affect rhabditid
the
resident
bacterial
flora
in
nematodes;
compost
(i)
bacteria
and casing material (Hayes et al., 1969; Eger, 1972); (ii) 'foreign'
in or on the body of nematodes
which are probably
introduced
by flies,
from rotting vegetable matter. As sources
especially fungus gnats (Sciaridae),
material
of
of casing
(especially
peat) and of fly infestations differ, the bacterial
flora at each farm may be different and so may have differential
effects on
nematode
This may determine
the effects of saprobes
on
populations.
mushroom
reports on
yield and, in part, explain why there are conflicting
whether or not rhabditid
nematodes
are pests in the mushroom
industry.
Interactions
between
and mushroom
are
bacteria/nematodes
mycelium
et
A.
creates
a
bacteriostatic
environment
al., 1969;
complex.
(Hayes
bisporus
Eger, 1972; Barron, 1988) and may impart selectivity on bacterial populations
in the compost and/or the casing material.
the quantity
This may determine
and quality of bacterial populations
in compost or casing and ultimately
the
size of the populations
of bacteria-feeding
rhabditid nematodes.
Furthermore,
saprobic rhabditid nematodes are known to encourage bacterial populations
by
in the form of their excretory
nutrition
(i) providing
products and/or dead
nematode tissues (Novogrudsky,
1948; Ingham et al., 1985; Schiemer,
1987),
bacteria in the substrate
and consequently
and (ii) by spreading
providing
them with fresh and new food resources (Cayrol & B'Chir,
1972; Griffiths,
1986; Poinar & Hansen,
1986).
I thank Mr Rodney Edmondson
for statistical advice, Mr Paul Richardson
for supervision
of Vice-Chancellors
and the Committee
and Principals
of
British Universities
for an Overseas Research Student Award.
RÉSUMÉ
Influencedes bactérieset de la températuresur la reproductionde Caenorhabditis elegans (Nematoda:
Rhabditidae)infestantle champignonde couche(Agaricus bisporus)
Dix espèces de bactéries associées à Caenorhabditiselegans- un nématodes Rhabditide saprobionte infestant le champignon de couche (Agaricusbisporus)- ont été isolées et identifiées. En
cultures monospécifiques, cinq espèces de bactéries (Acinetobacter
calcoaceticus
var. anitratus, A. calcoaceticusvar. lwoffi, Enterobactercloacae,Pseudomonasmaltophiliaet Serratia liquefaciens)permettent
la croissance et la reproduction de C. eleganspendant plusieurs générations. Bacilluscereuset Pseudomonassp. permettent la croissance et la reproduction des nématodes mais conduisent à des
populations plus faibles. E. amnigenuset P. aeruginosapeuvent permettre la croissance et la reproduction pendant les 2 ou 3 premières générations; Bacillussp. peut permettre la croissance, mais
non la reproduction. Le pouvoir reproducteur de la femelle parthénogénétique de C. elegans
81
varie avec la température et la source bactérienne de nourriture. Les équations volumétriques
ont été ajustées aux données de la fécondité du nématode. Les températures optimales ont été
précisées. La température influence significativement la durée d'une génération du nématode
mais l'espèce de la bactérie n'a qu'une faible influence. La signification des relations entre C.
eleganset ses bactéries associées dans les cultures de champignon de couche est discutée.
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