Table 1 contd.
PHAG
STRAIN CODES C2* C3* C4* CTl CT2 CU* RH
-----------------------------------------------------
PDDCC 2607-69 - - - - - - -
PDDCC 34496-73 + + + - - - -
NCPPB 1109 + + + - - - -
NCPPB 2979 PH + + - - - -
NCPPB 2581 + + + - - - -
NCPPB 797 + PH + - - - -
NCPPB 2980 + + + - - - -
PDDCC 2624 + + PH - - - -
NCPPB 255 + + + - - - -
NCPPB 1953 + + + - - - -
CS2 - - - + + - -
CS14
(NCPPB 35-52) - - - PH PH - -
CS29 - - - + + - -
CS30 - - - + + - -
CS31 - - - + + - -
CS34 - - - + + - -
PDDCC 2594-69 - - - - - PH -
NCPPB 2113 - - - - - + -
NCPPB 854 - - - - - + -
NCPPB 3067 - - - - - - PH
----------------------------------------------------------------
Table 1. Strains and phages used in the study. (*: phage used to
prepare the stock suspension, **: CS: Western Australian Department
of Agrlculture South Perth, Australia (Riley, 1987, Riley and
Ophel, 1992), NCPPB: National Collection of Plant Pathogenic
Bacteria, Plant Pathology Laboratory, Hatching Green, Harpenden,
Hertfordshire, UK; PDDCC: Plant Disease Division Culture
Collection, Plant Disease, D.S.I.R., Auckland, New Zealand;
PH: Propagation host; +: confluent lysis or congruent plaques; -:
no reaction).
Phage isolation and partial characterisation .
Phages were isolated to C.toxicus and purified
following the methodology described by Bradley et al. (1961)
from toxic ryegrass seeds and livestock feed. Phages utilising
other plant pathogenic coryneform bacteria (Table 1) were also
isolated from an organic mulch. Characteristics of the mulch were
previously described (Kurtboke et al., 1993b).
Host ranges of the phages were detected using 20 strains of
plant pathogenic coryneform actinomycetes and 11 strains of
C.toxicus obtained from WADA (Table 1). Details of the
strains were previously described (Riley, 1987; Riley and Ophel,
1992a).
Particle morphology was studied with a transmission electron
microscope (JEOL-2000 FX II) operated at 80 kV.
Effects of selected herbicides on conversion rates of
non-toxigenic C.toxicus isolates into toxigenic strains by
bacteriophage.
Three tentatively identified non-toxigenic C.toxicus
isolates from toxic ryegrass seeds were used to test the effect
of herbicides on the conversion of the strains into toxigenic ones.
Broth cultures of 523M medium (100 ml) were inoculated with one of
the isolates (10^8 cfu/ml) and incubated at 26 C for 2 dd as
described by Ophel et al. (1993). After 2 dd of incubation a
mixture of a phage and herbicide was added to the growing cultures.
The phages were either phiCT1 or phiCT2 (10^8 pfu/ml) and the
herbicides included Simazinet (registered trademark), (Ciba-Geigy
Australia Ltd.), Glean (registered trademark), (Dupont Australia
Ltd.), Hoegrass (registered trademark), Trifluralin (registered
trademark), (Hoechst Australia Ltd.), Sertin (registered
trademark), (Schering Australia Pty. Ltd.). Herbicide
concentrations ranged from 20 to 400 ulg/ml (Roslycky, 1982).
Controls contained the phage but no herbicides. Cultures were
further incubated for 5 dd at 25 C and aliquots (200 ul) of the
Iysates ( 10^9 pfu/ml) were plated onto 523M agar and incubated for
14 dd (Ophel et al., 1993). Colonies were grouped into types
according to the morphological appearances as described by Ophel
et al. (1993). Three replicates were used for each treatment
and results were analysed using the Student's 't' test. The
toxicity of C.toxicus isolates was tested before and after
herbicide and phage applications using the rapid technique
described by Riley and Ophel (1992b).
RESULTS
Detection, isolation and identification of C.toxicus from the
plates treated with and without phage.
On plates without phage, colonies resembling
Curtobacterium flaccumfaciens, Clavibacter michiganensis
(subspp. insidiosus, michiganensis, nebraskensis and
sepedonieus) and Bacillus spp. formed confluent
growth and the enumeration and isolation of C.toxicus was
difficult. On the other hand, as a result of the reduction in the
number of the bacteria masking the growth of C. toxicus on
the plates treated with phage, detection of the bacterium was much
easier (Table 2). Most of the tentatively identified isolates from
phage treated plates were susceptible to the phage specific to
C. toxicus and biochemical tests also confirmed that these
isolates were C. toxicus (Table 3).
Partial characterisation of C.toxicus phage.
Phages phiCT1 and phiCT2 isolated to C.toxicus
formed small circular clear plaques 1 mm in diameter on the
host species .
-------------------------------------------------------------------
Ryegrass Seeds Livestock Feed
Treatment cfu/plate cfu/g dry wt cfu/plate cfu/g dry
wt
-------------------------------------------------------------------
Control 4.2+-1.20 0.14x10^4 5.9+-1.12 0.2x10^4
(38.86+-1.50) (1.4x10^4) (33 9+-1.53) (1.13x10^4)
Phage 15.3+-1.33 0.51x10^4 11.08+-1.60 0.37x10^4
(11.2+1-.60) (0.4x10^4) (13.8+-1.45) (0.46x104)
-------------------------------------------------------------------
Table 2. number of colony forming units (cfu) of C.toxicus
and of other bacteria (in brackets) in isolation plates treated
or not with phage. (The 't test is significant at P<0.001 for
the reduction of bacteria and at P<0.01 (ryegrass) and P<0.02
(livestock feed) for the increase of C.toxicus on
isolation plates treated with phage.
-------------------------------------------------------------------
Ryegrass Seeds Livestock
Feed
Organisms Control Phage Control Phage
Clavibacter toxicus 5 25 3 18
Clavibacter spp. 11 3 11 3
Arthrobacter spp. 2 - - -
Curtobacterium spp. 1 - 1 -
Rhodococcus spp. 2 - - -
Other bacteria 9 3 15 9
Total No. of isolates 30 30 30 30
-------------------------------------------------------------------
Table 3. Distribution of isolates from plates with or without phage
(control) according to biochemical Riley and Ophel, 1992a) and
phage typing criteria. (-: none isolated).
-------------------------------------------------------------------
Negatively stained particles of phages phiCT1, and phiCT2 belonged
to Siphoviridae (B1) morphotype (Francki et al.,
1991) with icosahedral heads 52 and 50 nm in diameter
respectively. Rigid tails of the phages were 6.35 and 6.25 nm wide
and 145 and 1.40 nm long respectively (Fig. 1). Phages phiCTl and
phiCT2 were species specific and only lysed C.toxicus
strains (Table 1).
Effects of selected herbicides on the conversion rate . of
non-toxigenic C.toxicus isolates into toxigenic ones in the
presence of the bacteriophage.
Colony morphology.
Colony morphology designated as Type 2 fitted the
description of Ophel et al. (1993) and had a glassy
appearance. The colonies characterised by this morphology grew very
slowly (10-15 dd) and were resistant to phages phiCT1 and phiCT2.
They were extremely sticky and resembled melted cheese. Colonies
that grew 5-7 dd after the Iysate was plated and showed normal
morphology were designated as Type 3 following the description of
Ophel et al. (1993). These colonies were resistant to
species specific phages phiCT1 and phiCT2.
Figure 1. Morphology of phages phiCTl (a) and phiCT2 (b). Bar
= 50 nm.
Toxin production.
On the basis of treatments with and without herbicides,
all Type 2 isolates were found to be toxic. However, herbicide
treated host-phage systems yielded greater numbers of toxic Type 2
colonies in comparison with the non-herbicide treated host-phage
systems. This increase was observed only when high concentrations
of herbicide (200-400 ug/ml) were applied (Table 4). Increase in
colony forming units (cfu) of toxic colonies of C.toxicus
was observed with each type of the herbicide and was
independent of the type of the herbicide used (Table 4).
DISCUSSION
Although there are studies characterising the population of C.
toxicus in grain, knowledge of the ecology of this bacterium
in such substrates is still poor. This is due to the dominance of
other saprophytic bacteria on isolation plates which overgrow and
inhibit the development of slow growing C.toxicus (McKay and
Ophel, 1993). The method of preincubating samples with phage
utilising unwanted bacterial taxa on isolation plates successfully
reduced the numbers of bacteria masking the development of slow
growing C.toxicus and
hence facilitated the detection and isolation of the bacterium. The
methodology described here may be used for the rapid isolation and
culturing of the ARGT bacterium once it is detected in natural
substrates using other techniques such as Randomly Amplified
Polymorphic DNA - Polymerase Chain Reaction RAPD-PCR). Use of this
methodology would increase our knowledge of the ecology of the ARGT
bacterium and its phage in toxic fields, grain and livestock feed.
In addition it would provide information on the host-phage
interactions, essential for further exploitation of phage
susceptibility for selective isolation purposes (Kurtboke and
Williams, 1991).
-------------------------------------------------------------------
PHAGE
Herbicide Concentration
(ug/ml) phiCTl phiCT2
C.T.2 C.T.3 C.T.2 C.T.3
-------------------------------------------------------------------
Control 2 30 3 32
Simazine (R) 20 2 28 4 36
50 2 29 3 33
100 2 37 2 32
200 4 32 4 33
400 8* 24 5 34
Glean (R) 20 2 24 5 36
50 3 36 4 39
100 3 28 5 30
200 4 33 4 33
400 4 27 4 26
Hoegrass(R) 20 2 27 5 36
50 2 33 5 37
100 2 28 4 36
200 3 34 4 33
400 4 36 5 40
Trifluralin(R) 20 3 33 3 34
50 3 27 2 32
100 3 30 3 38
200 4 33 7 33
400 6* 32 8* 36
Sertin (R) 20 2 32 3 35
50 2 31 2 31
100 2 34 4 36
200 3 36 4 34
400 4 35 6 31
-------------------------------------------------------------------
Table 4. Number (cfu/plate) of colony types (C.T) 2 and 3 after
plating Iysates from the host phage systems treated with different
herbicides. Grouping according to colony morphology (Ophel et al.,
1993).(R) = registered trademark.
-------------------------------------------------------------------
The importance of phage typing for the identification of plant
pathogenic bacteria has already been stressed by Gross and Vidaver
(1979). The species specific phages isolated from two different
sources in this study facilitated the rapid tentative
identification of the ARGT bacterium. The isolated phages showed
great similarities in their morphology and resembled the phage
isolated by Riley and Gooden (1991) and Ophel et al. (1993).
The relative frequency of morphological phage types may have
significance in the detection of phage responsible for the toxin
production by C.toxicus when it is infected with a
bacteriophage since McKay and Ophel (1993) noted that the phage
isolates to C.toxicus were indistinguishable on the basis of
DNA restriction patterns.
Although further chemical tests are required to confirm the
production of the toxin, such as high-performance liquid
chromatography (Cockrum and Edgar, 1985), results obtained with the
semiquantitative method of Riley and Ophel (1992b) indicated that
herbicide application at high concentrations can increase the
numbers of toxic colonies and the frequency of toxigenic
conversion.
The capability of a temperate phage to lysogenise a sensitive
host is controlled by three factors: the genetic composition of the
phage, the genetic composition of the host, and the environment
(Gold, 1959). During the first minutes after infection with a
temperate phage lysis or lysogenisation take place. This depends on
many conditions such as cation concentration in the environment,
changes in temperature and multiplicity of infection (Gold, 1959).
These factors can also affect a carrier-type lysogeny (Gold, 1959).
However, a thorough study on the environmental parameters and their
effect on the carrier-type lysogenicity of C.toxicus phage
has not so far been conducted in Australia. Further studies in the
Southern and Western Australian grain belt would provide more
information on the effects of herbicides and of other environmental
parameters on the toxigenic conversion of C.toxicus.
ACKNOWLEDGEMENTS. Parts of this study were conducted in
collaboration with the WADA, Division of Animal Industries. I thank
Dr.S.S. Sutherland (WADA) for her cooperation and for supplying
materials and type strains used in this study. I am also grateful
to Assoc.Prof.K.Sivasithamparam at the University of Western
Australia (UWA) and Dr.R.Gilmour (WADA Plant Industries) for their
support and for allowing me to use their facilities. Mr.S.Parry
(UWA) assisted with the preparation of the electron micrographs.
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Copyright 1994 C. E. T. A.
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