Epidemiology of epiphytic Pseudomonas syringae on barley
by Dimitrios G Georgakopoulos
A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Plant
Pathology
Montana State University
© Copyright by Dimitrios G Georgakopoulos (1987)
Abstract:
Epiphytic populations of P. syringae from 24 barley cultivars and lines planted in Montana in 1986
were determined by dilution plate assay of 10-leaf samples on BCBRVB, a modified King's B selective
medium. Leaf symptoms were recorded at each sampling. P. syringae colonies were tested for ice
nucleation activity (INA) by a dropfreezing technique and the percentage of INA+ bacteria determined.
Populations were low in the beginning of the study and increased up to log 6 cfu/leaf by the end of the
growing season. Populations from some entries were consistently 100% INA+ bacteria. There was no
correlation between leaf symptoms and population levels. Significant differences in population levels
were observed among the entries. Six entries were reexamined in the field in Arizona during the winter
of 1987, and in Montana during the summer of 1987, and the differences in population levels, and
no-correlation of symptoms and population seemed to persist. The second time, populations were again
almost 100% INA+ bacteria, but the third time they were lower. An experiment on diurnal population
changes showed only small changes in a 24-hour period. Dissemination experiments included a study
of plant-to-plant dissemination and two studies of the movement of marked strains. Plant-to-plant
dissemination was studied by planting a 1:8 mixture of a high-population line with a low-population
cultivar and comparing the population of P. syringae on the "low" cultivar in the mixture with those of
the control (" low" cultivar alone). No significant differences were observed. The marked strain
dissemination studies included the creation of double marked strains by spontaneous mutation and the
inoculation with these of barley cultivars and lines. In the first study, the inoculum did not survive very
well epiphytically. In the second study, one line was inoculated with a marked INA+ strain and another
line with a 1:1 mixture of marked INA+ and INA- strains.
In both cases the inoculum survived epiphytically, and the INA- strain did not eliminate the INA+
strain, or vice-versa. The INA+ strain was disseminated short distances during sprinkler-irrigation, and
up to 70 m during rain. EPIDEMIOLOGY OF EPIPHYTIC
PSEUDOMONAS SYRINGAE
ON BARLEY
by
Dimitrios G. Georgakopoulos
A thesis submitted in partial fulfillment
of the requirements for the degree
of
Master of Science
in
Plant Pathology
MONTANA STATE UNIVERSITY
Bozeman, Montana
November 1987
ii
APPROVAL
of a thesis submitted by
Dimitrios G. Georgakopoulos
This thesis has been read by each member of the thesis committee
and has been found to be satisfactory regarding content, English
usage, format, citation, bibliographic style and consistency, and is
ready for submission to the College of Graduate Studies.
Date
Approved for
Fv-?/7
Date
Head, Major Department
Approved for the
^
9. /ff?
Graduate Dean
iii
STATEMENT OF PERMISSION TO USE
In presenting this thesis in partial fulfillment of the
requirements for a Master's degree at Montana State University, I
agree that the Library shall make it available to borrowers under
rules of the Library.
Brief quotations from this thesis are allowable
without special permission, provided that accurate acknowledgment of
source is made.
Permission for extensive quotation from, or reproduction of this
thesis may be granted by my major professor, or in his absence, by the
Dean of Libraries when, in the opinion of either, the proposed use of
the material is for scholarly purposes.
Any copying or use of the
material in this thesis for financial gain shall not be allowed
without my written permission.
Signature
Date
/2
%
—
iv
TABLE OF CONTENTS
Page
LIST OF TABLES................................................
vi
LIST OF FIGURES...............................................
ix
ABSTRACT................. ......................... '..........
xii
INTRODUCTION.....................................................
1
LITERATURE REVIEW................................................
3
MATERIALS AND METHODS.........................................
Variability in
syringae population size among
barley cultivars......................................
Plant Material........................
Planting.... .........................................
Leaf Sampling.........................
Leaf Samples Processing...........................
Bacterial Colony Identification..........
Collection of P^_ syringae Isolates....................
Analysis of Results...................................
Dirunal Population Changes.................................
Plant-to-Plant Dissemination...............................
Planting..............................................
Leaf Sampling.......................... ...............
Leaf Samples Processing...............................
Analysis of Results.................
1986 Dissemination Experiment withMarked Strains...........
Marking Procedure.....................................
Planting..............................................
Inoculum Production and Inoculations.................
Leaf Sampling...................
Leaf Samples Processing...............................
1987 Dissemination Experiment withMarkedStrains.........
Marking Procedure.....................................
Doubling Times and INA of DoubleMarked Strains.......
Planting............................. ................
Inoculum Production and Inoculations..................
Leaf Sampling.........................................
Leaf Samples Processing.........
14
1414
15
15
16
16
17
19
19
19
19
20
21
21
21
22
2424
24
25
25
26
27
28
28
V
TABLE OF CONTENTS— Continued
Page
Air Dissemination of
syringae.... ....................
Use of an Air Pump...... ................. ............
Display of Petri Dishes...............................
RESULTS..............
Variability in Pi syringae population sizes
among barley cultivars................................
Dirunal Population Changes...............................
Plant-to-Plant Dissemination.............................
1986 Experiment with Marked Strains......................
1987 Experiment with Marked Strains...........
Doubling Times............ .................... :......
Epiphytic Survival of the Marked Strains.......... .
Air Dissemination of P. syringae.........................
28
29
29
32
32
74
75
75
80
81
84
85
DISCUSSION........................
89
LITERATURE CITED...........
92
APPENDIX.... ........................................
List of Media Used
10
102
Vi
LIST OF TABLES
Table
1.
Page
List of the 24 barley lines and cultivars examined for
epiphytic populations of F_^ syringae in the field,
Bozeman, 1986........... ................ ...... ..........
15
Antibiotics and concentrations (ppm) tested for marking
isolates of P. syringae, 1986, 1987...........
23
List of
syringae isolates used in the experiments
to create antibiotic-resistant (marked) strains..........
23
Comparison of the epiphytic populations of I\ syringae.
on the 24 entries tested in the field, Bozeman, 1986.....
34
5.
P. syringae populations
on ARl,Bozeman, 1986............
34
6.
P. syringae populations
on AR2, Bozeman, 1986...........
35
7.
P. syringae populations
on AR3, Bozeman, 1986............
35
8.
P. syringae populations
on AR4, Bozeman, 1986............
36
9.
P. syringae populations
on AR5, Bozeman, 1986............
36
10.
P. syringae populations
on AR6, Bozeman, 1986............
37
syringae populations on AR7, Bozeman, 1986............
37
2.
3.
4.
11.
12.
P. syringae populations
on AR8, Bozeman, 1986............
38
13.
p. syringae populations
on AR9, Bozeman, 1986............
38
14.
p.
syringae populations
on ARID, Bozeman, 1986............
39
15.
p.
syringae populations
on ARll, Bozeman, 1986............
39
16.
P^ syringae populations
on ARl2, Bozeman, 1986............
40
17.
p.
syringae populations
on ARl3, Bozeman, 1986............
40
18.
P.
syringae populations
on ARl4, Bozeman, 1986............
41
19.
p.
syringae populations
on AR15, Bozeman, 1986............
41
vii
LIST GE TABLES— Continued
Table
Page
20.
P_L syringae populations on AR16, Bozeman, 1986...........
42
21.
P.
42
22.
P_i. syringae populations bn 222-1, Bozeman, 1986...........
43
23.
Pi syringae populations on 222-9, Bozeman, 1986..........
43
24.
P,
syringae populations on BOLD, Bozeman, 1986...........
44
25.
P . syringae populations on STEPTOE, Bozeman, 1986........
44
26.
Pi
syringae populations on KLAGES, Bozeman, 1986.........
45
27.
Pi syringae populations on CLARK, Bozeman, 1986..........
45
28.
P.
46
29.
Percentages of INA+ bacteria in the population of
Pi syringae, Bozeman, 1986................................
59
30.
P^i syringae populations on AR4, Arizona,
1987............
60
31.
P.
syringae populations on AR5, Arizona,
1987............
60
32.
P.
syringae populations on AR6, Arizona,
1987............
60
33.
Pi syringae populations on AR13, Arizona,
1987...........
61
34.
Pi syringae populations on AR15, Arizona,
1987...........
61
35.
P; syringae populations on CLARK, Arizona, 1987..........
61
36.
Percentages of INA+ bacteria in the population of
P. syringae, Arizona, 1987.,..............................
62
37.
P.
syringae populations on AR4, Bozeman,
1987............
62
38.
P. syringae populations on AR5, Bozeman,
1987............
63
39.
Pi. syringae populations on AR6, Bozeman,
1987............
64
40.
P. syringae populations on AR 13, Bozeman,
1987...........
65
41.
P. syringae populations on AR15, Bozeman,
1987...........
66
42.
P. syringae populations on CLARK, Bozeman, 1987..........
67
syringae populations on AR17, Bozeman, 1986...........
syringae populations on ERSHABET, Bozeman, 1986.......
viii
LIST OF TABLES— Continued
Table
43.
Percentages of INA+ bacteria in the population of
P. syringae, Bozeman, 1987......... ...................
44.
Comparison of tile epiphytic populations of P^ syringae
on the 6 selected entries during 1986 and 1987 in .
Arizona and Bozeman.......... '.........................
45.
Biochemical characteristics of the P. syringae isolates
collection from Bozeman, 1986..........................
46.
Diurnal change in epiphytic populations of P^ syringae
on AR13 (7/24-25/1987)................. ...............
47.
Results of Klett # versus population (log cfu/ml)
correlation. Cultures suspended in water of 7 isolates
from the 1986 collection, grown at 2loC, were used.....
48.
Results of Klett # versus population (log cfu/ml)
correlation. Liquid cultures in NBG of 4 marked strains
(1987 experiment) were used............................
49.
Doubling times of P^ syringae marked strains and
their wild type parents....................... .........
50.
Populations of
P. syringae on
' '
Populations of
AR13, Bozeman,
I
51.
52.
total, marked INA+, and marked INAAR15, Bozeman, 1987............ ........
I'
‘
■
total and marked INA+ P. syringae on
1987.... .............. ....... ..........
Dissemination of rifampicin-streptomycin marked INA+
P. syringae....... ....... ............... .............
ix
LIST OF FIGURES
Page
Figure
1.
2.
Petri dish disple
of ARl3 and AR15,
the inoculated fields
Area under the ,pc
Bozeman, 1986....,
for AR17, repetition I,
30
33
Bozeman, 1986............
• 47
Bozeman, 1986............
47
Bozeman, 1986............
48
Bozeman, 1986............
48
Bozeman, 1986............
49
Bozeman, 1986............
49
Bozeman, 1986............
50
Bozeman, 1986............
50
Bozeman, 1986............
51
, Bozeman, 1986.......... .
51
, Bozeman, 1986.......... .
52
, Bozeman, 1986.......... .
52
, Bozeman, 1986..........
53
, Bozeman, 1986.......... .
53
, Bozeman, 1986:.........
54
, Bozeman, 1986....,......
54
19.
, Bozeman, 1986..........
55
20.
I, Bozeman, 1986.........
55
X
LIST OF FIGURES— Continued
Figure
Page
21.
P^_ syringae populations
on 222^-9, Bozeman, 1986...........
56
22.
P.
on BOLD, Bozeman, 1986.........
56
23.
P . syringae populations on STEPTOE, Bozeman, .1986........
57
24.
P.
on KLAGES, Bozeman, 1986.--- .....
57
25.
P . syringae populations on CLARK,.Bozeman,- 1986..........
58
26.
P . syringae populations
on ERSHABET, Bozeman, 1986.......
58
27.
P. syringae. populations
on AR4, Bozeman, 1987............
68
28.
P. syringae populations
on AR5, Bozeman, 1987............
68
29.
P. syringae populations
on AR6, Bozeman, 1987............
69
30.
P. syringae populations
on ARl3, Bozeman, 1987......
69
31.
P. syringae populations
on AR15, Bozeman, 1987...........
70
32.
P. syringae populations
on CLARK, Bozeman, 1987..........
70
33.
p. syringae populations
Bozeman, 1986........
on 24 barley cultivars and lines,
34.
35.
36.
37.
38.
39.
40.
syringae populations
syringae populations
^l
P. syringae populations on six barley cultivars and lines,
Bozeman, 1987............................. ..............
^^
Diurnal change in epiphytic populations of P . syringae
on AR13 (7/24-25/87)............
74
Results of the first plant-to-plant dissemination
experiment..........
7^
Results of the second plant-to-plant dissemination
experiment......................
7^
Populations of marked Pjl syringae on KLAGES,
Bozeman, 1986............................................
7^
Populations of marked P\_ syringae on CLARK,
Bozeman, 1986....................
7^
Populations of marked P jl syringae on 222-1,
Bozeman, 1986.......................
7^
xi
LIST OF FIGURES— Continued
Figure
41.
Page
Populations of marked Pi syringae on 222-9,
Bozeman, 1986.....
79
.
42.
43.
Populations of total, marked INA+, and marked !NAP. syringae on AR15, Bozeman, 1987......................
86
Populations of total and marked INA+ Pi syringae on
AR13, Bozeman, 1987...........................
86
xii
ABSTRACT
Epiphytic, populations of Pl syringae from 24 barley cultivars and
lines planted in Montana in 1986 were determined by dilution plate
assay of 10-leaf samples on BCBRVB1 a modified King's B selective
medium. .Leaf symptoms were recorded at each sampling. P,;syringae
colonies were tested for ice nucleation activity (!NA) by a drop­
freezing technique ani the percentage of INA+jbacteria determined.
Populations were low in the beginning of the study and increased up to
log 6 cfu/leaf by the end of the growing season. Populations from
some entries were consistently 100% INA+ bacteria. There was ho
correlation between Iqaf symptoms and population levels. Significant
differences in population levels were observed among the entries. Six
entries were reexamined! in the field in Arizona during the winter of
1987, and in Montana ^uring the summer of 1987, and the differences in
population levels, and no-correlation of symptoms and population
seemed to persist. The second time, populations were again almost
100% INA+ bacteria, but the third time they were lower. An experiment
on diurnal population changes showed only small changes in a 24-hour
period. Dissemination experiments included a study of plant-to-plant
dissemination and two studies of the movement of marked strains.
Plant-to-plant dissemination was studied by planting a 1:8 mixture of
a high-population line with a low-population cultivar and comparing
the population of Pl syringae on the "low" cultivar in the mixture
with those of the control (" low" cultivar alone). No significant
differences were observed. The marked strain dissemination studies
included the creation of double marked strains by spontaneous mutation
and the inoculation with these of barley cultivars and lines. In the
first study, the inoculum did not survive very well epiphytically. In
the second study, one line was inoculated with a marked INA+ strain
and another line with a 1:1 mixture of marked INA+ and INA- strains.
In both cases the inoculum survived epiphytically, and the INA- strain
did not eliminate the INA+ strain, or vice-versa. The INA+ strain was
disseminated short distances during sprinkler-irrigation, and up to
70 m during rain.
I
INTRODUCTION
The ability of the bacterium Pseudomonas syringae to cause ice
crystals to form in supercooled liquids and water vapor (ice nucleation activity, INA) has led to a number of studies during recent
years.
Most focus on the relationship between INA and frost damage on
a number of crops, where the bacterium lives epiphytically.
Consider­
able controversy has arisen concerning the release of a strain of P.
syringae developed by recombinant DNA techniques for biological con­
trol of frost damage.
However, less effort has been devoted to the
possible involvement of ice nucleating bacteria in atmospheric
phenomena such as the condensation of rain and ice crystals in clouds,
thus affecting precipitation.
Ice nucleating bacteria, especially P.
syringae, are the most efficient ice nuclei in nature, active at -I to
-2°C as opposed to dust particles (-15°C) which are considered the
main source of atmospheric ice nuclei.
It has been proposed that a "bioprecipitation cycle" may exist in
nature:
ice nucleating bacteria leave the ,plant surface, enter the
atmosphere, catalyze the formation of rain in the clouds and thus
create more moisture for plant growth and more "substrate" for
bacterial growth.
The accelerating desertification process in dry
areas of the world, such as the Sahara and the Sahel in Africa, and
decrease of precipitation in South America has been attributed to
overgrazing, burning and injudicious farming practices.
An
2
explanation for this observation could be the break caused in the
"bioprecipitation cycle", because these acts destroy the vegetative
substrates of bacterial growth, resulting in. a decrease in condensa­
tion and ice nucleation in the atmosphere.
Subsequently, a drastic
reduction of precipitation occurs, accompanied by greater precipita­
tion runoff.
This occurs due to land erosion that results from the
destruction of natural plant communities.
Could the selection of
crops that support high populations of ice—nucleating bacteria affect
and even counteract this process?
The following research was done to answer two primary questions:
1.
Can barley support a high population of Pl syringae and are
there differences in the population size among cultivars?
2.
Does P. syringae enter the atmosphere from a barley field?
Barley, a major crop, in Montana, was. chosen as the model plant
because it is drought-tolerant and one of the most important crops in
arid areas of the world.
The answers to these questions may provide
additional evidence for the existence of a "bioprecipitation cycle" in
nature, and perhaps facilitate future research.
3
LITERATURE REVIEW
The leaf surface is a favorable environment for the survival and
growth of microorganisms.
Epiphytic microorganisms are microorganisms
that live and multiply on the leaf surface. ' The survival and popula­
tion dynamics of the epiphytic microflora depends on a number of
factors, such as temperature, relative humidity, water on leaves,
nutrients, host, but also interspecific interactions (parasitism,
competition, antibiotic production) (Blakeman, 1982; Hirano and Upper,
1983; Morris and Rouse, 1985).
Bacteria form a major component of the
epiphytic microflora; many are saprophytic, belonging to the genera
Erwinia, Pseudomonas, Xanthomonas, Flavobacterium, Lactobacillus,
Bacillus and many others, not identifiable at the species level
(Blakeman, 1982). Crosse in 1959 was the first to report that
phytopathogenic bacteria are a component of the microflora of
apparently healthy leaves.
He isolated Pseudomonas syringae pv.
morsprunorum in large numbers from healthy cherry leaves and stems and
suggested that these populations could provide inoculum for the infec­
tion of stems and branches.
His technique was leaf and stem washings,
a technique largely used in studies of epiphytic bacteria.
It con­
sists of shaking individual leaves, or leaves pooled in samples, or
other plant material in water, for some time, and subsequent dilution
plating.
Leaf washing is the best method for quantitative studies.
/
Populations are usually expressed in terms of log 10 of colony-forming
4
units per gram fresh or dry weight of tissue, per unit area, or per
leaf.
A concern in quantitation of epiphytic bacteria is associated
with the utilization of bulked samples.
Crosse found that epiphytic
populations of Pseudomonas syringae pv. morsprunorum vary greatly from
leaf to leaf and from branch to branch in cherry.
Hirano et al. in
1982 reported that for any given canopy at any given time, total
epiphytic bacterial populations and selected components thereof can be
described by the lognormal distribution (i.e. the logarithm of
bacterial populations on individual leaves is normally distributed).
Other methods for studying epiphytic bacteria are microscopy and
leaf imprinting.
Microscopic techniques have been useful primarily
for determination of the spatial distribution or preferential
localization of bacteria on leaf surfaces.
Leaf imprinting has been
successful in isolating or detecting a.specific component of the
epiphytic microflora.
Both methods, however, are qualitative (Hirano,
1983).
Cells of epiphytic bacteria, both saprophytic and pathogenic
adhere on the leaf surface.
Haas and Rotem (1976) inoculated cucumber
leaves with precise numbers of the pathogen Pseudomonas syringae pv.
lachrymans. One minute after inoculation, leaves were shaken for ten
minutes and bacterial populations counted with dilution plating.
They
showed that a constant proportion of bacteria (7%), independently of
inoculum concentration, were removable, the great majority being ■
adsorbed on the leaf surface.
They also showed that this adsorption
does not involve specific sites on the leaf.
In a similar study, but
using different techniques, Leben and Whitmoyer (1979) showed that not
5
only pathogenic but also saprophytic bacteria adhere on the leaves.
Preece and Wong (1981) further demonstrated that pathogens attach
themselves much more effectively to their host plants (52-92%
attachment) than to non-hosts (11-30%).
Only about 20% of saprophytic
bacteria became attached to leaf surfaces.
Mew and Kennedy in 1971
published similar results for Pseudomonas syringae pv. glycinea on
soybean leaves.
By scanning electron microscopy (Mariano and McCarter, 1985) and
leaf imprints (Luisetti and Gaignard1 1984), it was shown that
bacterial epiphytic populations are localized as microcolonies on
sites more or less hidden:
epidermal cell junctions, along veins,
around the base of trichomes, and occasionally within stomates.
It is
believed that bacterial adsorption involves the adhesive properties of
extracellular polysaccharide (Blakeman, 1982).
The environment on the leaf surface fluctuates very rapidly.
Changes can be quick and unpredictable; e.g. temperature, relative
humidity, leaf wetness, or more gradual; e.g. stage of the leaf.
Bacterial populations respond to these changes, both in number and
composition of the microbial community.
Of all factors, the most
influencing the growth and survival of microorganisms is relative
humidity (RH) at the plant surface.
Epiphytic bacterial populations
tend to increase when plants are wet (after rain, overhead irrigation,
or high RH in controlled situations).
Free water is essential for .
bacterial growth, because nutrients that affect growth are dissolved
in it.
It can also be important for the movement of epiphytic
bacteria, either by their own motility or mechanical dissemination
6
such as aerosols, or leaf runoff water (Hirano and Upper,
1983;
Khodair and Ramadan!, 1984; Blackeman, 1985).
There is little question that epiphytic phytopathogenic bacteria
provide inoculum for disease.
A general observation is that increased
inoculum results in increased disease incidence.
But quantitative
relationships such as the minimum population size required for disease
development, have been established in only a few cases;
Erwinia
amylovora and fire blight; ice nucleation-active bacteria and frost
damage; Pseudomonas syringae pv. syringae and brown spot on snap
beans; and
P. syringae pv. coronafaciens and halo blight on oats
(Hirano et al., 1981; Hirano, 1983; Lindow, 1983; Lindemann et al.,
1984).
Pseudomonas syringae is a major pathogen on many crops.
Several
studies on epiphytic Pl syringae and plant disease have been
published, both on annual and perennial crops.
The bacterium lives as
epiphyte on many species (Lindow and Upper, 1977; Lindow et al., 1978;
Lindow, 1983a) beyond its host range as a pathogen.
Epiphytic popula­
tions of P. syringae are influenced from the same factors mentioned
for all epiphytic bacteria, relative humidity and free moisture on the
leaves being the most important.
Cool temperatures seem to be the
most suitable for epiphytic growth of this bacterium. In the case of
(
P. syringae pathovars that are ice nucleation-active, frosts also
result in an increase in epiphytic populations.
Sources of inoculum
can be seeds, plant debris, dormant tissues, or weeds.
Dispersal is
also favored by moisture; rainsplash, rain and irrigation-generated
aerosols and even airborne bacteria are effective ways of
7
dissemination (Leben et al., 1970; Ercolani et al., 1974; Venette and
Kennedy, 1975; Smitley and McCarter, 1982; Gross et al., 1983; Hirano,
1983; Baca and Moore, 1984; Latorre et al., 1985; Wimalajeewa and
Fleet, 1985).
Pseudomonas syringae causes two diseases on cereals:
on oats and leaf blight on wheat and barley.
halo blight
Leaf blight was first
observed in South Dakota in 1965, on spring and winter wheat, and it
was first reported by Otta in 1972.
generally from the boot to the
On wheat, symptoms appear
early heading stage as numerous, very
small, water soaked spots on the flag leaf and oh the first and second
leaf below it.
Within 2-3 days these spots will expand and often
coalesce into large, greyish-green dessicated areas (Otta, 1974).
The
disease has been reported also in Montana (Scharen et al., 1976; Sands
et. al., 1977) and Minnesota (Sellam and Wilcoxon, 1976).
It is not
one of the major diseases of wheat and barley, but it can cause yield
losses as reported by Scharen et al., in 1976.
Leaf necrosis and the
leaf spot stage of basal glume rot of wheat, incited by Pseudomonas
atrofaciens have a similar symptomatology.
In bis 1977 article, Otta
found little, if any, difference between isolates of P^ syringae and
P. atrofaciens.
Reports indicate differences in susceptibility of
wheat cultivars to the bacterium (Otta, 1974, Sellam and Wilcoxon,
1976, Scharen et al., 1976).
However, epiphytic populations of P.
syringae did not differ significantly on seedlings of susceptible,
moderately susceptible, and resistant wheat cultivars under controlled
conditions, according to Fryda and Otta (1978).
The same authors
reported that the bacterium moved from the seed to the seedling and
8
survived on healthy leaves under greenhouse, growth chamber, and field
conditions.
These results indicate that
syringae can survive as an
epiphyte on wheat and that seedborne Pl syringae can be an important
source of inoculum.
Research on epiphytic bacteria became more important after the
discovery by Maki et al. (1974), that isolates of Pl syringae from
decaying alder leaves were found to be ice nucleation-active at very
warm (-1.8 to -3.8°C) temperatures.
Many pathovars of Pl syringae,
certain strains of Erwinia herbicola, P. fluorescens, P. viridiflava,
and Xanthomonas translucens are also ice nucleation active (Lindow et
al., 1978b; Lindow, 1983a; Kim et al., 1987).
The principle of ice nucleation is based on the fact that water
does not necessarily freeze at the melting point.
It can be
supercooled to several degrees below O0C and still be in the liquid
phase.
It will freeze only upon the presence of a suitable catalyst
for the liquid-solid phase transition.
nuclei.
These catalysts are called ice
The mechanism of ice nucleation involves the ordering of
water molecules into an ice-like lattice around a nucleus with lattice
structure similar to ice (Lindow, 1983a).
Other materials possessing
ice nucleation activity are dust particles (active below -10 to 15°C), silver iodide, used as a cloud seeding agent (-8°C), and
crystals of several organic compounds (-5°C) (Mason and Hallet,
Zettlemoyer et al., 1961; Lindow 1983a).
1957;
But ice nucleation-active
bacteria and especially Pl syringae are the most efficient ice nuclei,
active at -1.8°C.
9
The ice nucleation-active factor has been identified and purified
for P. syringae and Pl fluorescens.
It is a protein located on the
outer cell membrane, of 153kD molecular weight for Pl syringae and
ISOkD for Pl fluorecens (Wolber et al.j 1986, Corotto et al., 1986).
These two proteins have very similar structures and properties.
The
genes coding for these proteins have also been cloned in Escherichia
coli and sequenced.
The amino acid sequence predicted from the DNA
sequence consists of interlaced 8, 16, and 48-amino acid repeats (in
ascending order of fidelity).
The repeated unit is hydrophilic and.
particularly rich in serine and threonine.
The primary sequence
suggests that the protein folds into a regular structure built up from
the 48-amino acid repeat, and that this structure presents H-bending
side chains in a manner which mimics an ice lattice.
The fact that
the 48-amino acid repeat is built up from 3 less perfect 16-amino acid
repeats, which are in turn built up from two least perfect 8-amino
acid repeats, suggests that the protein structure is formed by a
hierarchy of folded domains (Orser et al., 1984; Green and Warren,
1985; Corotto et al., 1986; Wolber and Warren,- 1986).
Other reports
indicate that phospholipids are also determinants of the ice nucleation activity CKozloff et al., 1984; Govindarajan and Lindow,
1984).
In vitro cultural conditions, such as medium composition, solid
versus liquid growth medium, aeration, and growth temperature were
found to affect the ice nucleation efficiency of cells of many ice
nucleation—active strains of Pl syringae and E,.herbicola, as well as
the temperature at which ice nucleation is expressed in these cells
(Maki et al., 1974; Paulin and Luisetti, 1978; Lindow et al., 1978a,b;
10
Yankofsky et al., 1981; Lindow et al., 1981; Lindows 1983a; Hirano,
1985).
The presence of epiphytic ice nucleation-active (!NA) bacteria on
frost sensitive plants increases their sensitivity to frost damage at
temperatures.slightly below O0C.
Normally plant tissue can supercool
to -7°C without the formation of ice, but epiphytic INA bacteria
catalyze the formation of ice in, or on plant tissue, causing
mechanical disruption of cell membranes (Arny et al., 1976).
Even before the discovery of the role of INA bacteria in frost
damage, reports indicated that many diseases induced by
syringae
require, or are favored by, ice formation on plants prior to disease
development (Panagopoulos and Crosse, 1964; Weaver, 1978; Sule and
Seemuller, 1987).
As most bacteria, including P^. syringae, cannot
invade plant tissue, it is possible that Pl syringae evolved with the
capacity to predispose plant tissue to ice damage and subsequent
penetration and disease development (Lindow, 1983a).
Populations of INA Pl syringae undergo seasonal variations, as
observed for all epiphytic bacteria.
vegetative tissue.
They are usually low in young,
Colonization and survival on plants also vary with
the host (Lindow, 1985).
Hirano et al. (1984), reported large diurnal
changes (up to 2.8 log cfu/leaf) of Pl syringae populations on bean
leaflets, as well as diurnal changes in their ice nucleation activity.
The host seems to affect not only the population size but also the ice
nucleation activity and pathogenicity of Pl syringae (Gross et al.,
1984; Lindow, 1986; Baca et al., 1987).
11
After the discovery of.INA bacteria, frost damage was regarded as
a "plant disease" that can be "cured" by eliminating INA bacteria from
the plant surface.
Three strategies have been used:
application of
chemicals (bactericides and ice nucleation inhibitors), selection and
use of naturally occurring antagonistic bacteria, and use of
genetically engineered ice nucleation deficient ("ice-minus")
bacteria.
Bactericides and ice nucleation inhibitors (usually salts of
heavy metals that do not kill the bacteria but inactivate their ice
nucleation activity) provided significant frost control in
experimental applications on several crops.
It seems that they are
more effective as protectants (before bacterial populations establish
on the leaf surface),because even dead bacteria can nucleate ice
formation as long as the cell is intact (Lindow 1982, 1983b).
The degree of competition among epiphytic microorganisms on the
leaf surface is insufficient to prevent buildup of significant popula­
tions of INA bacteria.
Thus, it was attempted to select for bacteria
antagonistic to the INA ones, and alter the epiphytic microbial
community, in order to reduce the populations of INA bacteria during
periods of low temperatures, and therefore reduce the probability of
frost injury.
Antagonistic bacteria that have been tried as in vivo
competitors of INA bacteria, include non-INA strains of El herbicola,
P. fluorescens and Pl putida with variable results.
The mechanism of
antagonism seems to be site exclusion rather than production of
antimicrobial compounds (Lindow, 1981; Lindow et al., 1983a,b; Cody et
al., 1987). . ■
12
The most recent approach to prevent frost injury of plants by
application of antagonistic bacteria, concerns the use of genetically
engineered "ice-minus" Pl syringae and Pl fluorescens, with
considerable .controversy arising.about the safety of such a release in
the environment.
The proposed advantage of "ice-minus" bacteria
versus natural antagonists lies in their potential for establishment
on the leaf surface:
being near-isogenic with the wild types, they
should occupy the same sites on the leaf, use the same nutrients, and
outnumber the naturally occurring INA bacterial populations, (Lindow,
1985; Lindemann et al., 1985a; Lindemann and Suslow, 1987).
Recently
it was reported that the use of "ice-minus".bacteria reduced frost
damage on plants up to 80% (Time 11/9/87, data not published).
Recent work has shown that significant numbers of bacteria,
including species of !NA, can leave the plant surface, enter the
atmosphere, and disseminate from one point of a field to another.
Such phenomena occur not only during wet conditions (rain, overhead
irrigation) but also during dry days.
Bacterial concentrations are
higher in the atmosphere over plants than over soil, suggesting that
plant canopies constitute a major source of airborne bacteria includ­
ing INA (Lindemann et al., 1981; Lindemann et al., 1982; Andersen and
Lindow, 1985; Dow and Maki, 1985; Lindemann and Upper, 1985).
results were obtained by Bovallius et al (1978a).
Similar
The same authors
(1978b), and Mandrioli et al (1984), give evidence for long range
transport of biological particles, including bacteria, in the atmos­
phere, over distances as far as 1800 km and as high as 6 km.,
13
Earlier work indicated that biological ice nuclei in the
atmosphere originated from decomposing vegetation (Schnell and Vali5
1972; Schnell and Vali5 1973; Schnell and Vali5 1976) but these nuclei
were not further characterized or identified as bacteria.
The
demonstrated presence of microbes in the atmosphere in raindrops and
snow flakes, along with the discovery of the ice nucleating properties
of P. syringae (Maki et al., 1974), led Vali and Schnell (1976) to
suggest that INA bacteria may play a more or less important role in
atmospheric precipitation processes.
Parker (1970) reported the
presence of organic substances of biological origin in raindrops and
clouds (vitamins and other nutrients) and suggested that the clouds
might be viewed biologically, as atmospheric ecosystems having
significant numbers of functioning microorganisms.
In 1978, Maki and
Willoughby conducted successful ice nucleation experiments in
controlled cloud chambers by using freeze-dried cultures of INA P.
syringae and P^ fluorescens isolated from decomposing plant material,
water from streams and lakes, and from snow and rain.
Sands et al.
(1982) reported the isolation of INA Pl syringae from raindrops in
rainstorms at elevations from 180 to 2500 m above cropland, and
suggested that these epiphytic bacteria "are components of a cycle
involving rainfall induction, followed by enhancement of vegetation,
leading to increased production of INA bacteria".
They named this
phenomenon "bioprecipitation cycle" and suggested that the enhancement
or decrease of this cycle "may result in increased vegetation and
biomass productivity in a geographical area or decreased productivity
and desertification".
14
MATERIALS AND METHODS
Variability in P. syringae Population
Size Among Barley Cultivars
The scope of these experiments was to determine possible
differences in epiphytic population sizes of
syringae among barley
cultivars, and to select for one or more cultivars supporting high
epiphytic populations of the bacterium.
The susceptibility to
bacterial leaf blight of the plant material examined was also
investigated by recording leaf blight symptoms throughout the course
of the experiments, and correlating symptoms to populations of P.
syringae.
Plant Material
The epiphytic growth of Pl syringae was studied on 24 barley
lines and cultivars.
Twenty of these were six-row lines that
originated from a breeding program for dryland barley at the
University of Arizona, Tucson.
The other four were commonly grown
barley cultivars in Montana (Table I).
The epiphytic populations of
P. syringae were monitored on all entries during the summer of 1986
and on six selected during the winter of 1987 (Marana Agricultural
Experiment Station, Arizona) and the summer of 1987 in Bozeman.
15
Table I
List of the 24 barley lines and cultivate examined for
epiphytic populations of
syringae in the field, Bozeman,
1986.
ARl
AR2
AR3
AR4
AR5
AR6
AR7
AR8
AR9
ARlO
ARll
AR12
AR13
AR14
ARl 5
AR16
ARl 7
222-1
.222-9
BOLD
STEPTOE
KLAGES
CLARK
ERSHABET
Planting
All entries were planted in four randomized replications.
consisted of four rows, three m long and 30 cm apart.
received five g of seed planted with a cone seeder.
Plots
Each row
The seed was
previously sterilized in water at 51°C for 10 minutes.
Planting for
the 1986 Bozeman experiment was done on May 28, for the 1987 Arizona
experiment in November 1986, and for the 1987 Bozeman experiment on
May 31, 1987.
not irrigated.
The plots of the 20 dryland lines in Bozeman, 1986 were
The plots of the four "Montana" cultivars and all
plots in Bozeman, 1987 were irrigated once or twice a week by
sprinkler irrigation.
The plots in Arizona, 1987 were irrigated by
flood irrigation.
Leaf Sampling
Hirano et al. (1984) reported that epiphytic population sizes of
P. syringae on bean leaves change with the time of the day.
Thus, a
standard time of sampling (8-10 a.m.) was established in order to
minimize the possible, effect of this factor on the results.
From each
entry and replication, 5 flag and 5 lower leaves were sampled at
random with a pair of forceps sterilized in 70% ethanol.
The leaves
16
were put in a Ziploc plastic bag, transported to the laboratory and
stored in a cold room at 4°C until processing.
The time between
Sampling and processing never exceeded 2 1/2 hours.
During every
sampling, and for each entry and replication, symptoms were
recorded on the flag leaf by using a scale from 0-5.
Leaf Samples Processing
In every plastic bag containing 10 leaves, 50 ml of sterile
distilled water were added.
The bag was shaken briefly by hand, left
for 15 minutes, and then shaken again.
Three to five tenfold serial
dilutions were performed by using an automatic pipette (Pipetman), and
plastic sterile pipette tips.
From each dilution, 0.1 ml was plated
on a BCBRVB plate (Sands, et al., 1980) a modified King's B (King,
1954) selective medium, which mainly allows the growth of fluorescent
pseudomonads.
Bacterial Colony Identification
Plates were incubated for five days in the dark at 21°C.
Then,
for each leaf sample (entry and replication) the number of colonies
that produced a fluorescent pigment under long wave ultraviolet light
was counted, at the plate and dilution where colonies grew normally,
and expressed their typical characteristics.
At least 20% of the
colonies of that plate were tested for oxidase reaction and ice
nucleation activity (INA).
Fluorescent and oxidase-negative colonies
were initially characterized as P. syringae-1ike (Palleroni, 1984;
Sands, et al., 1970, 1980).
colonies
From the number of P^ syringae-like
at a dilution, the number of P^ syringae-like colony forming
17
units (cfu) per leaf was calculated.
The INA of the colonies was
tested with a variation of Lindow's drop-freezing technique with an
aluminum foil "boat" (Lindow et al., 1978a).
A piece of aluminum
foil was pressed against the surface of an ELISA plate, sprayed with
an inert paraffin (Pledge, SiC. Johnson and Son, Inc.) and wiped with
a piece of tissue paper, in order to create uniform indentations and a
hydrophobic surface.
A 0.03 ml sterile distilled water droplet was
placed in each indentation with an automatic pipette and sterile
pipette tips.
One droplet per colony was inoculated with a P.
syringae-like colony, until it became cloudy (concentration of
bacteria 10^-10^/ml).
A few droplets were not inoculated.
The
aluminum foil "boat" was placed on a liquid (water-ethylene glycol
1:1) circulating cooling bath (model RM 20, Brinkmann Co.) set at -4°
C.
After 5 minutes the number of frozen inoculated droplets were
recorded.
The solid or liquid state of the droplets was determined
visually and physically by touching with a bacteriological loop..
For
each plate tested, the percentage of INA positive (!NA+) colonies was
calculated.
Thus, the percentage of epiphytic INA+ P^ syringae-like
bacteria for each leaf sample (entry and replication) was
determined.
'
Collection of P. syringae Isolates
From the 1986 population study, 48 colonies from all entries
were purified by streaking on King's B medium.
After 5. days of
incubation in the dark at 21°C, they were tested for fluorescent
pigment production, oxidase activity, as previously, and for !NA.
For
.18.
the latter, 10 single colonies were tested, per isolate, with the
aluminum "boat" technique.
Test tubes, containing 4 ml of Kings' B
broth were inoculated with one single colony each.
After three days
of incubation in the dark at 21°C, 2 ‘ml of an 80% solution of glycerol
in sterile water was added in each tube and the tubes were stored in
the freezer at -IO0C.
All isolates were tested for arginine dehydrolase activity
(Thornley, 1960), hypersensitivity in tobacco leaves (Klement, 1963)
and utilization of alpha-ketoglutarate and D (-)tartrate, from 2-day
old cultures at 28°C in the dark.
For the first test, cultures were
stabbed into tubes of Thorley's medium. 2A, plugged with a layer of
sterile mineral oil, and incubated for three days at 28°C.
syringae gives a negative reaction to this test.
P.
An oxidase-positive,
fluorescent saprophytic Pseudomonas sp. was used as a positive con­
trol.
The second test consists of injecting an aqueous suspension of
bacteria into the intercellular space of a tobacco leaf cv. Burley
with a 26 1/2 gauge needle and syringe.
The same oxidaserpositive
Pseudomonas sp. was injected as a negative control.
collapse of the tissue) were recorded after 24 hours.
Results (complete
The third and
fourth tests.consist of streaking aqtieous bacterial suspensions on
plates of Ayers' medium, supplemented with,D (-)tartrate and alphaketoglutarate (Ayers et al.» 1919).
Results were recorded after 3, 7,
arid 14 days. . Positive tests were repeated once.
As controls,
bactefial suspensions were streaked.on plates of Ayers' medium alone,
and Ayers' medium supplemented with glucose.
=V
■■■.=■■■■■. -
"
■
P. syringae utilizes .
19
alpha-ketoglutarate, but not D (-)tartrate (Palleroni, 1984, Sands et
al., 1970, 1980).
Analysis of Results
For every entry and replication in all experiments, the mean
population was determined as the area under the population curve,
divided by the total time of sampling, in days (Figure 2, see
Results).
Population values were converted to logarithmic.
The
statistical analysis was performed by using the AVMF mode of the
MSUSTAT program.
Diurnal Population Changes
Leaf samples were taken every four hours from AR13 in four
repetitions starting at 8 a.m. on July 24, 1987 and ending at 8 a.m.
on July 25, 1987.
as previously.
Sampling, plating, and incubation, were performed
Colony identification was performed by fluorescence
and oxidase reaction.
Plant-to-Plant Dissemination
The scope of this experiment was to determine if Pl syringae
moves from plant to plant.
Planting
A 1:8 mixture of the entries ARl3 and CLARK was planted in 1987.
The plot consisted of 40 rows, three m long and 30 cm apart, planted
with a cone seeder.
Each row received five g of seed mixture.
As
20
control plots of AR13 and Clark (planted separately), the same plots
for the epiphytic populations study were used.
The seed was
previously sterilized in water at 51°C for 10 minutes.
The reasons
for choosing AR13 and Clark were the substantial difference in the
mean populations of Pl syringae that they supported during the 1986
experiment, and the difference in appearance:
AR13 is an early, high-
population, six-row line while Clark is a later, low-population, tworow cultivar.
Also, AR13 has wider leaves and fewer tillers, while
Clark has narrower leaves and more tillers.
Leaf,Sampling
Leaf samples were taken from 8-10 a.m.
From the plot planted
with the seed mixture, four plants of AR13 were chosen at random and,
pulled out.
Their leaves then were cut with a pair of forceps
sterilized in 70% ethanol, counted, and put in a Ziploc plastic bag.
The leaves of the two Clark plants that were flanking each ARl3 plant
were sampled in the same way.
As controls, the leaves of four plants
of AR13 and four plants of Clark from the control plots, chosen at
random, were sampled.
The samples were transported in the laboratory,
stored in a cold room at 4°C, and processed within 2.1/2 hours.
The
experiment was performed twice.
.Leaf Samples Processing
In every plastic bag containing one sample, 100 ml of sterile
distilled water were added.
Serial dilutions, plating, incubation of
the plates and colony identification were performed as in the
experiment on diurnal population changes.
21
Analysis of Results
For each plant sampled, the population of Pl syringae per leaf
was determined, since the number of leaves per plant was recorded.
The population values were transformed to logarithmic, and the popula­
tions on the Clark plants from the treatment plot were compared with
the populations on the Clark plants from the control plots.
1986 Dissemination Experiment with Marked Strains
The scope of this experiment was to create strains of Pl syringae
resistant to two antibiotics (double-marked) and to test their ability
to survive epiphytically in the field.
Marking Procedure
The procedure to create double-marked strains of Pl syringae was
performed in three rounds:
1st Round: The selection for marked strains was performed with
the "disk" method.
A sterile filter paper disk, 1/2 inch in diameter
(Schleicher and Schuell, Inc.) was immersed in a filter-sterilized
solution of an antibiotic and placed in the middle of a Petri dish
containing King's B medium, plated with a suspension of a Pl syringae
strain (from 24-hour culture on King's.B slants at 21°C).
The plates
were incubated at 21°C in the dark for five days and spontaneous
antibiotic-resistant mutants appeared as single colonies in the zone
of inhibition around the paper disk.
The antibiotics used were
rifampicin (0.1, I, 10, 100, 1000 ppm), erythromycin (10, 100, 1000
22
ppm), and streptomycin (10, 100, 1000 ppm) (Table 2).
isolates of
Sixteen
2nd Round:
syringae were used in this round (Table 3).
Colonies selected from the 1st.round were suspended
in sterile distilled water and plated on Petri dishes containing
King's B medium, amended with 1000 ppm rifampicin, or 1000 ppm
streptomycin, in order to select for resistant strains to these high
concentrations of the antibiotics.
The plates were incubated in the
dark at 21°C for five days.
3rd Round;
In this round, the double-marking was attempted:
selection of strains resistant to two different antibiotics.
the
Colonies
selected from the 2nd round were again suspended in sterile distilled
water and plated on Petri dishes containing King's B medium amended
with one of the following:
rifampicin (1000 ppm), streptomycin (1000
ppm), tobramycin (100, 500 ppm), tetracycline (100, 500 ppm),
trimethoprim (50, 100, 500 ppm), kasugamycin (50, 100, 500 ppm), and
novobiocin (50, 100, 500 ppm).
They were incubated in the dark at
21°C for five days.
Planting
Four entries (Klages, Clark, 222-1, and 222-9) were planted in a
field of approximately 0.4 hectares
west of Bozeman, Montana.
one for each entry.
at
the A.H. Post Research Farm,
The field was divided in four equal parts,
Planting was performed with a cone planter and
each cultivar was planted at a rate of approximately one g of seed/m.
The seed was previously sterilized in water at 51®C for 10 minutes.
23
Table 2.
Antibiotics and concentrations (ppm) tested for marking
isolates of Pi syringae, 1986, 1987.
1986 ..
1987
I
I
1st
Round
("Disk"
Method)
I Streptomycin: 10,100,1000
I Erythromycin: 10,100,1000
I Rifampicin: 0.1,1,10,100,1000
I
I
11st
IStreptomycin: 500
(Round
|Rifampicin: 100
I(Plating)I
2nd
Round
(Plating)
IStreptomycin: 1000
IRifampicin: 1000
12nd
IStr.: 500-Rif.: 100
IRound
I Str.: 500-Kan.: 10
|Rif.: 100-Kan.: 10
[Double
[Marking
I (Plating)I
3rd
Round
Double
Marking
(Plating)
IStreptomycin: 1000
IRifampicin: 1000
ITobramycin: 100,500
ITetracyclin: 100,500
!Trimethoprim: 50,100,500
lKasugamycin: 50,100,500
!Novobiocin: 50,100,500
I
I
I
I
I
Table 3.
I
I
I
I
I
I
I
I
i
i
i
I
I
’
I
I
I
I
I
I
I
List of P. syringae isolates used in the experiments to
create antibiotic-resistant (marked) strains.
1986 Experiment: DGl13, DG154, DG167, DG173, DG175
DG178, DGl84, DGl87, DG198, DG201
DG205, DG206, DG214, DG218, DG219
DG260
1987 Experiment: DG100,
DG109,
DG118,
DG124,
DG130,
DG136,
DG143,
DGI49,
DG101,
DGl12,
DG119,
DGl25,
DG131,
DG138,
DG144,
DG150,
DG102
DG114
DG120
DG126
DG132
DG139
DG145
DG151
DG103,
DGl15,
DG121,
DG127,
DG133,
DG140,
DG146,
DG152,
DG104,
DGl16,
DG122,
DG128,
DG134,
DG141,
DGl47,
DG153
DG105
DG117
DG123
DG129
DG135
DG142
DG148
24
Inoculum Production and Inoculations
Test tubes containing 5 ml of a liquid medium with nutrient broth
and glycerol (hereafter abbreviated NBG) were inoculated, each one
with one double-marked strain.' They were incubated at room
temperature (25°C) in a shaker.
After 48 hours, I ml from each .
culture was pipetted in a 2-liter Erlenmeyer flask containing I I of
the same liquid medium (one culture per flask).
Flasks were put in a
shaker at room temperature, and after 24 hours all cultures were mixed
with 60 liters of distilled water (non-sterile), resulting in an
inoculum concentration of approximately I x IO6 cfu/ml (determined
with serial dilutions).
All four entries were inoculated the
evening of the same day, from 8:45-11:00 with a backpack sprayer.
Klages and Clark were at the boot stage; 222-1 and 222-9 were at the
early heading stage.
Leaf Sampling
Leaf samples were taken as in the epiphytic population study.
From each entry, 3 samples were taken from sites chosen at random
and maintained throughout the experiment.
Samples were taken in the
morning of the day of inoculation, in order to determine the
background population of Jh syringae naturally resistant to the two
antibiotics of the double-marked strains (if any).
Leaf Samples Processing
Leaf samples were processed as in the study for epiphytic
populations. .The medium used was King's B amended with 100 mg/1
25
cychloheximide (antifungal compound. Sigma Co.) and the antibiotics to
which the strains of the inoculum were resistant.
incubated for seven days at 21°C in the dark.
The plates were
Colonies of Pl syringae
were identified as in the:study.for.diurnal population changes.
1987 Dissemination Experiment with Marked Strains
The scope of this experiment was to create double marked strains
of Pl syringae, inoculate barley cultivars, and follow the
dissemination of the strains through the air (over distance).
Marking Procedure
In this experiment, the double-marking of Pl syringae isolates
was performed in two rounds:
1st Round:
■
'
The selection for marked strains was performed by
direct plating of bacterial suspensions in sterile distilled water on
Petri dishes containing King's B medium amended with rifampicin (100
ppm), or streptomycin (500 ppm), or kanamycin (10, or 20 ppm).
The
bacterial suspensions originated, from 24-hour cultures of 48
p. syringae isolates (Table 3) on King's B slants at 28 C in the dark.
The plates were incubated at 21°C
2nd Round:
in the dark for five days.
Colonies selected from the 1st round were suspended
in sterile distilled water and plated on Petri dishes with King's B
medium amended with rifampicin (100 ppm) and streptomycin (500 ppm),
or rifampicin (100 ppm) and kanamycin (10 ppm), or kanamycin (10 ppm)
and streptomycin (500 ppm) in order to.select for double-marked
strains.
The plates were incubated as previously stated (Table 2).
26
Doubling Times and INA of
Double-Marked Strains
The doubling times (DT) of the double-marked strains
were compared with the doubling times of the parental strains, in
order to select for one or more that would have DT as close as
possible to their parental strains, and thus survive better
epiphytically.
This study was performed by using a Klett-Summerson photoelectric
colorimeter with a red filter.
This instrument estimates the
bacterial concentration in a liquid culture or suspension by measuring
the optical density.
So, it was necessary to determine the regression
between bacterial concentration and Klett units.
seven strains of
In order to do this,
syringae (DG100, DG101, DG103, DG104, DG105,
DGl46, DGl48) were grown on King's B slants for 24 hours at 21 and
28°C.
Sterile distilled water suspensions of these cultures were
prepared and five-fold dilutions were performed in all.
Each dilution
was plated on King's B plates and a reading on the Klett was taken
immediately after.
The experiment was repeated in the same way by
using liquid cultures in room temperature of four double-marked
strains in NBG.
These cultures were each grown in a 500 ml conical
flask with a side-arm, special for growth rate studies (Bellco),
containing 100'ml of NBG, under constant shaking.
The doubling times of 32 double marked strains, isolated from
single colonies, and of their parental strains were calculated.
The
cultures were first grown in test tubes containing 5 ml of NBG, in
room temperature, under constant shaking.
After 48 hours, I ml from
27
each culture was pipetted into a 500 ml side-armed flask, containing
100 ml of NBG.
temperature.
The flasks were put on a wrist-action shaker, at room
Readings on the Klett colorimeter were taken every two
hours, after bacterial growth was visible ("cloudy" cultures).
The INA of all double-marked strains was tested from 48 hour
cultures on King's B medium amended with the necessary antibiotics.
Aqueous suspensions of approximately 10® cells/ml were prepared, and
eight droplets from each strain (0.03 ml) were tested with the
standard method of the aluminum foil "boat".
Planting
Two fields at the Horticultural Research Farm, Bozeman, Montana,
one 0.12, and the second 0.09 hectares (approximately) were planted,
the larger with AR13 arid the smaller with AR15, on May 31st, and June
3rd, 1987, at a rate of 5 g seed/3 m.
The seed was previously
sterilized in water at 51°C for 10 minutes.
A cone planter was used.
Sprinkler irrigation was provided once or twice a week.
Inoculum Production and Inoculations
They were performed as in the 1986 experiment with marked
strains.
AR13 was inoculated with an INA+ marked strain (inoculum
concentration 1.8 x IO7 cells/ml), and ARl5 was inoculated with a 1:1
mixture of an INA+ arid an INA- strain, carrying different markers
(inoculum concentration 2.6 x IO7 cells/ml).
irrigated prior to inoculations.
field was left uninoculated.
Both fields were
One piece, at the SW corner of every
28
Leaf Sampling
Leaf samples were taken from 8-10 a.m. from four sites, selected
in random in every field, with the standard methodology.
was also taken from the uninoculated plots.
order to determine any background
One sample
Samples were taken in
syringae population naturally
resistant to the antibiotics of the double-marked strains, as in the
1986 experiment.
Leaf Samples Processing
The leaf samples were taken in the laboratory, stored in the cold
room at A0C, and processed within 90 minutes, with the standard
methodology (addition of sterile distilled water, shaking, serial
dilutions).
Dilutions from AR13 samples were plated onto BCBRVB and
King's B amended with the marking antibiotics and cycloheximide, in
order to determine the populations of total and marked P^ syringae.
Similarly, dilutions from AR15 samples were plated onto BCBRVB and
King's B with cycloheximide the appropriate marking antibiotics for
each strain sprayed on AR15.
21°C in the dark.
The plates were incubated for 5 days at
Fluorescence, oxidase reaction and INA tests were
used to identify colonies of P^ syringae.
Air Dissemination of P. syringae
The scope of this experiment was to detect any aerial
dissemination of Pl syringae from the inoculated fields of AR13 and
AR15, especially the conditions under which this occurred, and the
distance of migration.
A total of 30 samples to detect airborne
29
bacteria, were taken from 7/29/87 until 9/4/87, by using mainly two
techniques:
Use of an Air Pump
An LVM H O electric air pump, powered from a car battery was used
to sample airborne marked Pl syringae (the INA+ strain), approximately
30 cm above the canopy level.
The output of the pump was connected to
a plastic tube carrying a Millipore filter with a 0.2-microns membrane
at the end.
Similar devices (Anderson 2000 viable airborne particles
sampler) have been used in other studies (Venette and Kennedy,
Lindemann et al., 1982).
1975,
The membrane filters were put in test tubes
containing 5 ml of sterile distilled water, sonicated for five minutes
in a ME 4.6 Ultrasonic cleaner (Mettler Electronics Corp.).
Serial
dilutions were then performed and plated on King's B amended with the
appropriate antibiotics, in order to isolate the INA+ marked strain.
Only two samples were taken with this technique.
Display of Petri Dishes
Twenty-two sites at various distances from the fields of AR13 and
AR15 were selected and King's B Petri dishes, amended with the
appropriate antibiotics (for the isolation of the INA+ marked strain)
and cycloheximide were displayed, fixed on stakes or fence posts
(Figure I).
I.
Such samples were taken under five types of conditions:
Petri dishes during the day (morning or afternoon) for up to
two hours (five samples).
1 5 (6 3 )
1 0 ( 1 0 )
1 8 ( 1
10)
1 1 ( 0 )
1 6 ( 6 2 )
1 7 ( 1 4 1 )
1 2 ( 1 0 )
1 3 ( 0 )
1 4 (1 0 )
1 9 (5 9 )
9 (0 )
N<-
6 (
2 0 (10 0 )
2 1 (8 0 )
2 2 (7 0 )
Figure I.
Petri dish display sites around the inoculated fields of AR15 and AR13.
closest distance to the fields, in m.
In parenthesis,
31
2.
Petri dishes overlayed with 10 ml of sterile distilled water,
during the day (morning or afternoon) for up to seven hours
(12 samples).
3.
Petri dishes in the evening, during and after irrigation, .for
up to two hours (five samples).
A.
Petri dishes during rain (day) for up to 10 hours (two
samples).
5.
Petri dishes, with or without water, overnight, displayed
after sunset and collected before sunrise, in order to avoid
• the effect of ultraviolet light (four samples)..
In cases where water was still present in the plate after collec­
tion, plates were transported carefully to the laboratory and dried in
the clean air hood.
This experiment was designed to study only the dissemination of
the INA+ marked strain of Eh syringae.
32
RESULTS
Variability in P. syringae Population
Sizes Among Barley Cultivars
Significant differences in the mean population of epiphytic P.
syringae were observed among the 24 entries examined, which were
classified as low, intermediate, and high, in regard to the mean P.
syringae population (Table 4).
Populations were low (log 0-3
cfu/leaf) in all entries except AR13, before heading.
An increase in
population sizes was generally observed throughout the time of the
experiment, and at the end they reached log 3-6 cfu/leaf (Figures 2,
3-26, 33, 34, Tables 5-28).
Differences among the entries were also observed in the
percentages of INA+ bacteria in the populations of P^ syringae (Table
29).
Some entries supported almost consistently 100% INA+ bacteria
(AR6, AR13) while others supported lower percentages (Steptoe, Clark).
There was no correlation between population levels and leaf
blight symptoms (r = 0.13, r^ = 0.02).
Symptoms were low (1-2 of the
symptom rating scale) and appeared mostly at the end of the growing
season.
Six entries:
AR4, AR5, AR6, AR13, AR15, and Clark were selected
for further study, in order to see if the differences in epiphytic
populations of Pl syringae would be consistent.
These
8
7
4
3
2
I
O
O
Figure 2.
10
20
dcye
30
Area under the population curve for AR17, repetition I, Bozeman, 1986.
40
lo g
5
c fu /le a f
6
LO
34
Table 4.
Comparison of the epiphytic populations of
syringae on
the 24 entries tested in the field, Bozeman 1986.
I
I
LOW
Entry
AR9
AR5*
AR8
222-1
AR17
Mean
(log cfu/leaf)
2.12
2.18
2.31
2.42
2.67
INTERMEDIATE
HIGH
I
Entry
Clark*
AR 10
222-9
AR7
BOLD
ARll
AR2
AR16
ARl 2
ARl 4
ARl
AR4*
STEPTOE
A
AB
AB
AB
ABC
|
Mean
(log cfu/leaf).
2.92
2.95
3.05
3.11
3.19
3.41
3.52
3.59
3.60
3.65
3.71
3.76
3.77
ABCD
ABCD
ABCD
ABCD
ABCD
ABCD
ABCD
ABCD
ABCD
ABCD
ABCD
ABCD
ABCD
Entry
Mean
(Log cfu/leaf)
AR3
KLAGES
AR15*
AR6*
ERSHABET
AR13* :
4.17 BCDE
4.17 BCDE
4.40 CDE
4.46 CDE
4.72
DE
5.53
E
LSD (by t, 5% Sign Level) = 1.07
*Selected for further study.
Table 5.
P^ syringae populations on ARl, 1986, Bozeman.
Date
Day
6/27
6/27
7/1
7/1
7/8
7/9
7/11
7/15
7/16
7/17
7/18
7/23
7/26
8/1
8/5
0
0
4
4
11
12
14
18
19
20
21
26
29
35
39
Repetition
I
II
III
IV
II
III
■IV
I
II
III
IV
I
II
I
II
Symptoms
0
0
0
0
0
0
0
0
0
0
I
0
0
0
0
log cfu/leaf
100
100
4.46
2.64
—
—
0.74
3.18
4.08
2.74
4.08
4.60
4.08
4.23
5.23
6.00
6.65
5.98
% INA+
■
100
100
100
0
100
67
100
100
100
100
100
100
35
Table 6.
P . syringae populations on AR2, 1986, Bozeman.
Symptoms
log cfu/leaf
% INA+
0
4.36
0
1.59
2.52
3.34
3.11
3.00
2.70
2.59
3.83
3.52
4.74
5.81
5.11
4.81
5.40
5.90
100
—
100
67
100
100
100
100
100
100
60
80
100
100
100
100
40
Uate
Day
Repetition
6/26
6/27
6/30
7/1
7/5
7/8
7/9
7/11
7/15
7/16
7/17
7/18
7/23
7/26
7/30
7/31
8/1
8/5
0
I
4
5
9
12
13
1;
5
19
20
21
22
27
30
34
35
36
40
I
II
III.
IV
I
II
III
IV
I
II
Ill
IV
I
II
IV
III
I
III
Table 7.
P l syringae populations on AR3, 1986, Bozeman.
Date
Day
6/26
6/30
6/30
7/1
7/5
7/8
7/9
7/11
7/15
7/16
7/17
7/18
7/23
7/26
7/31
8/5
0
4
4
5
9
12
13
15
19
20
21
22
27
30
35
40.
. .
.
Repetition
I
II
III
■ IV
I
II
III
IV
I
II
III
IV
I
II
III
II
0
.0
0
0
0
0
.o
0
, 0
0
0
0
. 0
0
0
0
0
0
■
Symptoms
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
- 2
log cfu/leaf .
2.04
0
’1.34
1.59
4.32
4.48
4.08
3.70
4.86
4.57
4.70
3.45
6.18
6.45
6.26
6.70
% INA+
100
—
.
50
100
100
100
100
100
100
100
100
100
100
100
100
100
‘
36
Table 8.
Date
6/27
6/30
6/30
6/30
7/8
7/9
7/11
7/15
7/16
7/17
7/18
7/23
7/26
7/31
8/1
8/5
Table 9 .
syringae populations on AR4, 1986, Bozeman.
Day
.
0
3
3
3
11
12
14
18
19
20
21
26
29
34
35
39
Repetition
I
IT
III
IV
II
III
IV
I
II
III
IV
I
II
III
I
III
.
.Symptoms
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
log cfu/leaf
0
0
0
1.64
3.23
3.26
3.86
4.79
4.26
4.34
3.97
, 5.70
5.85
6.88
5.88
5.78
% INA+
—
—
75
100
100
100
83
100
100
100
80
100
20
100
20
P l syringae populations on AR5, 1986, Bozeman.
Date
Day
6/26
6/27
7/1
7/3
7/5
7/8
7/9
7/11
7/15
7/16
7/17
7/18
7/23
7/26
7/30
0
I
5
7
9
12
13
15
19
20
21
22
27
30
34
Repetition
Symptoms
I
II
III
IV
I
II
III
IV
I
II
III
IV
I
II
IV
0
0
0
0
0
0
0
0
0
0
0
0
0
.o
0
'
log cfu/leaf
0 .
0
0 •
1.70
0
0.74
3.89
2.45
3.04
2.34
2.89
2.83
2.93
3.70
4.70
% INA+
■
——
—
60
—
100
83
13
100
0
40
80
80
■100
. 83
37
Table 10.
syringae populations on AR6, 1986, Bozeman.
Date
Day
6/26
6/30
7/1
7/1
7/5
7/8
7/9
7/11
7/15
7/16
7/17
7/18
7/23
8/5
0
4
5
5
9
12
13
15
19
20
21
22
27
40
Repetition
Symptoms
log cfu/leaf
% INA+
I
II
III
IV
I
II
III
IV
I
II
III
IV
I
II
0
0
0
0
2.04
4.89
5.30
0
3.83
5.00
4.83
4.97
4.92
5.20
4.79
5.00
5.34
5.74
100
86
100
P
6
0
0
0
0
0
0
0
0
.
—
100
100
loo
100
100
100
100
100
100
80
syringae populations on AR7, 1986, Bozeman.
Table 11.
Date
Day
Repetition
6/26
6/27
6/27
7/1
7/5
7/8
7/9
7/11
7/15
7/16
7/17
7/18
7/23
7/26
7/30
8/1
0
I
I
5
. 9
12
13
15
19
20
21
22
27
30
34
36
I
II
III
IV
I
Il
III
IV
I
II
III
IV
I
II
IV
I
Symptoms
0
0
0
0
0
0
P
0
0
0
0
0
0
0
■ 0
0
■
log cfu/leaf
0
0
0
1.34
1.64
1.74
4.34
4.11
3.23
3.26
4.79
3.59
4.85
5.32
5.26
5.81
%
INA+
—
—
100
100
80
100
38
73
75
100
100
100
100
100
100
38
Table 12.
P l syringae populations on AR8, 1986, Bozeman.
Date
Day
6/27
6/30
7/1
7/1
7/8
7/9
7/11
7/15
7/16
7/17
7/18
7/23
7/26
7/30
7/31
8/5
0
3
4
4
11
12
14
18
19
20
21
26
29
33
34
40
Table 13.
Date
Repetition
Symptoms
I
II
III
IV
II
III
0
0
0
0
'
0
.0
0
0
0
0 .
iv
I
II
III
IV
I
II
IV
III
II
.o
•
6
0
0
0
0
log cfu/leaf
%
INA+
0
3.52
. 0
0.74
1.45
1.52
1.70
1.45
4.18
2.92
0
4.20
5.26
3.54
5.15
4.40
100—
100
100
60
100
20
17
80
—
100
63
75
100
80
P l syringae populations, on AR9, 1986, Bozeman.
Day
Repetition
Symptoms
log cfu/leaf
% INA+
. •. :
6/26
6/26
6/27
6/30
7/5
7/5
7/8
7/9
7/11
7/15
7/16
7/17
7/18
7/23
7.26
0
0
I
4
9
9
12 .
13
15
19
20
21
22
27
30
II
I
III
IV
I
II
II
III
IV
I
- II
III
IV
■ I
II
0
0
0
0
0
.0
0
0
0
0
0
0
0
I
2
...-O
2.04
0
0
1.23
0
3.28
2.52
2.23
2.34
4.45
4.18
3.23
2.70
4.48
100
—
—
67
—
100
17
40
100
100
100
100
100
50
Table 14.
P_L syringae populations on ARID, 1986, Bozeman
Date
. Day
Repetition
6/26
6/26
6/30
7/1
7/5
7/5
7/8
7/9
7/11
7/15
7/16
7/17
7/18
7/23
0
0
4
5
9
9
12
13
. 15
19
20
21
22
27
Il
I
III
IV
II
I
II
III
IV.
I
II
III
IV
I
Table 15.
Symptoms
0
0
0
0
0
0
0
0
0
0
0
0
I
■ 0
log cfu/leaf
0
0
1.74
2.52
4.62.
2.52
3.74
3.70
3.45
3.92
3.59
0
4.38
.5.54
% INA+
-100
67
100
100
100
- 100
100
100
100
—
100
100
syringae populations on ARlI, 1986, Bozeman.
Date
Day
6/27
6/27
6/30
7/3
7/8
7/9
7/11
7/15
7/16
7/17
7/18
7/23
7/26
7/30
7/31
8/5
0
0
3
6
11
12
14
18
19
20
21
26
29
33
34
39
Repetition
I
II
III
IV
II
III
IV
I
II
III
IV
I
II
IV
III
III
Symptoms
0
0
. 0
0
0
0
0
0
0
0
0
0
I
0
0
0
log cfu/leaf
0
0
0
2.00
2.70
2.83
3.64
3.79
3.23
4.23
3.64
5.60
5.90
6.04
6.18
6.54
% INA+
—
——
100
0
100
67
100
86
50
20
100
60
100
71
100 .
40
Table 16.
Date
6/27
6/30
7/1
7/1
7/8
7/9
7/11
7/15
7/16
7/17
7/18
7/23 .
7/26
Table 17.
Date
6/26
6/27
6/27
6/30
7/5
7/8
7/9
7/11
7/15
7/16
7/17
7/18
7/23
7/26
7/31
8/1
8/5
P^_ syringae populations on ARl2, 1986, Bozeman.
Day .
Repetition
Symptoms
log cfu/leaf
% INA+
0
3
4
4
11
12
14
18
19 .
20
21
26
29
I
II
III
IV
II
III
IV
I
II
III
IV
I
II
0
0
0
0
0
0
0
0
0
0
0
0
0
2.59
2.79
1.70
4.89
4.11
2.08
3.45
4.97
4.86
2.97
2.79
4,85
4.98
100
100
0
100
100
100
100
100
20
50
100
100
60
P. syringae populations on ARl3, 1986, Bozeman.
Day
0
I
I
4
9
12
13
15
19
20
21
22 ■
27
30
35
36
40
Repetition
I
II
III
IV
I
II •
III
IV
I
II
III
IV
I
II
III
I
II.
Symptoms
0
0
0
0
0
0
0
0
0
0
0
2
I
2
I
0
I
log cfu/leaf
5.60
4.53
5.69
. 4.04
4.64
5.23
5.28
5.11
5.11
5.40
5.83
5.74
6.54
6.81
6.81
6.60
6.88
% INA+
100
100
100
100
100
100
100
25
100
100
100
100
100
100
100
100
100
Al
Table 18.
syringae populations on ARIA, 1986, Bozeman.
Date
Day
6/26
6/27
7/1
7/1
7/5
7/8
7/9
7/11
7/15
7/16
7/17
7/18
7/23
7/26
7/31
0
I
5
5
9
12
13
15
19
20
21
22
27
30
35
Table 19.
Repetition
I '
II
III
IV
I
II
III
IV
I
II
III
IV
I
II ■
III
Symptoms
log cfu/leaf
0
0
0
0
0
0
0
I
0
0
0
I
0
0
0
0
0
0
2.7A
3.15
3.28
5.A6
3.83
3.59
A.3A
A. 28
5.00
5.15
5.08
6.15
% INA+
__
—
—
100
100
100
100
17
100
100
75
100
50
100
100
P. syringae populations on AR15, 1986, Bozeman.
Date
Day
6/26
6/30
7/1
7/1
7/5
7/8
7/9
7/11
7/15
7/16
7/17
7/18
6.23
7/26
8/5
0
A
5
5
9
12
13
15
. 19
20
21
22
27
30
AO
Repetition
I
II
III
IV
I
II .
Ill
IV
I
II
III
IV
I
II
II
Symptoms
0
0
0
0
0
0
0
2
2
I
2
2
I
2
2
log cfu/leaf
A.00
1.86
2.70
2.6A
3.20
A.7A
A.AO
A.52
A. 86
5. AS
5.08
5.08
5.51
5.90
6.AS
% INA+
o
100
60
100
100
100
67
100
100
100
100
100
100
100
100
80
42
Table 20.
syringae populations on AR16, 1986, Bozeman.
Date
Day
Repetition
6/26
6/27
6/30
7/3
7/5
7/8
7/9
7/11
7/15
7/16
7/17
7/18
7/23
7/26
7/30
0
I
4
7
9
12
13
15
19
20
21
22
27
30
34
I' .
II
III
IV
I
II
III
IV
.I
II
III
IV
I
.II
IV
Table 21.
Symptoms
0
0
0
0 .
0
0
0
0
0
0
0
0.
0
0
0
% INA+
log cfu/leaf
__
0
0
1.64
2.34
3.72
2.70
5.43
3.79
3.89
4.04
4.48
2.97
4.78
4.98
4.78
— -
100
100
100
100
100
100
71
0
91
80
100
100
100
syringae populations on AR17* 1986, Bozeman.
-
Date
Day .
Repetition
Symptom's
log cfu/leaf.
6/26
6/26
6/30
7/1
7/5
7/5
7/8
7/9
7/11
7/15
7/16
7/17
7/18
7/23
7/26
7/30
7/31
8/1
0
0
4 .
5
9
9
12
13
15
19 '
20
21
22
27
30
34
35
36
II
I
III
IV
II
I
II
III
IV
I
II
III
IV
I
II
IV
III
I
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
I
0
I
0
0
1.59
0
2.23
0.74
1.45
2.38
2.15
3.86
3.86
3.00
2.15
3.65
4.98
3.20
5.74
4.70
% INA+
__
——
100
——
100
100
100
100
100
100
100
0
83
83
100
0
100
100
43
Table 22.
P l syringae populations on 222-1. 1986, Bozeman.
Date
Day
6/26
6/26
6/27
6/30
7/5
7/5
7/8
7/9
7/11
7/15
7/16
7/17
7/18
7/23
7/26
7/30
7/31
0
0
I
4
9
9
12
13
15
19
20
21
22
27
30
34
35
Table 23.
Date
6/26
6/26
6/30
6/30
7/5
7/5
7/8
7/9
7/11
7/15
7/16
7/17
7/18
7/23
7/26
7/30
7/31
8/5
Repetition
I
II
III
IV
II
I
II
I
IV
I
II
III
IV
I
II
IV
III
Symptoms
0
0
0
0 ■
0
0
0.
0
0
0
0
0
0
0
0
.0
0
log cfu/leaf
0
0
0
0.74
1.04
0
0
2.34
3.00
3.34 ■
2.23
4.08
3.64
2.98
4.81
4.90
5.08
%
INA+
100
50
100
70
72
50
100
100
100
100
100
100
Pl syringae populations on 222-9, 1986, Bozeman.
Day
0
0
4
4
9
9
12
13 ‘
15
19
20
21
22
27
30
34
35
40
Repetition
Symptoms
log cfu/leaf
II
I
III
IV
II
I
II
III
IV
I
II
III
IV
I
II
IV
III
III
0
0
0
0
0
0
0
0
0
0
0
0
0
2
I
0
0
0
0
0
3.18
0
1.23
1.52
1.74
2.59
3.08
3.11
3.59
4.26
3.92
3.81
5.81
5.40
5.57
5.74
"% INA+
____
62
■ —.
100
100
100
100
100
100
100
50
100
100
100
80
71
100
44
Table 24.
I\ syringae populations on BOLD. 1986, Bozeman.
Date
Day
6/26
6/27
6/27
7/1
7/5
7/8
7/9
7/11
7/15
7/16
7/17
7/18
7/23
7/26
7/31
8/1
0
I
I
5
9
12
13
15
19
20
21
22
27
30
35
36
Table 25.
Repetition
I
II
III
IV
I
II
III
IV
I
II
III
IV
I
II
III
I
Symptoms
log cfu/leaf
% INA+
0
0
0
0
0
0
0
0
0
0
0
0
0
0
I
0
2.70
0
0
1.04
2.74
2.64
0
4.11
4.89
3.20
4.52
4.04
4.85
4.70
6.23
5.88
100
100
100
40
100
0
100
100
75
100
100
100
0
P. Syringae populations on STEPTOE, 1986, Bozeman.
Date
Day.
7/3
7/3
7/3
7/3
7/8
7/9
7/11
7/15
7/16
7/17
7/18
7/23
7/26
7/30
7/31
8/1
8/5
. o
0
0
0
5
6 .
8
12
12
14
15
20
23
27
28
29
33
Repetition
IV
III
II
I
II
III
IV
I
II
III
IV
' I
II
IV
III
I
I
Symptoms
log cfu/leaf
0
2
0
I
0
0
0
0
0
0
0
I
2
2
2
I
I
2.53
4.96
2.52
4.59
2.74
4.74
3.54
3.95
3.45
0
3.26
5.32
4.95
6.60
4.23
5.88
5.28
% INA+
31
0
20
17
60
14
63
0
20
— —
86
0
0
60
29
0
13
45
Table 26.
Pjl syringae populations on KLAGES, 1986, Bozeman. •
Date
Day
7/3
7/3
7/3
7/3
7/8
7/9
7/11
7/15
7/16
7/17
7/18
7/23
7/26
7/30
7/31
8/1
8/5
0
0
0
0
5
6
8
12
13
14
15
20
23
27
28
29
33
Repetition
Symptoms
IV
III
II
I
II
III
IV
■ I
II
III
IV
I
II
IV
III
I
I
0
0.
0
0
0
0
0
0
0 .
0
0
0
0
I
I
0
I
,
log cfu/leaf
% INA+
3.45
3.11
4.00
4.00
4.04
4.20
3.56
4.92
4.15
3.64
3.00
4.65
4.90
5.54
4.90
4.54
4.70
100
100
100
100
100
78
100
0
86
100
100
100
100
100
100
71
80
syringae populations on CLARK, 1986, Bozeman.
Table 27.
Date
Day
7/3
7/3
7/3
7/3
7/8
7/9
7/11
7/15
7/16
7/17
7/18
7/23
7/26
7/30
7/31
8/1
8/5
0
0
0
0
5
6
8
12
13
14
15
20
23
27
28
29
33
Repetition
II
I
IV
III
II
III
IV
I
II
III
IV
I
II
IV
III
I
I
Symptoms
-
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
log cfu/leaf
2.04
2.34
0
1.92
0
3.41
. 2.74
4.45
2.70
2.96
3.86
5.70
0
6.15
0
6.00
4.49
% INA+
100
0
-50
—
90
.0
60
50
100
40
100
-100
—
100
62
46
Table 28.
P. syringae populations on ERSHABET, 1986, Bozeman.
Date
Day
7/3
7/3
7/3
7/3
7/8
7/9
7/11
7/15
7/16
7/17
7/18
7/23
7/26
7/30
7/31
8/1
8/5
0
0
0
0
5
6
8
12
13
14
15
20
23
27
28
29
33
Symptoms
Repetition
II
I
IV
III
II
III
IV
I
II
III
IV
I
II
IV
III
I
I
I
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
•
entries were studied in Arizona.
log cfu/leaf
% INA+
3.11
5.00
4.86
0.74
3.45
4.54
3.72
3.08
5.52
5.26
4.04
5.88
6.30
6.78
5.65
5.11
5.08
100
100
100
100
80
63
.100
0
50
57
60
80
0
0
40
67
86
Results were taken for only three
days and differences were observed, but were not statistically sig­
nificant among five out of six entries (Tables 30-35, 44).
The
majority of bacteria in the epiphytic populations of P. syringae were
100% INA+ (Table 36).
The symptoms recorded in Arizona ranged from 1-
3 but again no correlation was found between symptoms and population
sizes (r = -0.18, r^ = 0.03).
When the. results in Arizona were taken,
all entries except Clark had headed.
Statistically significant differences, in epiphytic P^ syringae
population, sizes were also observed among the 6 entries in Bozeman,
1987 (Tables 37-42, 44).
It is important to note that these
differences were quite consistent during the 3 experiments.
In
Bozeman, 1987 no correlation was observed again between symptoms and
population sizes (r = 0.08, r^ = 0.01).
Differences were observed in
log ofu/leaf
47
log cfu/l eaf
. syringae populations on ARl, Bozeman, 1986.
Figure 4.
P. syringae populations on AR2, Bozeman, 1986.
48
Figure 5.
P. syringae populations on AR3, Bozeman, 1986.
Figure 6.
P. syringae populations on AR4, Bozeman, 1986.
. syringae populations on AR5, Bozeman, 1986.
log ofu/leaf
,
log ofu/leaf
49
Figure 8.
P. syringae populations on AR6, Bozeman, 1986.
50
Figure 9.
Figure 10
P. syringae populations on AR7, Bozeman, 1986.
P. syringae populations on AR8, Bozeman, 1986.
log cfu/1 eof
51
P. syringae populations on AR9, Bozeman, 1986.
log ofu/1 eof
LI.
Figure 12.
P. syringae populations on ARlO, Bozeman, 1986.
log cfu/l eof
log cfu/l oaf
52
Figure 14.
P . syringae populations on AR12, Bozeman, 1986
log ofu/leof
53
Icsg cfu/leof
syringae populations on AR13, Bozeman, 1986
Figure 16.
P, syringae populations on AR14, Bozeman, 1986
log cfu/leaf
54
log cfu/loof
P. syringae populations on AR15, Bozeman, 1986.
Figure 18.
P. syringae populations on AR16, Bozeman, 1986.
log cfu/leaf
55
log cfu/Ieaf
P. syrlngae populations on ARl7, Bozeman, 1986.
Figure 20.
P. syringae populations on 222-1, Bozeman, 1986.
P. syringae populations on 222-9, Bozeman, 1986.
log CfuZIeof
'
log cfuZleof
56
Figure 22.
P. syringae populations on BOLD, Bozeman, 1986
log cfu/l eof
57
log cfu/l eaf
P. syringae populations on STEPTOE, Bozeman, 1986.
Figure 24.
P. syringae populations on KLAGES, Bozeman, 1986.
log ofu/leaf
58
log cfu/leaf
P. syringae populations on CLARK, Bozeman, 1986.
Figure 26.
P
syringae populations on ERSHABET, Bozeman, 1986.
59
Table 29.
ENTRY
ARl
AR2
AR3
AR4
AR5
AR6
AR?
AR8
AR9
ARlO
ARll
ARl 2
ARl 3
ARl 4
AR15
ARl 6
AR17
222-1
222-1
BOLD
STEPTOE
KLAGES
CLARK
ERSHABET
TOTAL
Percentages of INA+ bacteria in the population of P.
syririgae, Bozeman, 1986.
100
12
12
14
8
3
11
9
6
7
10
6
9
16
9
12
9
9
8
11
9
99-80
79-60
I
I
2
I
4
2
I
2
I
I
2
2
I
I
12
5
5
I
2
I
3
212
26
59-40
19-i
I
;
I
'I
I
I
2
I
2
.3
I
I
2
I
2
I
I
I
I
2
I
I
2
I
I
I
2
I
3 .
2
2
3 ■
2
I
I
33
39-20
I
I
I
I
I
I
I
2
2
8
I
2
3
4
3
3
17
12
26
the percentages of INA+ bacteria in the population of P^ syringae.
This time, percentages ranged from 0-100% in. all entries (Table 43).
It is interesting to note that similar results were obtained in
Bozeman, 1986 for the four cultivars that were sprinkler-irrigated
(Steptoe, Klages, Clark, Ershabet) (Table 29).
The 48 fluorescent and
oxidase negative strains, isolated during the Bozeman, 1986 study, all
gave a negative arginine dehydrolase reaction, utilized alphaketoglutarate, but not D (-)tartrate.
syringae group.
This places them in the P.
Futhermore, two strains were. !NA- and two gave a
negative hypersensitivity reaction in tobacco leaves (Table 45).
60
Table 30.
syringae populations on AR4, 1987, Arizona.
Repetition
Symptoms
log cfu/leaf
I
II
I
III
IV
II
III
IV
0
0
0
0
0
0
0
0
1.00
2.81
0.70
2.00
0
2.65
2.51
1.30
Date
Day
2/28
2/28
3/1
3/1
3/1
3/2
3/2
3/3
0
0
I
I
I
2
2
3
Table 31.
P l syringae populations on AR5, 1987, Arizona.
Repetition
Symptoms
log cfu/leaf
Date
Day
2/28
2/28
3/1
3/1
3/1
3/2
3/2
3/3
0
0
I
I
I
2
2
3
Table 32.
P l syringae populations on AR6, 1987, Arizona.
Date
Day
2/28
2/28
3/1
3/1
3/1
3/2
3/2
3/3
0
0
I
I
I
2
2
3
I
II
I
III
IV
' II
III
IV
Repetition
I
II
I
III
IV
II
III
IV
0
0
0
0
I
0
0
I
Symptoms
I
I
I
0
0
I
0
0
0
2.18
0.70
0.70
0
1.70
0
1.74
log cfu/leaf
4.26
3.36
4.20
4.15
3.81
5.08
4.08
4.26
% INA+
100
100
100
100
—
89
0
75
% INA+
— —
90
0
100
—
100
—
100
% INA+
100
100
100
95
100
100
94
100
61
Table 33.
syringae populations on AR13, 1987, Arizona.
Date
Day
2/28
2/28
3/1
3/1
3/1
3/2
3/2
3/3
O
O
I
I
I
2
2
3 .
Repetition
I
II.
I
III ..
IV
TI
III
IV
NT=Not tested
Symptoms
2
2
3
2
.2
3 ■
2
log cfu/leaf
1.90
0.70
3.04
2.81
1.90
3.43
NT*
3.43
% INA+
100
100
100
100
100
100
'—
100
■:
syringae populations on AR15, 1987, Arizona.
Table 34.
Date
Day
Repetition
2/28
2/28
3/1
3/1
3/2
3/2
0
0
I
I
2
•2
I
II
I
IIT
TI
III
Symptoms
I
2
I
2
2
2
log ofu/Teaf,
1.54
1.00
2.00
0
1.48
2.08
% INA+
86
100
100
—
67
0
A fourth repetition of AR15 was not planted.
syringae populations on CLARK, 1987, Arizona.
Table 35.
Date
Day
2/28
2/28
3/1
3/1
3/1
3/2
3/2
3/3
0
0
I
I
I
.2
2
3
Repetition
‘ I
II
I
III
IV
II
III
IV
Symptoms
0
0
Q
0
0
0
0
0
log cfu/leaf
0.70
0
0
0
0.70
2.74
1.18
3.32
% INA+
0
—
—
—
100
91
100
100
.
6.2
Table 36.
Percentages of'INA+ bacteria -in the population of P1
syringae, Arizona 1987.
ENTRY
9S -80 :
100
4 .
3
6
7
2
3
ARA
AR5
AR6
AR13
AR15
CLARK
25
TOTAL
79-60
I
I
2
I
I
I
I
6
. 2
•
59-40
:.
19-0
39-20
1
0
-• :
I
I.
-
3
•
•
P. syringae populations on AR4, 1987, Bozeman.
Table 37.
Date
7/6 ■
7/13
Day
Repetition
0
i
II
III
IV
. o
0
0
0
.7
I
II
. HI
IV
0
0
0
0
7/20
14
7/27
21
8/10
35
",
■
.
,
:
0
o
i
6
0
0
0
0
■ 0
NT
0
1.70
3.00
■ 2.65
' 0
0 /
4.85
\ NT ■
5.33 ..
,
4.95
b
•
0
: 0 V
0
.I
II •
. . Ill
; IV
NT=Not Tested
.
'0
b
o .
- 0
■ I. .
II
III
IV
•’ I
- II '
III •
IV
log cfu/leaf
Symptom's
•
'
- •
, 6.59
5.95 •
6.88 .
5:
.88
• % INA+
..■■
—
-T
——
• Mean/SE
0
-,0.57+0.80
—
-;
ibq .
50
0
—
—.
1.41+1.42
o
—
56,
0
5.04+0.21
80
' .40.
60
14
6.32+0.42
63
Day
Date
7/6
7/13
Bozeman.
P. syringae populations on AR5, 1987, :
CO
CO
Table
.
0
7
Repetition
I
II '
III
IV
Symptoms
log cfu/leaf
0
0
0
0
0
0
0,
I
II
III
IV
0
0
0
0
0
2.48
0
2.81
.0
7/20
14
I
II
III
IV
0
0
0
0
2.70
2.18
2.18
2.00
7/21
21
I
II
III
IV
0
0
0
0
4.74
NT
4.34
4.28
I "
II
III
IV
0
0
0
0
0
5.95
6.58
4.88
8/10
35
NT=Not Tested
% INA+
Mean/SE
0
—
—
—
——
1.32+1.33
0 .
—
0
0
100
0
0
2.26+0.26
0
4.45+0.20
—
0
0
— —
100
78
71
4.35+2.59
64
P. syringae populations on AR6, 1987, Bozeman.
Table 39.
Date
7/6
7/13
7/20
Day
0
7
14
Repetition
Symptoms
I
II
III
IV
0
0
0
0
I
II
III
IV
0
0
0
0
I
II
III '
IV
0
0
0
0
log cfu/leaf
.
.
2.70
0
2.70
2.90
% INA+
0
Mean/SE
2.08+1.20
—
0
0
—
—
NT
NT
1.70
0
—
3.18
3.18
0.
2.70
33
0
—
0
2.27+1.32
0.85+0.85
0
7/27
21
‘
I
II
III
IV
0
0
0
0
5.88
5.00
6.00
5.30
0
0
0
0
5.55+0.41
8/10
35
I
II
III
IV
0
I
0
0
4.70
NT
3.70
3.70
100
— 0
0
4.03+0.47
NT=Not Tested
65
P. syringae populations on AR13, 1987, Bozeman.
Table 40.
Date ' Day
7/6
7/13
0
7
Symptoms
log cfu/leaf
% INA+
Mean/SE
I
II
III
IV
0
0
0
0
2.00
4.54
0
0
33
57
1.64+1.87
I
II
III
IV
0
0
0
0
0
NT
0
3.70
—
100
Repetition,
—
—
1.23+1.74
—
—
7/20
14
I
II
III
IV
0
0
0
b
NT
3.60
3.00
NT
——
0
0
——
3.30+0.30
7/27
21
I
II .
Ill
IV
I
I
I
0
7.84
7.28
7.85
7.34
0
38
41
67
7.58+0.27
8/10
35
I
II
III
IV
I
I
I
I
5.88
NT
5.00
5.40
60
5.42+0.36
NT=Not Tested
—
100
100
66
P. syringae populations on ARl5, 1987, Bozeman.
Table 41.
Date
Day
Repetition
Symptoms.
log cfu/leaf
0
0
0
0
% INA+.
Mean/SE
'—
—
0
7/6
0
I
II
IIJ
IV
0
0
0
0
7/13
7
I
II ■
' III
IV
0
0
0
0
0
3.00
0
2.83
—
29
"
78
1.46+1.46
7/20
14
I
II
III
IV
.0
0
0
0
0
3.40
0
2.70
——
0
—
0
1.52+1.54
7/27
21
I
II
III
IV
I
0
0
0
5.23
6.08
NT
5.41
14
0
—
9
5.57+0.30
8/10
35
I
II
III
IV
I
2
I
2
7.11
7.20
6.93
6.30
17
71
18
10
6.89+0.35
NT=Not Tested
67
Table 42.
Date
7/6
Day
0
P. syringae populations on CLARK, 1987, Bozeman.
Repetition
Symptoms . log cfu/leaf
I
II •
III
IV
0
0
0
0
.
% INA+
Mean/SE
1.70
0
o .
0
0
——
—
——
0.49+0.74
7/13
7
I
II
III
IV
0
0
0
0
0
2.00
0
0
——
P
—
—
0.50+0.87
7/20
14
I
II
III
IV
0
0
0
0
0
0
0
1.70
—
—
—
0.42+0.74
0
7/27
21
I
II
III
IV
0
0
0
0
4.60
4.48
4.95
4.98
75
0
0
40
4.75+0.22
8/10
35
I '
II
III
IV
0
0
0
0
4.95
5.54
4.48
6.65
50
29
17
0
5.41+0.81
P . syringae populations on AR4, Bozeman, 1987.
log cfu/l«»f
'
log cfu/taaf
68
Figure 28.
P. syringae populations on AR5, Bozeman, 1987.
69
Figure 29.
P. syringae populations on AR6, Bozeman, 1987.
Figure 30.
P . syringae populations on AR13, Bozeman, 1987.
log cfuZI eaf
log ofu/leaf
70
Figure 32.
P. syringae populations on CLARK, Bozeman, 1987.
71
8
7
•
.
•
!
.
:
,
6
•
,
i :
: .
!
•
o
5
OJ
•
.
.
CD
* s
• . I
•
4
u
;
'
;
: : : :
.
I
£
3
1
•
. ;
•
S
•
•
’
.
.
.
:
"
.
•
:
: •
■ I *
" i
•
•
.
:
:
.
•
•
•
.
.
! i I :
•
•
‘
2
i
5
•
:
•
’
I
:
•
•
•
,
•
•
•
2
I
:
:
•
X
5
•
•
•
:
•
:
•
-
I
:
- - - - - - - - L _ ---- -- - - -
0
0
10
20
.
30
40
days
Figure 33.
P. syringae populations on 24 barley cultivars and lines,
Bozeman, 1986.
8
•
7
-
•
•
6
o
_Q)
.
5
•
•
X
£
4
o
CD
£
3
•
-
2
•
•
:
!
•
I
•
0
0
10
20
30
40
days
Figure 34.
P. syringae populations on six barley cultivars and lines,
Bozeman, 1987.
72
Table 43.
Percentages of INA+ bacteria in the population of P.
syringae, Bozeman 1987.
100
99-80
79-60
AR4
AR5
AR6
AR13
AR15
CLARK
I
2
I
3
I
I
2
TOTAL
7
ENTRY
Table 44.
I
39-20
19-0
4
8
12
3
8
7
42
■, 3
2
2
I •
2
I
2
I
I
8
7
5
2
Comparison of the epiphytic populations of P. syringae on
the six selected entries during 1986 and 1987 in Arizona
and Bozeman.
Bozeman 1987
Mean
Entry • ■
CLARK
AR4
AR5
AR15
AR6
AR13
59-40
2.64
2.93
2.94
3.09
3.29
4.53
Sign, level 5%
Bozeman 1986
Cultivar
Mean
Arizona 1987
Cultivar
Mean
A
AB
AB .
AB
AB
B
AR5
CLARK
AR15
AR4
AR13
AR6
0.88
1.08
1.35
1.62
2.73
4.15
A
A
A
A
A
B
AR5
CLARK
AR4
AR15
AR6
ARl 3
2.18
2.92
3.76
4.40
4.46
5.53
A
AB
ABC
BC
. BC
C
73
Table 45.
Biochemical characteristics of the P. syringae isolates
collection from Bozeman, 1986.
Utilization
Isolate
DGlOO
alpha-
of
D (-)
Oxidase
Fluor.
Argin.
Hyper-
Reaction
-
Pigment
+
Dehyd.
-
Sensit.
+
INA
+
ketogl.
+
+
+
+
+
+
+
+
+
+
-
+
+
-
DGlOl
-
+
-
DG102
-
+
-
+
+
+
tar.
-
DG104
-
+
+
D G 105
-
+
-
+
-
+
+
+
+
-
-
+
-
+
+
+
+
+
-
+
-
+
+
-
+
+
-
+
-
+
+
4
-
+
-
+
+
+
-
+
+
-
D G l 03
DG109
DG112
DG114
D G l 15
DG116
-
DG118
-
+
-
+
+
+
D G l 19
DG120
-
+
-
+
-
+
+
+
+
DG121
-
+
+
+
+
-
DG122
-
+
-
+
+
-
D G 1 23
-
+
+
+
+
+
+
+
+
D G l 17
DG124
D G 125
DG126
+
-
+
+
-
+
-
+
-
+
-
•f
-
+
+
-
D G 1 27
-
+
+
+
+
-
DG128
-
-
+
+
+
-
D G 1 29
-
+
+
-
+
+
+
+
-
+
+
+
-
+
+
-
D G l 20
DG131
-
+
-
+
+
DG132
-
+
-
+
+
-
+
+
+
-
+
+
+
-
+
-
+
+
+
-
+
-
+
+
+
+
+
-
+
+
+
+
+
-
+
+
-
+
+
+
DG133
-
+
DG134
-
+
DG135
DG136
-
DGl 3 8
-
DG139
D G l 40
-
-
-
DG141
-
+
-
+
+
+
-
D G 142
-
+
+
+
+
+
-
-
+
+
+
D G 143
-
+
-
+
+
+
-
+
-
+
+
+
-
-
-
+
-
D G 14 4
D G 145
D G 146
+
-
-
+
-
+
+
D G 14 8
+
-
+
-
+
+
D G 149
-
+
-
+
+
+
DG150
-
+
+
-
+
+
+
+
-
+
-
+
+
+
+
+
-
-
+
-
+
+
+
-
D G l 47
DGl 5 1
DG152
DG153
-
74
Diurnal Population Changes
Epiphytic
syringae populations increased from 8 a.m. until 12
noon by I log approximately, but remained stable from 12 noon until 8
a.m. the next day (Table 46, Figure 35).
Hirano, et al. (1984)
reported large fluctuations of up to log 2.8 cfu/leaf of epiphytic P1
syringae populations on bean leaves in a 26-hour period.
Table 46.
Diurnal change in epiphytic populations of P1 syringae on
AR13 (7/24-25/1987).
Mean + S.E. (log cfu/leaf)
Time
8
12
4
8
12
4
8
5.88
7.01
7.06
6.99
7.25
7.48
7.11
AM
PM
PM
PM
PM
AM
AM
+
+
+
+
+
+
+
0.13
0.31
0.30
0.73
0.58
0.48
0.22
V-
8
I0
0
Figure 35.
4
8
12
hours
16
20
24
Diurnal change in epiphytic populations of P1 syringae on
AR13 (7/24-25/87).
75
Plant-to-Plant Dissemination
No significant difference between the mean population of P.
syringae per leaf on Clark in the.mixture and on Clark in the control
plots was observed, by testing with the Student's t test, in both
experiments (5% level of significance):
- expt.,1:
t - -0.22.
. %(0.025,6) = -2.45
- expt. 2:
t = 0.25
. t (0.025,6) =1.86
It is not clear from these experiments if plant-to-plant •
dissemination of Pl syringae occurred, because the "control" plants of
Clark supported as high populations of Pl syringae as the "treatment"
plants of Clark (Figures 36,37).
1986 Experiment with Marked Strains
During the first round of selection, performed with the paper
disk method, from 16 Pl syringae strains, seven developed
streptomycin-resistant colonies around the 1000 ppm disk (DG154,
DG173, DG178, DGl84, DGl87, DG205, DG214), and two developed
rifampicin resistant colonies around the 1000 ppm disk (DG173, DGl84).
No zone of inhibition appeared around the disks with any concentration
of erythromycin, thus no selection for marked strains was possible
with this antibiotic.
In the second round .(direct plating), DG173
developed resistant colonies at 1000 ppm rifampicin, and DG173, DG178,
76
Figure 36.
Results of the first plant-to-plant dissemination experi­
ment. Solid bars: AR13 plants; open bars: CLARK plants.
7
Figure 37.
Results of the second plant-to-plant dissemination experi­
ment. Solid bars: AR13 plants; open bars: CLARK plants.
77
DGI87 developed resistant colonies to 1000 ppm streptomycin.
In the
third round for selection of double-marked strains, the previous four
strains developed resistant colonies to both rifampicin and
streptomycin at 1000 ppm each.
They all grew normally on trimethoprim
(all concentrations), novobiocin (50, 100 ppm), kasugamycin (50 ppm)
and did not grow at all on tobramycin (all concentrations),
tetracyclin (all concentrations), novobiocin (500 ppm), and
kasugamycin (100, 500 ppm); thus, no selection for double-marked
strains with the previous antibiotics as second markers was
possible.
No background I\_ syringae populations naturally resistant to 1000
ppm of streptomycin and rifampicin were detected.
The survival of the
four selected double-marked strains (inoculated all four in a mixture)
on Klages, Clark, 222-1, 222-9 is shown on Figures 38-41.
establishment on the leaf surface was rather poor.
Their
Moreover they
showed very slow growth in vitro on King's B amended with rifampicin
and streptomycin, and it was necessary to incubate the plates for
seven days until colonies of I mm in diameter developed.
These
colonies were not fluorescent, because in seven days the pigment
diffuses into the medium until it cannot be detected with ultraviolet
light.
Thus, the identification of the colonies was based only on
their oxidase reaction (always negative) and INA (positive colonies
were often found).
Populations of marked P. syringae on KLAGES, Bozeman, 1986.
log ofu/leaf
*
log ofu/leaf
78
Figure 39.
Populations of marked P. syringae on CLARK, Bozeman, 1986.
Populations of marked P. syringae on 222-1, Bozeman, 1986
log cfu/leaf
’
log cfu/leaf
79
Figure 41.
Populations of marked P. syringae on 222-9, Bozeman, 1986
•80
1987 Experiment with Marked Strains
Selection of Marked Strains.
During the first round of selection
for marked strains (direct plating, method), from 50 isolates of P.
syringae tested, one developed resistant colonies to 500 ppm of
streptomycin (DG151); three developed resistant colonies to 10 ppm of
kanamycin (DG151, DG173, DGl17); one developed resistant colonies to
20 ppm of kanamycin (DG151); and five developed resistant colonies to
100 ppm of rifampicin (DG102, DG118, DG126, DG129, DG151).
The
colonies on 20 ppm of kanamycin were smaller than those on 10 ppm,
indicating slower growth.
For this reason they were not selected.
In
the second round for double marking, six strains developed colonies
resistant to 10 ppm of kanamycin and 100 ppm of rif ampicin (DG102,
DG117, DG118, DG126, DG129, DG151); two strains developed colonies
resistant to 10 ppm of kanamycin and 500 ppm of streptomycin (DG151,
DG173); and one strain developed colonies resistant to 100 ppm
rifampicin and 500 ppm streptomycin (DG151).
Of 32 double-marked strains tested, only two rifampiclnstreptomycin (RS)-marked strains were INA+ (151-4RS, 151—5RS); every
other double marked strain was INA-. ' This made possible the use of a
1:1 mixture of near-isogenic strains, differing only in the markers
they carry and their !NA, to inoculate the cultivar ARl5, and see if
this difference in INA would have an impact in, the epiphytic survival
of that strain on barley.'
81
Doubling Times
The results of the experiments to determine the relation between
Klett units and bacterial population/ml (e.fu/ml) are shown in Tables
47 and 48.
There is a linear relation between the logarithm of
bacterial population and the logarithm of Klett units.
This relation,
calculated from the data of Table 47 is:
y = 1.05x + 6.99, with r = 0.96
and from the data of Table 48:
y = 1.377x + 6.625, with r = 0.97
where y = log (cfu/ml) and x = log Klett.
These two equations are very similar and show that the relation
between bacterial population and Klett units for Pl syringae is
independent of strain, incubation temperature and growth medium.
The
second equation was used to convert Klett units to bacterial
populations for the study of doubling times of the double-marked
strains.
The doubling times of 32 double-marked strains and their wildtype parents are shown in Table 49.
Two strains:
the INA+ 151-4RS
and the INA- 151-13KS were selected for further work because their
doubling times are very close to the parental DG151.
The selection
criteria concerning the INA- strain included fluorescence:
other
strains that had doubling times closer to DG151 were not selected
because of their weak fluorescence.
The line AR13 was inoculated
with 151-4RS and the line AR 15 with a 1:1 mixture of 151-4RS
and 151-13KS.
82
No background
syringae populations naturally resistant to
either 100 ppni rifampicin and 500 ppm streptomycin, or 10 ppm
kanamycin and 500 ppm streptomycin were detected.
Table 47.
Results of Klett # versus population (log cfu/ml)
correlation. Cultures "suspended in water of 7 isolates
from the 1986 collection, grown at 21°C were used.
21°C
I
Strain
DG104
I
Klett #
I
157
49
9
0
I
DG148
DGl46
28°C
I
I
I
I
I
I
I
I
I
I
log cfu/ml
9.35 •
8.65 .
7.95 .
7.25
Klett #
212
96
22
4
log cfu/ml
. 9.49
8.80
8.10
7.40
58
13
3
8.69
7.99
7.29
211
62
14
.0
9.46
8.76
8.06
7.36
90
6
2
8.70
8.00
7.31
40
9
4
8.85
8.16
7.46
113
59
10
,2
9.68
8.99
8.29
7.59
76
14
4
8.81
8.11
7.42
104
61
10
4
9.57
8.87
8.17
7.47
86
39
7
2
9.39
8.69
8.00
7.30
NT
'
NT
NT
NT
76
15
3
8.89
8.19
7.49
I
DG103
I
I
I
DG105
I
I
I
I
I
DGlOl
I
I
I
209 ■
76
15
5
. 9.45
8.75
8.05
7.35
I
DGlOO
I
I
I
I
NT=Not Tested
83
Table 48.
Results of Klett # versus population (log cfu/ml)
correlation. Liquid cultures in NBG of 4 marked strains
(1987 experiment) were used. KS = Kanamycin-Streptomycin
resistant.
Strain
Klett #
173-AKS
' 81
58
40
4
8.90
9.17
9.04
7.24
15I-JKS
48
.28
10
■5
9.00
8.57
8.28
7.66
Population (log fcfu/ml)
:
15I-DKS
12
8.03
15I-KKS
. 2
7.00
Table 49.
Doubling times of P. syringae marked strains and their
wild-type parents (preceded by DG). RS = RifampicinStreptomycin resistant, KS = Kanamycin-Streptomycin
resistant, RK = Rifampicin-Kanamycin resistant.
Strain
DG151
151- IRS
151- 2RS
151- 3RS
151- 4RS
151- 5RS
151- 5KS
151- 6K(20)S
151- 7K(20)S
151-IOKS
151-12KS
151-13KS
151-14KS
151-15KS
151-16KS
151-17KS
15I-DKS
151-JKS
15I-KKS
51-1RK
*Selected INA+
**Selected INA-
Doubling Time (hrs)
1.11
0.97
1.11
1.43
1.19
1.18
1.30
1.18
1.13
1.39
0.97
1.02
1.34
1.29
1.12
1.10
1.42
1.64
1.62
1.41
+
+
+
+
+
+
+
+
+
+
+
.+
+
+
+
+
+
+
+
+
0.08
0.29
0.12
0.44
0.05*
0.22
0.21
0.30
0.25
0.30
0.11
0.10**
0.26
0.04
0.10
0.16
0.36
0.14
0.11
0.38
Strain
151-2RK
151-3RK
151-4RK
.151-5RK
DG126
126-RK
DG102
102-RD
DGliS
118-IRK
118-2RK
118-3RK
II8-4RK
DGl 17
117-RK
DG129
129-RK
DG173
173-KS
Doubling Time (hrs)
1.29
1.04
1.11
1.17
1.70
1.38
1.36
1.20
1.61
1.63
1.83
1.30
1.70
.1.68
1.59
1.36
1.58
1.98
1.58
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
0.36
0.25
0.21
0.26
0.06
0.14
0.16
0.12
0.16
0.09
0.32
0.18
0.07
0.15
0.20
0.16
0.18
0.20
0.31
84
Epiphytic Survival of the Marked Strains
Both marked strains survived much better on the leaf surface than
the strains used in the 1986 experiment.
AR15 (Table 50, Figure 42).
Both strains survived on
This is in accordance with previous
research showing that exclusion of an INA+ pseudomonad from an INAone, and vice-versa, is based on the inoculum level and time of appli­
cation of each one, rather than the ice nucleation ability (Lindemann
et al., 1985; Lindow and Panopoulos, 1986; Lindemann and Suslow,
1987).
43).
Strain 151-4RS survived equally well on AR13 (Table 51, Figure
In four cases the marked strains were found on leaf samples from
the uninoculated plots.
This shows that dissemination in the field
from plant to plant does exist, something not proven during the plantto-plant dissemination experiment..
Air Dissemination of P. syringae
Attempts to isolate the INA+ marked strain of F\_ syringae (1514RS) from the atmosphere above the canopy with an air pump were not
successful.
This could be due to either the absence of the bacterium
from the atmosphere or a technical deficiency of the system.
On the contrary, the strain 151-4RS was isolated six times from
the atmosphere, when Petri dishes were displayed around the inoculated
fields (Table 52).
In four cases, bacteria were disseminated during
and after irrigation in distances up to 10 m.
Once, the marked P.
syringae was isolated during dry conditions on the border of the field
(0 m).
But it moved as far as 70 m during a rainy day.
These data
suggest that dissemination of P^ syringae occurs more frequently
85
during wet conditions (sprinkler irrigation and rain), probably
because of the formation of aerosols over the canopy.
Table 50:
Populations of total, marked INA+, and marked !NAPseudomonas syringae on AR15, Bozeman, 1987.
Date
Day
7/7
7/8
7/9
7/10
7/12
7/14
7/16
7/21
7/22
8/7
8/11
9/4
0
I
2
3
5
7
9
14
15
31
35
59
Total
Mean/SE of log cfu/leaf
Marked INA+
•Marked INA-
4.30+0.78
3.09+0.97
3.02+0.12
3.70+1.00
0
3.55+1.09
2.73+0.72
3.44+0.28
4.37+0.77
5.83+0.63
5.98+0.22
NT .
4.25+0.60
3.37+0.62
2.92+0.74
3.70+0.63
3.33+0.66
3.58+0.28
2.50+1.61
3.03+0.51
3.65+0.36
4.28+0.41
3.94+0.41
3.03+0.82*
4.10+0.48
2.16+0.46
1.69+1.06
3.51+0.65
2.69+1.75
. 2.64+1.56
2.28+1.37
1.35+0.93
• 1.78+1.41
1.97+1.52
2.43+0.20
NT
*0n dry leaves
NT=Not Tested
Table 51.
Populations of total and marked INA+ Pseudomonas syringae
on ARl3, Bozeman, 1987.
Date
Day
7/8
7/9
7/10
7/12
7/14
7/16
7/21
7/22
8/7
8/11
9/4
0
I
2
4
6
8
13
14
30
34
58
*0n dry leaves
NT=Not Tested
Mean/SE of log cfu/leaf
Marked INA+
Total
4.83+0.77
4.30+0.70
5.21+0.37
5.56+0.25
5.97+0.79
3.79+1.09
5.18+0.00
4.81+0.19
6.26+0.15
5.91+0.37
NT
3.73+0.49
3.07+0.64
3.17+0.22
2.88+1.67
3.92+0.88
4.26+0.51
3.99+0.47
4.22+0.61
3.89+0.63
3.27+1.56
3.95+0.99*
86
o total P.8
Figure 42.
D INfi- P.8
Populations of total, marked INA+, and marked INAP, syringae on AR15, Bozeman, 1987.
■ totoI P.e
Figure 43.
Populations of total and marked INA+ P. syringae on
AR13, Bozeman, 1987.
87
When plates were overlayed with water,, in order to prolong their
exposure without drying, the selectivity for the marked INA+ P.
syringae was lost:
the medium.
both fungi and non—fluorescent bacteria grew on
This was not observed when plates were exposed for up to
two hours, either when water was not added, or during sampling under
irrigation; it was observed, though, when samples were taken during
rain.
Therefore, it was necessary to verify that the fluorescent,
oxidase-negative, and INA+ bacteria that were isolated, were indeed
the strain 151-4RS.
To prove this* 10-20% of the colonies counted
were tested in the laboratory for. growth on King's B amended with
rifampicin and streptomycin.
In cases where "foreign" P. syringae
colonies were identified by'this test, the number of true colonies of
the strain 151-4RS was corrected by extrapolation.
It is certain that
bacterial cells were dividing, as long as water was present in the
plate, either when plates were displayed around the inoculated fields,
or when they were dried in the clean air hood.
However, this
experiment was designed to study the dissemination of marked P.
syringae over distance, not the sizes of bacterial populations that
moved.
Therefore, colony counts were not corrected for the time that
water was present in the plate.
88
Table 52.
Dissemination of rifampicin-streptomycin marked INA (+)
P. syringae.
Date
Conditions
Site (^Colonies)
8/8
Irrigation, PM
1(6), 4(275), 6(355)
8(29), 11(4), 12(95)*
8/14
Irrigation, PM
2(1), 6(600), 9(21)*
8/16
Irrigation, PM
6(13), 7(16), 8(1)*
8/19
Irrigation, PM
6(1)*
8/21
Dry, AM, Water
8(1)
8/24
AM, Rain
6(77), 7(182), 11(500)*
16(18), 22(10).
*Plates were dried in the clean air hood.
89
DISCUSSION
Ice nucleating bacteria are frequently found in the atmosphere
and have been implicated in rain formation.
This study was not
intended to prove this, but to give some evidence for the existence of
a "bioprecipitation cycle" in nature.. During irrigation and
especially during rain, a marked strain of
70 m from a field inoculated with it.
syringae moved as far as
Therefore, long or short
distance dissemination of the bacterium is possible, at least under
certain conditions such as sprinkler irrigation or rain.
Although the
marked strains of P. syringae established well on the leaves, their
number remained rather stable, log 3-4 cfu/leaf, as opposed to natural
populations of the bacterium which.showed an increasing trend.
That
may be the reason why during dry conditions the marked strain was
found only once in the atmosphere.
These data suggest that a "bioprecipitation cycle" in nature is
not impossible:
conditions.
P^ syringae can be disseminated under certain
Rain is formed around ice nuclei in the clouds, and P.
syringae is the most effective ice nucleus in nature.
This study also showed significant differences in epiphytic popu­
lations of the bacterium among barley cultivars.
These differences
appear to be quite stable in different areas and times.
these differences is unknown.
The nature of
It seems possible then to "grow" Pl
syringae on cultivars that support high numbers, of the bacterium in
arid areas of the world, and perhaps slow or ultimately reverse the
90
desertification process in these areas.
There is also evidence for a
"dew condensation^ ability of these bacteria (Cary and Lindow, 1986).
That, too, if true, could be a very important additional source of
moisture in arid areas.
At least the barley cultivars and lines used in this study seem
resistant to bacterial leaf blight, as opposed to wheat.
syringae
lives also epiphytically on wheat,- but the fact that barley is
resistant to the bacterium and more drought tolerant than wheat, makes
it more suitable as a "nursery" of Pl syringae in arid areas.
Another interesting point made in this study is the lower
percentages of INA+ bacteria in Pl syringae populations observed
during the summer of 1987, and on four cultivars that were sprinklerirrigated in the 1986 study.
An explanation for that can be the
presence of water on the leaves:
water is necessary for the survival
of the bacterium and the increase of the population.
So, a selection
pressure might exist under dry conditions for INA+ strains in the
population, resulting in accumulation of water by dew condensation.
This could explain the high percentages of INA+ bacteria on 20
entries in Bozeman, 1986 and even Arizona, 1987 where flood, irriga­
tion was applied (which in no case results in water accumulation on
the leaves).
In Bozeman, 1987, water was abundant and this selection
pressure was removed, resulting in lower percentages of INA+ bacteria.
It has been hypothesized that the ice nucleation activity of
certain bacteria including Pl syringae facilitates the infection of
plant tissue by causing frost damage; another "ecological advantage"
of this activity may be the accumulation of water on the leaf to the
91
advantage of the bacteria.
A fact supporting this hypothesis is that
ice nucleating bacteria have been isolated in areas of the world such
as the Middle East and North Africa, where frost never occurs.
LITERATURE CITED
.93
LITERATURE CITED
Andersen, G.L., and Lindow, S.E. 1985. Local differences in epiphytic
bacterial population size and supercooling point of citrus
correlated with type of surrounding vegetation and rate of
bacterial immigration. Phytopathology 75:1321.
Arny, D.C., Lindow, S.E., and Upper, C.D. 1976. Frost sensitivity of
Zea mays increased by application of Pseudomonas syringae.
Nature 262:282-284.
Ayers, S.H., Rupp, P., and Johnson, W.T. 1919. A study of the alkali­
forming bacteria in milk. U.S. Dept. Agric. Bull. 782.
Baca, S., Canfield, M.L., and Moore, L.W. 1987. Variability in ice
nucleation strains of Pseudomonas syringae isolated from diseased
woody plants in Pacific Northwest nurseries. Plant Disease
71:412-415. _
_________ , , and Moore, L.W. 1984. Overwintering of Pseudomonas
syringae on two grass species used as cover crops in three
Pacific. Northwest nurseries. Phytopathology 74:1135.
Blakeman, J.P. 1982. Phylloplane interactions. Pages 307-333 in:
Phytopathogenic Prokaryotes, Vol. I. M.S. Mount and G.H. Lacy
eds. Academic Press, New York.
____________, 1985. Ecological succession of leaf surface
microorganisms in relation to biological control. Pages 6-30 in:
Biological Control on the Phylloplane.
C.E. Windels and S.E.
Lindow eds. The American Phytopathological Society, St. Paul,
Minnesota.
Bovallius, A., Bucht, B., Roffey, R., and Anas, P. 1978a. Three-year
investigation of the natural airborne bacterial flora at four
localities in Sweden. Appl. Env.■ Micr. 35:847-852.
____________, Bucht, B., Roffey, R., and Anas, P. 1978b. Long-range
air transmission of bacteria. Appl. Env. Micr. 35:1231-1232.
Cary, J.W., and Lindow, S.E. 1986. The effect of leaf water variables
on ice nucleating Pseudomonas syringae in beans. Hortscience
21:1417-1418.
94
Cody, Y.S., Gross, D.C., Proebsting, E.Li, and Spotts, R.A. 1987.
Suppression of ice nucleation-active Pseudomonas syringae by
antagonistic bacteria in fruit tree orchards and evaluations of
frost control. Phytopathology 77:1036-1044.
Corotto, L.V., Wolber, P.K. and Warren, G.J. 1986. Ice nucleation
activity of Pseudomonas fluorescens: mutagenesis,
complementation analysis and identification of a gene product.
The EMBO Journal 5:231-236.
Crosse, J.E. 1959. Bacterial canker of stone fruits.
IV.
Investigation of a method for measuring the inoculum potential of
cherry trees. Ann. App. Biol. 47:306-317.
Dow, E.J., and Maki, L.R. 1985. Ice nucleating bacteria in the
atmosphere. P. 279 in: Abstracts of the Annual Meeting 1985,
American Society for Microbiology.
Ercolani, G.L., Hagedorn, D.J., Kelman, A., and Rand, R.E. 1974.
Epiphytic survival of Pseudomonas syringae on hairy vetch in
relation to epidemiology of bacterial brown spot of bean in
Wisconsin. Phytopathology 64:1330-1339.
Fryda, S.J., and Otta, J.D. 1978. Epiphytic movement and survival of
Pseudomonas syringae on spring wheat. Phytopathology 68:10641067.
Govindarajan, A.G., and Lindow, S.E. 1984. Phospholipid requirements
for expression of ice nuclei in bacterial membranes and in vitro.
Plant Phys. 75 (suppl. I):43.
Green, R.L. and Warren G.J. 1985. Physical and functional repetition
in a bacterial ice nucleation gene. Nature 317:645-648*.
Gross D.C., Cody, Y.S., Proebsting, E.L., Randamaker, G.K., and
Spotts, R.A. 1983. Distribution, population dynamics and
characteristics of ice nucleation-active bacteria in deciduous
fruit tree orchards. Appl. Env. Micr. 46:1370-1379.
Gross, D.C., Cody, Y.S., Proebsting, E.L., Randamaker, G.K., and
Spotts, R.A. 1984. Ecotypes and pathogenicity of ice nucleationactive Pseudomonas syringae isolated from deciduous fruit tree
orchards. Phytopathology 74:241-248.
Haas, J.H., and Rotem, J. 1976. Pseudomonas lachrymans adsorption,
survival, and infactivity following precision inoculation of
, leaves. Phytopathology 66:992-997.
Hirano, S.S., Nordheim, E.V., Arny, D.C., and Upper, C.D. 1982.
Lognormal distribution of epiphytic bacterial populations on leaf
surfaces. App. Env. Micr. 4^:695-700.
95
____________, Rouse, D.I., and Upper, C.D. 1984. Epidemiology of
bacterial brown spot and ecology of Pseudomonas syrlngae pv.
syringae on Phaseolus vulgaris. Pages 24-27 in: Proceedings of
the 2nd working group on Pseudomonas syringae pathovars. C.G.
Panagopoulos, P.G. Psallidas and A.A. Alivizatos eds. The
Hellenic Phytopathological Society, Athens, Greece.
Hirano, S.S., and Upper, C.D. 1983. Ecology and epidemiology of
foliar bacterial plant pathogens. Ann. Rev. Phytopathol. 21:243269.
____________, and Upper, C.D. 1985. Ecology and physiology of
Pseudomonas syringae. Biotechnology 3 :1073-1078.
Khodair, A.A., and Ramadani, A.S. 1984. Rainfall washing effect on
bacteria and fungi, and their recolonization of aerial surfaces
of some wild plants in Saudi Arabia. J. Coll. Sci., King Saud
Univ. 15:365-377.
Kim, H.K., Orser, C., Lindow, S.E., and Sands, D.C. 1987. Xanthomonas
campestris pv. translucens strains active in ice nucleation.
Plant Disease 71:994-997.
King, E.O., Ward, M.K., and Raney, D.E. 1954. Two simple media for
the demonstration of pyocyanin and fluorescin. J. Lab. Clin.
Med. 44:301-307.
Klemnt, Z. 1963. Rapid detection of the pathogenicity of
phytopathogenic Pseudomonads. Nature 199:299-300.
Kovacs, N. 1956. Identification of Pseudomonas pyocyanea by the
oxidase reaction.
Nature 178:703.
Kozloff, L.M., Lute, M„ and Westaway, D. 1984. Phosphatidylinositol
as a component of the ice nucleating site of Pseudomonas syringae
and Erwinia herbicola. Science 226:845-846.
Latorre, B.A., Gonzalez, J.A., Cox, J.E., and Vial, F . 1985.
Isolation of Pseudomonas syringae pv. syringae from cankers and
effect of free moisture on its epiphytic populations on sweet •
cherry trees. Plant Disease 69:409-412.
Leben, C., Schroth, M.N., and Hildebrand, D.C. 1970. Colonization and
movement of Pseudomonas syringae on healthy been seedlings.
Phytopathology 60:677-680.
Leben, C., and Whitmoyer, R.E. 1979.
Can. J. Micro. 25:896-901.
Adherence of bacteria to leaves.
Lindemann, J., Arny, D.C., Hirano, S.S., and Upper, C.D. 1981.
Dissemination of bacteria, including Pseudomonas syringae, in a
bean plot. Phytopathology 71:890.
96
____________, Arny1 D.C., and Upper, C.D. 1984. Epiphytic populations
of Pseudomonas syringae pv. syringae on snap bean and nonhost
plants and the incidence of bacterial brown spot disease in
relation to cropping patterns. Phytopathology 74:1329-1333.
Lindemann1 J., Constantinidou1 H.A., Barchet1 W.A., and Upper. C.D.
1982. Plants as sources of airborne bacteria, including ice
nucIeation-active bacteria. Appl. Env. Micr. 44:1059—1063
, Joe, L., and Moayeri1 A. 1985a. Reciprocal competition
between IMA+ wild-type and INA- deletion mutant of Pseudomonas on
strawberry blossoms. Phytopathology 75:1361.
____________, and Suslow, T.V. 1937. Competition between ice
nucleation—active wild type and ice nucleation-deficient deletion
mutant strains of Pseudomonas syringae and P^ fluorescens biovar
I and biological control of frost injury on strawberry blossoms.
Phytopathology 77:882-886.
, Suslow1 T.V., Joe, L., and Moayeri1 A. 1985b. Efficacy
of INA- deletion mutant strains of Pseudomonas for biological
control of frost injury to strawberry blossoms. Phytopathology
75:1343.
, and Upper, C.D. 1985. Aerial dispersal of epiphytic
bacteria over bean plants. Appl. Env. Micr. 50:1229-1232.
Lindow1 S.E. 1981. Frost damage to pear reduced by antagonistic
bacteria, bactericides, and ice nucleation inhibitors.
Phytopathology 71:237.
, 1982. Epiphytic ice nucleation-active bacteria. Pages
335-362 in: Phytopathogenic Prokaryotes, Vol. I. M.S. Mount and
G.H. Lacy eds. Academic Press, New York.
, 1983a. The role of bacterial ice nucleation in frost
injury to plants. Ann. Rev. Phytopathol, 21: 363-384.
, 1983b. Methods of preventing frost injury caused by
epiphytic ice nucleation active bacteria. Plant Disease 67:327332. '
, 1985. Ecology of Pseudomonas syringae relevant to the
field use of ice-deletion mutants constructed in vitro for plant
frost control. Pages 23-35 in: Engineered Organisms in the
Enviroment: ' Scientific Issues. Proceedings of a crossdisciplinary symposium held in Philadelphia, Pennsylvania 10-13
June 1985. H.O. Halvorson1 D. Pramer1 and M. Rogul eds.
American Society for Microbiology, Washington, D.C.
97
, 1986. Epiphytic fitness and host preference among ice
nucleation active strains of Pseudomonas syringae.
Phytopathology 76:1068-1069.
____ , S.E., Arny, D.C., and Upper, C.D. 1977. Distribution of
epiphytic ice nucleation-active strains of Pseudomonas syringae.
Proceedings of the American Phytopathologies! Society 4:107.
Lindow, S.E., Arny, D.C., and Upper, C.D. 1978b. Erwinia herbicola:
a bacterial ice nucleus active in increasing frost injury to
corn. Phytopathology 68:523-527.
_____________ , S.E., Arny, D.C., and Upper, C.D. 1983a Biological
control of frost injury: an isolate of Erwinia herbicola
antagonistic to ice nucleation active bacteria. Phytopathology
73:1097-1102.
____________, Arny, D.C., and Upper, C.D. 1983b. Biological control
of frost injury: establishment and effects of an isolate of
Erwinia herbicola antagonistic to ice nucleation active bacteria
on corn in the field. Phytopathology.73:1102-1106.
, Hirano, S.S., Barchet, W.R., Arny, D.C., and Upper, C.D.
1982. Relationship between ice nucleation frequency of bacteria
and frost■injury. Plant Physiol. 70:1090—1093.
Luisetti, J., and Gaignard, J.L. 1984. Variations in the distribution
of Pseudomonas persicae epiphytic populations. Pages 17-18 in:
Proceedings of the 2nd working group on Pseudomonas syringae
pathovars. C.G. Panagopoulos, P.G. Psallidas, and A.S.
Alivizatos eds. The Hellenic Phytopathoiogical Society,- Athens,
Greece.
Naki, L.R., Galyan, E.L., Chang-chien, M., and Caldwell, D.R. 1974.
Ice nucleation induced by Pseudomonas syringae: Appl. Micr. 28:
456-459.
.
'/-V '
___ _______ and Willoughby, ■K.J.-: 197.8. Bacteria ,as biogenic sources
of freezing nuclei:. J. Appl. Meteorol: ■17:1049-1053.
Mandrioli, P.., Negrini., M.G.,..Cesari,'G., and, Morgan, G. 1984. .
'Evidence for long range transport of biological and anthropogenic
aerosol particles’in the atmosphere. Grana 23:43-53.
Mariano, R., and McCarter, S.M.'1985. Scanning electron microscopy
observation of Pseudomonas syringae pv. S^ pyringae and P.
syringae pv. tomato on tomato and epiphytic weed hosts.
Phytopathology 75:1381. ■'
•.
Mason, B.J.: and Hallett, J. 1957.
179:357-359.
Ice-forming nuclei.
Nature
98
Mew, T.W., and Kennedy, B.W. 1971. Growth of Pseudomonas glycinea on
the surface of soybean leaves. Phytopathology 61:715-716.
, and Kennedy, B.W. 1982. Seasonal variation in
populations of pathogenic Pseudomonads on soybean leaves.
Phytopathology 72:103-105.
Morris, C.E., and Rouse, D.I. 1985. Role of nutrients in regulating
epiphytic bacterial populations. Pages 63-82 in: Biological
Control on the Phylloplane.
G.E. Windels and S.E. Lindow eds.
The American Phytopathological Society, St. Paul, Minnesota.
Orser, C.S., Lotstein, R., Staskawicz, B.J., Dahlbeck, D., Lahue, E.,
Willis, D.K., Lindow, S.E., and Panopoulos, N.J. 1984. Molecular
genetics of bacterial ice nucleation. Pages 98-100 in:
Proceedings of the 2nd working group on Pseudomonas syringae
pathov'ars. C.G. Panagopoulos, P.G; PsalIidas, and A.S.
Alivizatos eds. The Hellenic Phytopathological Society, Athens,
Greece.
Otta, J.D. 1972. Wheat leaf mecrosis incited b y ■Pseudomonas syringae.
Phytopathology 62:1110.
, 1974. Pseudomonas syringae incites a leaf necrosis on
spring and winter wheats in South Dakota. Plant Dis. Rep.
58:1061-1064.
, 1977, Occurence and characteristics of isolates of
Pseudomonas syringae on winter wheat. Phytopathology 67:22-26.
Palleroni, N.J., 1984. Gram-negative aerobic rods and cocci. Family
I: Pseudomonadaceae. Genus I: Pseudomonas. Pages 141-199 in:
Bergey1S Manual of Systematic Bacteriology, Vol. I. N.R. Krieg,
and J.G. Holt eds. Williams and Wilkins, Baltimore.
Panagopoulos, C.G., and Crosse, J.E. 1964. Frost injury as a
predisposing factor in blossom blight of pear caused by
Pseudomonas syringae van Hall. Nature 202:1352.
Parker, B.C. 1979.
Life in the sky.
Natural History 79(8):54-59.
Paulin, J.P., and Luisetti, J. 1978. Ice nucleation activity among
phytopathogenic bacteria. Pages 725-731 in: Proceedings of the
4th International Conference on Plant Pathogenic Bacteria, Vol.
2. Station de Pathologie Vegetale et Phytobacteriologie, Angers,
France.
Preece, T.F., and Wong, W.C. 1981. Detectable attachment of bacteria
to intact plant and fungal surface's. Pages 399-410 in:
Microbial Ecology of the Phylloplane. J.P. Blakeman ed. Academic
Press, London. .
99
Rouse, D.J., Hirano, S.S., Lindemann, J., Nordheim, E.V., and Upper,
C.D. 1981. A conceptual model for those diseases with pathogen
population multiplication independent of disease development.
Phytopathology 71:901.
Sands, D.C., Kenfield, D.S., and Scharen, A.L. 1977. Observations of
leaf-spotting Pseudomonads on wheat and.barley. Proceedings of
the American Phytopathological Society 4:210.
, Langhans, V.E., Scharen, A.L., and de Smet, G. 1982.
The association between bacteria and rain and possible resultant
meteorological implications.
Idojaras 86:148-152.
____________, Schroth, M.N., and Hildebrand, D.C. 1970. Taxonomy of
Phytopathogenic Pseudomonads.
J. of Bacteriol, 101:9-23.
, Schroth, M.N., and Hildebrand, D.C. 1980. Pseudomonas.
Pages 36-44 in: Laboratory Guide for Identification of Plant
Pathogenic Bacteria. N.W. Schaad ed. The American
Phytopathological Society, St. Paul, Minnesota.
Scharen, A.L., Bergman, J.W., and Burns, E.E. 1976. Leaf diseases of
winter wheat in Montana and losses from them in 1975. Plant Dis.
Rep. 60:686-690.
SchnelI, R.C., and Vali, G. 1972. Atmospheric ice nuclei from
decomposing vegetation. Nature 236:163-165.
_______ , and Vali, G. 1973. World-wide source of leaf-derived
freezing nuclei. Nature 246:212—213.
____________, and Vali, G. 1976. Biogenic ice nuclei: Part I.
Terrestrial and marine sources. J. Atm. Sci. 33:1554-1564.
Sellam, M.A., and Wilcoxon, R.D. 1976. Bacterial leaf blight of wheat
in Minnesota. PI. Dis. Rep. 60:242— 245.
Smitley, D.R., and McCarter, S.M. 1982. Spread of Pseudomonas
syringae pv. tomato and role of epiphytic populations and
environmental conditions in disease development. Plant Disease
66:713-717.
Sule, S., and Seemuller, E. 1987. The role of ice formation in the
infection of sour cherry leaves by Pseudomonas syringae pv.
syringae. Phytopathology 77:173-177.
Thornley1 M.J. 1960. The differentiation of Pseudomonas from other
Gram-negative bacteria on the basis of apginine metabolism. J.
Appl. Bact. 23:37-52.
100
Valis G., Christensen, M., Fresh, R.W., Galyan, E.L., Maki, L.R., and
SchnelI, R.C. 1976. Biogenic ice nuclei. Part II: Bacterial
sources. J. Atm. Sci. 33:1565-1570.
Venette, J.R., and Kennedy, B.W. 1975. Naturally produced aerosols of
Pseudomonas glycinea. Phytopathology 65:737-738.
Weaver, D.J. 1978. Interaction of Pseudomonas syringae and freezing
in bacterial canker on excised peach twigs. Phytopathology
68:1460-1463.
Wimalaj eewa, D.L.S., and Flett, J.D. 1985. A study of populations of
Pseudomonas syringae pv. syringae on stonefruits in Victoria.
Plant Pathology 34:248-254*
Wolber, P.K., Deininger, C.A., Southworth, M.W., Vandekerckhove, J.,
van Montagu, M., and Warren, G.J. 1986. Identification and
purification of a bacterial ice nucleation protein. Proc. Natl.
Acad. Sci. USA 83:7256-7260.
____________, and Warren, G.J. 1986. Structural modeling of the ice
nucleation protein of Pseudomonas syringae. Biophys. J . 49:293a.
Yankofsky, S.A., Levin, Z., Bertold, T., and Sandlerman, N. 1981.
Some basic characteristics of bacterial freezing nuclei. J.
Appl. Meteorol. 20:1013-1019.
Zettlemoyer, A.C., Tcheurekdj ian, N., Chessick, J.J. 1961.
properties of Silver Iodide. Nature 192:653.
Surface
101
APPENDIX
102
LIST OF MEDIA USED
Inorganic chemicals and antibiotics were purchased from Sigma
Chemical Company. Organic chemicals were purchased from Difco
Laboratories. All media were autoclaved at 121°C and 20 Ib/scj inch
for 20 minutes.
.
.
'
King's medium B
Water
Proteose Peptone #3
K 2HPO4
MgSO4 .
Glycerol
Bacto-Agar
I I
20 S
I,.5 g
3 g
17 ml
15 g
BCBRVB
King's medium B
I I
Autoclave, cool to 50°C, then add a mixture.of the following
antibiotics in 70% ethanol:
Bacitracin
Vancomycin
Rifampicin
Cycloheximide
Benomyl .
10 mg
6 mg
0 .5 mg
100 mg
500 mg
BCRS (Modified King' s medium B used in the 1986,, 1987 dissemination
studies with marked strains).
1986 Study:
King's medium B
I I
Antibiotics added as previously:
1987 Study:
Rifampicin
Streptomycin
Cycloheximide
1000 mg
1000 mg
100 mg
Rifampicin
Streptomycin
Cycloheximide
100 mg
500 mg
100 mg
BCKS (Modified King' s medium B used in the 1987 dissemination study
with marked strains).
103
King's medium B
I I
Antibiotics added as previously:
Kanamycin
Streptomycin
Cycloheximide
10 mg
500 mg
100 mg
Thornley's medium 2A (for arginine dehydrolase, activity tests).
Water
Peptone
NaCl
R 2HPO4
Bacto-Agar
Phenol red
Arginine HCl
11.
1.0 g
5.0 g
6.3 g
3.0 g
0.01 g
.10.0 g
Adjusted to pH 7.2
Ayers et al. medium (for utilization of.carbohydrates).
Water
NH4H2PO4
KCl
MgSO4 .TH2O
Bromothymol blue
Cl.6% ale. sol.)' .
I l'
I-Os
0.2 g
0.2 g
'1.0 ml
'
After autoclaving. cool at 50oC; then add- a filter sterilized aqueous
solution of carbohydrate at a final concentration of. I g/1.
MONTANA STATE UNIVERSITY LIBRARIES
3
762 1002
O