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
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TABLE OF CONTENTS
Page
LIST OF TABLES...... vi
LIST OF FIGURES...... ix
ABSTRACT...... '...... xii
INTRODUCTION...... 1
LITERATURE REVIEW...... 3
MATERIALS AND METHODS...... 14
Variability in syringae population size among barley cultivars...... 14- Plant Material...... 14 Planting...... 15 Leaf Sampling...... 15 Leaf Samples Processing...... 16 Bacterial Colony Identification...... 16 Collection of P^_ syringae Isolates...... 17 Analysis of Results...... 19 Dirunal Population Changes...... 19 Plant-to-Plant Dissemination...... 19 Planting...... 19 Leaf Sampling...... Leaf Samples Processing...... 20 Analysis of Results...... 21 1986 Dissemination Experiment with Marked Strains...... 21 Marking Procedure...... 21 Planting...... 22 Inoculum Production and Inoculations...... 24- Leaf Sampling...... 24 Leaf Samples Processing...... 24 1987 Dissemination Experiment with Marked Strains...... 25 Marking Procedure...... 25 Doubling Times and INA of Double Marked Strains...... 26 Planting...... Inoculum Production and Inoculations...... 27 Leaf Sampling...... 28 Leaf Samples Processing...... 28 V
TABLE OF CONTENTS— Continued
Page
Air Dissemination of syringae...... 28 Use of an Air Pump...... 29 Display of Petri Dishes...... 29
RESULTS...... 32
Variability in Pi syringae population sizes among barley cultivars...... 32 Dirunal Population Changes...... 74 Plant-to-Plant Dissemination...... 75 1986 Experiment with Marked Strains...... 75 1987 Experiment with Marked Strains...... 80 Doubling Times...... :...... 81 Epiphytic Survival of the Marked Strains...... 84 Air Dissemination of P. syringae...... 85
DISCUSSION...... 89
LITERATURE CITED...... 92
APPENDIX...... 101
List of Media Used 102 Vi
LIST OF TABLES
Table Page
1. List of the 24 barley lines and cultivars examined for epiphytic populations of F_^ syringae in the field, Bozeman, 1986...... 15
2. Antibiotics and concentrations (ppm) tested for marking isolates of P. syringae, 1986, 1987...... 23
3. List of syringae isolates used in the experiments to create antibiotic-resistant (marked) strains...... 23
4. 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
11. syringae populations on AR7, Bozeman, 1986...... 37
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. syringae populations on AR17, Bozeman, 1986...... 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. syringae populations on ERSHABET, Bozeman, 1986...... 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 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...... '...... I 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 total, marked INA+, and marked INA- P. syringae on AR15, Bozeman, 1987...... ' ' I' ‘ ■ 51. Populations of total and marked INA+ P. syringae on AR13, Bozeman, 1987......
52. Dissemination of rifampicin-streptomycin marked INA+ P. syringae...... ix
LIST OF FIGURES
Figure Page
1. Petri dish disple the inoculated fields of ARl3 and AR15, 30
2. Area under the ,pc for AR17, repetition I, Bozeman, 1986...., 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. syringae populations on BOLD, Bozeman, 1986...... 56
23. P . syringae populations on STEPTOE, Bozeman, .1986...... 57
24. P. syringae populations 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 on 24 barley cultivars and lines, Bozeman, 1986...... ^l
34. P. syringae populations on six barley cultivars and lines, Bozeman, 1987...... ^ ^
35. Diurnal change in epiphytic populations of P . syringae on AR13 (7/24-25/87)...... 74
36. Results of the first plant-to-plant dissemination experiment...... 7^
37. Results of the second plant-to-plant dissemination experiment...... 7^
38. Populations of marked Pjl syringae on KLAGES, Bozeman, 1986...... 7^
39. Populations of marked P\_ syringae on CLARK, Bozeman, 1986...... 7^
40. Populations of marked Pjl syringae on 222-1, Bozeman, 1986...... 7^ xi
LIST OF FIGURES— Continued
Figure Page
41. Populations of marked Pi syringae on 222-9, Bozeman, 1986..... 79 . 42. Populations of total, marked INA+, and marked !NA- P. syringae on AR15, Bozeman, 1987...... 86
43. 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 nuclea- tion 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: halo blight on oats and leaf blight on wheat and barley. Leaf blight was first observed in South Dakota in 1965, on spring and winter wheat, and it was first reported by Otta in 1972. On wheat, symptoms appear generally from the boot to the 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. These catalysts are called ice nuclei. 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, 1957;
Zettlemoyer et al., 1961; Lindow 1983a). 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 nuclea-
tion 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. They are usually low in young, vegetative tissue. 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). Similar
results were obtained by Bovallius et al (1978a). 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 AR7 AR13 .222-9 AR2 AR8 AR14 BOLD AR3 AR9 ARl 5 STEPTOE AR4 ARlO AR16 KLAGES AR5 ARll ARl 7 CLARK AR6 AR12 222-1 ERSHABET
Planting
All entries were planted in four randomized replications. Plots consisted of four rows, three m long and 30 cm apart. Each row received five g of seed planted with a cone seeder. 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. The plots of the 20 dryland lines in Bozeman, 1986 were not irrigated. 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). From the number of P^ syringae-like
colonies 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. P. syringae gives a negative reaction to this test. 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. Results (complete collapse of the tissue) were recorded after 24 hours. The third and fourth tests. consist of streaking aqtieous bacterial suspensions on plates of Ayers' medium, supplemented with,D (-)tartrate and alpha- ketoglutarate (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. P. syringae utilizes .
= V
■■■.=■■■■■. - " ■ 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. Sampling, plating, and incubation, were performed
as previously. 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, two-
row 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). Sixteen
isolates of syringae were used in this round (Table 3).
2nd Round: 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: the
selection of strains resistant to two different antibiotics. 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 at the A.H. Post Research Farm,
west of Bozeman, Montana. The field was divided in four equal parts,
one for each entry. 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 .. I 1987 I 1st I Streptomycin: 10,100,1000 11st I Streptomycin: 500 Round I Erythromycin: 10,100,1000 (Round |Rifampicin: 100 ("Disk" I Rifampicin: 0.1,1,10,100,1000 I (Plating)I Method) I I I I I I 2nd I Streptomycin: 1000 12nd IStr.: 500-Rif.: 100 Round I Rifampicin: 1000 I Round I Str.: 500-Kan.: 10 (Plat- I [Double |Rif.: 100-Kan.: 10 ing) I [Marking I I (Plating)I I I I 3rd I Streptomycin: 1000 I I Round IRifampicin: 1000 I ’ I Double ITobramycin: 100,500 I Marking I Tetracyclin: 100,500 II (Plat- !Trimethoprim: 50,100,500 I I ing) lKasugamycin: 50,100,500 i I !Novobiocin: 50,100,500 i I I i
Table 3. 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, DG101, DG102 DG103, DG104, DG105 DG109, DGl12, DG114 DGl15, DGl16, DG117 DG118, DG119, DG120 DG121, DG122, DG123 DG124, DGl25, DG126 DG127, DG128, DG129 DG130, DG131, DG132 DG133, DG134, DG135 DG136, DG138, DG139 DG140, DG141, DG142 DG143, DG144, DG145 DG146, DGl47, DG148 DGI49, DG150, DG151 DG152, DG153 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. The plates were incubated for seven days at 21°C in the dark. 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 in the dark for five days.
2nd Round: 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. In order to do this,
seven strains of 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. The flasks were put on a wrist-action shaker, at room temperature. 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). Both fields were irrigated prior to inoculations. One piece, at the SW corner of every field was left uninoculated. 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. One sample was also taken from the uninoculated plots. Samples were taken in order to determine any background 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. The plates were incubated for 5 days at
21°C in the dark. 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, 1975,
Lindemann et al., 1982). 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). Such samples were taken under five types of conditions:
I. Petri dishes during the day (morning or afternoon) for up to
two hours (five samples). 1 5 ( 6 3 ) 1 6 ( 6 2 ) 17(141)
10(10) 12(10) 18(1 10) 14( 10) 19( 59) 11(0) 13(0)
9( 0) 6 ( N<-
2 0 ( 1 0 0 ) 2 1( 80)
2 2 ( 7 0 )
Figure I. Petri dish display sites around the inoculated fields of AR15 and AR13. In parenthesis, closest distance to the fields, in m. 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
6 5 4
3 log cfu/leaf LO
2
I O
O 10 20 30 40 dcye
Figure 2. Area under the population curve for AR17, repetition I, Bozeman, 1986. 34
Table 4. Comparison of the epiphytic populations of syringae on the 24 entries tested in the field, Bozeman 1986.
LOW I INTERMEDIATE | HIGH I I Entry Mean Entry Mean Entry Mean (log cfu/leaf) (log cfu/leaf). (Log cfu/leaf)
AR9 2.12 A Clark* 2.92 ABCD AR3 4.17 BCDE AR5* 2.18 AB AR 10 2.95 ABCD KLAGES 4.17 BCDE AR8 2.31 AB 222-9 3.05 ABCD AR15* 4.40 CDE 222-1 2.42 AB AR7 3.11 ABCD AR6* 4.46 CDE AR17 2.67 ABC BOLD 3.19 ABCD ERSHABET 4.72 DE ARll 3.41 ABCD AR13* : 5.53 E AR2 3.52 ABCD AR16 3.59 ABCD ARl 2 3.60 ABCD ARl 4 3.65 ABCD ARl 3.71 ABCD AR4* 3.76 ABCD STEPTOE 3.77 ABCD
LSD (by t, 5% Sign Level) = 1.07 *Selected for further study.
Table 5. P^ syringae populations on ARl, 1986, Bozeman.
Date Day Repetition Symptoms log cfu/leaf % INA+
6/27 0 I 0 4.46 100 6/27 0 II 0 2.64 100 7/1 4 III 0 — — 7/1 4 IV 0 0.74 100 7/8 11 II 0 3.18 100 7/9 12 III 0 4.08 100 7/11 14 ■IV 0 2.74 0 7/15 18 I 0 4.08 100 7/16 19 II 0 4.60 67 7/17 20 III 0 4.08 100 7/18 21 IV I 4.23 100 7/23 26 I 0 5.23 100 7/26 29 II 0 6.00 ■ 100 8/1 35 I 0 6.65 100 8/5 39 II 0 5.98 100 35
Table 6. P . syringae populations on AR2, 1986, Bozeman.
Uate Day Repetition Symptoms log cfu/leaf % INA+
6/26 0 I . . 0 0 6/27 I II . 0 4.36 100 6/30 4 III. 0 0 — 7/1 5 IV 0 1.59 100 7/5 9 I 0 2.52 67 7/8 12 II 0 3.34 100 7/9 13 III . o 3.11 100 7/11 1;5 IV 0 3.00 100 7/15 19 I , 0 2.70 100 7/16 20 II . 0 2.59 100 7/17 21 Ill 0 3.83 100 7/18 22 IV 0 3.52 60 7/23 27 I . 0 ■ 4.74 80 7/26 30 II 0 5.81 100 7/30 34 IV 0 5.11 100 7/31 35 III 0 4.81 100 8/1 36 I 0 5.40 100 8/5 40 III 0 5.90 40
Table 7. Pl syringae populations on AR3, 1986, Bozeman.
Date Day Repetition Symptoms log cfu/leaf . % INA+ ‘
6/26 0 I 0 2.04 100 6/30 4 II 0 0 — 6/30 4 III 0 ’ 1.34 50 7/1 5 ■ IV 0 1.59 100 7/5 9 I 0 4.32 100 7/8 12 II 0 4.48 100 7/9 13 III 0 4.08 100 7/11 15 IV 0 3.70 100 7/15 19 I 0 4.86 . 100 7/16 20 II 0 4.57 100 7/17 21 III 0 4.70 100 7/18 22 IV 0 3.45 100 7/23 27 I 0 6.18 100 7/26 30 II 0 6.45 100 7/31 35 III 2 6.26 100 8/5 40. II - 2 6.70 100 36
Table 8. syringae populations on AR4, 1986, Bozeman.
Date Day Repetition . Symptoms log cfu/leaf % INA+
6/27 . 0 I 0 0 6/30 3 IT 0 0 — 6/30 3 III . 0 0 — 6/30 3 IV 0 1.64 75 7/8 11 II 0 3.23 100 7/9 12 III 0 3.26 100 7/11 14 IV 0 3.86 100 7/15 18 I 0 4.79 83 7/16 19 II 0 4.26 100 7/17 20 III 0 4.34 100 7/18 21 IV 0 3.97 100 7/23 26 I 0 , 5.70 80 7/26 29 II 0 5.85 100 7/31 34 III 0 6.88 20 8/1 35 I 0 5.88 100 8/5 39 III 0 5.78 20
Table 9 . Pl syringae populations on AR5, 1986, Bozeman.
Date Day Repetition Symptoms log cfu/leaf % INA+
6/26 0 I 0 0 . ■ 6/27 I II ' 0 0 — — 7/1 5 III 0 0 • — 7/3 7 IV 0 1.70 60 7/5 9 I 0 0 — 7/8 12 II 0 0.74 100 7/9 13 III 0 3.89 83 7/11 15 IV 0 2.45 13 7/15 19 I 0 3.04 100 7/16 20 II 0 2.34 0 7/17 21 III 0 2.89 40 7/18 22 IV 0 2.83 80 7/23 27 I 0 2.93 80 7/26 30 II . o 3.70 ■100 7/30 34 IV 0 4.70 . 83 37
Table 10. syringae populations on AR6, 1986, Bozeman.
Date Day Repetition Symptoms log cfu/leaf % INA+
6/26 0 I 0 2.04 . 100 6/30 4 II 0 4.89 86 7/1 5 III 0 5.30 100 7/1 5 IV 0 0 — 7/5 9 I P 3.83 100 7/8 12 II 6 5.00 100 7/9 13 III 0 4.83 loo 7/11 15 IV 0 4.97 100 7/15 19 I 0 4.92 100 7/16 20 II 0 5.20 100 7/17 21 III 0 4.79 100 7/18 22 IV 0 5.00 100 7/23 27 I 0 5.34 100 8/5 40 II 0 5.74 80
Table 11. syringae populations on AR7, 1986, Bozeman.
Date Day Repetition Symptoms log cfu/leaf % INA+
6/26 0 I 0 0 6/27 I II 0 0 — 6/27 I III 0 0 — 7/1 5 IV 0 1.34 100 7/5 . 9 I 0 1.64 100 7/8 12 Il 0 1.74 80 7/9 13 III P 4.34 100 7/11 15 IV 0 4.11 38 7/15 19 I ■ 0 3.23 73 7/16 20 II 0 3.26 75 7/17 21 III 0 4.79 100 7/18 22 IV 0 3.59 100 7/23 27 I 0 4.85 100 7/26 30 II 0 5.32 100 7/30 34 IV ■ 0 5.26 100 8/1 36 I 0 5.81 100 38
Table 12. Pl syringae populations on AR8, 1986, Bozeman.
Date Day Repetition Symptoms log cfu/leaf % INA+
6/27 0 I ' 0 0 6/30 3 II 0 3.52 100- 7/1 4 III 0 . 0 — 7/1 4 IV 0 0.74 100 7/8 11 II 0 1.45 100 7/9 12 III .0 1.52 60 7/11 14 iv 0 1.70 100 7/15 18 I 0 1.45 20 7/16 19 II 0 4.18 17 7/17 20 III 0 . 2.92 80 7/18 21 IV . o 0 — 7/23 26 I • 6 4.20 100 7/26 29 II 0 5.26 63 7/30 33 IV 0 3.54 75 7/31 34 III 0 5.15 100 8/5 40 II 0 4.40 80
Table 13. P l syringae populations, on AR9, 1986, Bozeman.
Date Day Repetition Symptoms log cfu/leaf % INA+
. • . : 6/26 0 II 0 ...-O 6/26 0 I 0 2.04 100 6/27 I III 0 0 — 6/30 4 IV 0 0 — 7/5 9 I 0 1.23 67 7/5 9 II .0 0 — 7/8 12 . II 0 3.28 100 7/9 13 III 0 2.52 17 7/11 15 IV 0 2.23 40 7/15 19 I 0 2.34 100 7/16 20 - II 0 4.45 100 7/17 21 III 0 4.18 100 7/18 22 IV 0 3.23 100 7/23 27 ■ I I 2.70 100 7.26 30 II 2 4.48 50 Table 14. P_L syringae populations on ARID, 1986, Bozeman
Date . Day Repetition Symptoms log cfu/leaf % INA+
6/26 0 Il 0 0 6/26 0 I 0 0 -- 6/30 4 III 0 1.74 100 7/1 5 IV 0 2.52 67 7/5 9 II 0 4.62. 100 7/5 9 I 0 2.52 100 7/8 12 II 0 3.74 100 7/9 13 III 0 3.70 - 100 7/11 . 15 IV. 0 3.45 100 7/15 19 I 0 3.92 100 7/16 20 II 0 3.59 100 7/17 21 III 0 0 — 7/18 22 IV I 4.38 100 7/23 27 I ■ 0 . 5.54 100
Table 15. syringae populations on ARlI, 1986, Bozeman.
Date Day Repetition Symptoms log cfu/leaf % INA+
6/27 0 I 0 0 6/27 0 II 0 0 — 6/30 3 III . 0 0 —— 7/3 6 IV 0 2.00 100 7/8 11 II 0 2.70 0 7/9 12 III 0 2.83 100 7/11 14 IV 0 3.64 67 7/15 18 I 0 3.79 100 7/16 19 II 0 3.23 86 7/17 20 III 0 4.23 50 7/18 21 IV 0 3.64 20 7/23 26 I 0 5.60 100 7/26 29 II I 5.90 60 7/30 33 IV 0 6.04 100 7/31 34 III 0 6.18 71 8/5 39 III 0 6.54 100 . 40
Table 16. P^_ syringae populations on ARl2, 1986, Bozeman.
Date Day . Repetition Symptoms log cfu/leaf % INA+
6/27 0 I 0 2.59 100 6/30 3 II 0 2.79 100 7/1 4 III 0 1.70 0 7/1 4 IV 0 4.89 100 7/8 11 II 0 4.11 100 7/9 12 III 0 2.08 100 7/11 14 IV 0 3.45 100 7/15 18 I 0 4.97 100 7/16 19 . II 0 4.86 20 7/17 20 III 0 2.97 50 7/18 21 IV 0 2.79 100 7/23 . 26 I 0 4,85 100 7/26 29 II 0 4.98 60
Table 17. P. syringae populations on ARl3, 1986, Bozeman.
Date Day Repetition Symptoms log cfu/leaf % INA+
6/26 0 I 0 5.60 100 6/27 I II 0 4.53 100 6/27 I III 0 5.69 100 6/30 4 IV 0 . 4.04 100 7/5 9 I 0 4.64 100 7/8 12 II • 0 5.23 100 7/9 13 III 0 5.28 100 7/11 15 IV 0 5.11 25 7/15 19 I 0 5.11 100 7/16 20 II 0 5.40 100 7/17 21 III 0 5.83 100 7/18 22 ■ IV 2 5.74 100 7/23 27 II 6.54 100 7/26 30 II 2 6.81 100 7/31 35 III I 6.81 100 8/1 36 I 0 6.60 100 8/5 40 II.I 6.88 100 Al
Table 18. syringae populations on ARIA, 1986, Bozeman.
Date Day Repetition Symptoms log cfu/leaf % INA+
6/26 0 I ' 0 0 __ 6/27 I II 0 0 — 7/1 5 III 0 0 — 7/1 5 IV 0 2.7A 100 7/5 9 I 0 3.15 100 7/8 12 II 0 3.28 100 7/9 13 III 0 5.A6 100 7/11 15 IV I 3.83 17 7/15 19 I 0 3.59 100 7/16 20 II 0 A.3A 100 7/17 21 III 0 A. 28 75 7/18 22 IV I 5.00 100 7/23 27 I 0 5.15 50 7/26 30 II ■ 0 5.08 100 7/31 35 III 0 6.15 100
Table 19. P. syringae populations on AR15, 1986, Bozeman.
Date Day Repetition Symptoms log cfu/leaf % INA+
6/26 0 I 0 A.00 100 6/30 A II 0 1.86 60 7/1 5 III 0 2.70 100 7/1 5 IV 0 2.6A 100 7/5 9 I 0 3.20 100 7/8 12 II . 0 A.7A 67 7/9 13 Ill 0 A.AO 100 7/11 15 IV 2 A.52 100 7/15 . 19 I 2 A. 86 100 7/16 20 II I 5. AS 100 7/17 21 III 2 5.08 100 7/18 22 IV 2 5.08 100 6.23 27 I I 5.51 o 100 7/26 30 II 2 5.90 100 8/5 AO II 2 6.AS 80 42
Table 20. syringae populations on AR16, 1986, Bozeman.
Date Day Repetition Symptoms log cfu/leaf % INA+
6/26 0 I' . 0 0 __ 6/27 I II 0 0 — - 6/30 4 III 0 1.64 100 7/3 7 IV 0 . 2.34 100 7/5 9 I 0 3.72 100 7/8 12 II 0 2.70 100 7/9 13 III 0 5.43 100 7/11 15 IV 0 3.79 100 7/15 19 . I 0 3.89 71 7/16 20 II 0 4.04 0 7/17 21 III 0 4.48 91 7/18 22 IV 0 . 2.97 80 7/23 27 I 0 4.78 100 7/26 30 . II 0 4.98 100 7/30 34 IV 0 4.78 100
Table 21. syringae populations on AR17* 1986, Bozeman. - Date Day . Repetition Symptom's log cfu/leaf. % INA+
6/26 0 II 0 0 __ 6/26 0 I 0 0 —— 6/30 4 . III 0 1.59 100 7/1 5 IV 0 0 —— 7/5 9 II 0 2.23 100 7/5 9 I 0 0.74 100 7/8 12 II 0 1.45 100 7/9 13 III 0 2.38 100 7/11 15 IV 0 2.15 100 7/15 19 ' I 0 3.86 100 7/16 20 II 0 3.86 100 7/17 21 III 0 3.00 0 7/18 22 IV 0 2.15 83 7/23 27 I 0 3.65 83 7/26 30 II 0 4.98 100 7/30 34 IV I 3.20 0 7/31 35 III 0 5.74 100 8/1 36 I I 4.70 100 43
Table 22. Pl syringae populations on 222-1. 1986, Bozeman.
Date Day Repetition Symptoms log cfu/leaf % INA+
6/26 0 I 0 0 6/26 0 II 0 0 6/27 I III 0 0 6/30 4 IV 0 ■ 0.74 100 7/5 9 II 0 1.04 50 7/5 9 I 0 0 7/8 12 II 0 . 0 7/9 13 I 0 2.34 100 7/11 15 IV 0 3.00 70 7/15 19 I 0 3.34 ■ 72 7/16 20 II 0 2.23 50 7/17 21 III 0 4.08 100 7/18 22 IV 0 3.64 100 7/23 27 I 0 2.98 100 7/26 30 II 0 4.81 100 7/30 34 IV . 0 4.90 100 7/31 35 III 0 5.08 100
Table 23. Pl syringae populations on 222-9, 1986, Bozeman.
Date Day Repetition Symptoms log cfu/leaf "% INA+
6/26 0 II 0 0 6/26 0 I 0 0 ____ 6/30 4 III 0 3.18 62 6/30 4 IV 0 0 ■ —. 7/5 9 II 0 1.23 100 7/5 9 I 0 1.52 100 7/8 12 II 0 1.74 100 7/9 13 ‘ III 0 2.59 100 7/11 15 IV 0 3.08 100 7/15 19 I 0 3.11 100 7/16 20 II 0 3.59 100 7/17 21 III 0 4.26 50 7/18 22 IV 0 3.92 100 7/23 27 I 2 3.81 100 7/26 30 II I 5.81 100 7/30 34 IV 0 5.40 80 7/31 35 III 0 5.57 71 8/5 40 III 0 5.74 100 44
Table 24. I\ syringae populations on BOLD. 1986, Bozeman.
Date Day Repetition Symptoms log cfu/leaf % INA+
6/26 0 I 0 2.70 100 6/27 I II 0 0 6/27 I III 0 0 7/1 5 IV 0 1.04 100 7/5 9 I 0 2.74 100 7/8 12 II 0 2.64 40 7/9 13 III 0 0 7/11 15 IV 0 4.11 100 7/15 19 I 0 4.89 0 7/16 20 II 0 3.20 100 7/17 21 III 0 4.52 100 7/18 22 IV 0 4.04 75 7/23 27 I 0 4.85 100 7/26 30 II 0 4.70 100 7/31 35 III I 6.23 100 8/1 36 I 0 5.88 0
Table 25. P. Syringae populations on STEPTOE, 1986, Bozeman.
Date Day. Repetition Symptoms log cfu/leaf % INA+
7/3 . o IV 0 2.53 31 7/3 0 III 2 4.96 0 7/3 0 II 0 2.52 20 7/3 0 I I 4.59 17 7/8 5 II 0 2.74 60 7/9 6 . III 0 4.74 14 7/11 8 IV 0 3.54 63 7/15 12 I 0 3.95 0 7/16 12 II 0 3.45 20 7/17 14 III 0 0 —— 7/18 15 IV 0 3.26 86 7/23 20 ' I I 5.32 0 7/26 23 II 2 4.95 0 7/30 27 IV 2 6.60 60 7/31 28 III 2 4.23 29 8/1 29 I I 5.88 0 8/5 33 I I 5.28 13 45
Table 26. Pjl syringae populations on KLAGES, 1986, Bozeman. •
Date Day Repetition Symptoms log cfu/leaf % INA+
7/3 0 IV 0 , 3.45 100 7/3 0 III 0. 3.11 100 7/3 0 II 0 4.00 100 7/3 0 I 0 4.00 100 7/8 5 II 0 4.04 100 7/9 6 III 0 4.20 78 7/11 8 IV 0 3.56 100 7/15 12 ■ I 0 4.92 0 7/16 13 II 0 . 4.15 86 7/17 14 III 0 3.64 100 7/18 15 IV 0 3.00 100 7/23 20 I 0 4.65 100 7/26 23 II 0 4.90 100 7/30 27 IV I 5.54 100 7/31 28 III I 4.90 100 8/1 29 I 0 4.54 71 8/5 33 I I 4.70 80
Table 27. syringae populations on CLARK, 1986, Bozeman.
Date Day Repetition Symptoms log cfu/leaf % INA+
7/3 0 II 0 2.04 100 7/3 0 I 0 2.34 0 7/3 0 IV 0 0 - 7/3 0 III 0 1.92 50 7/8 5 II 0 0 — 7/9 6 III - 0 3.41 90 7/11 8 IV 0 . 2.74 .0 7/15 12 I 0 4.45 60 7/16 13 II 0 2.70 50 7/17 14 III 0 2.96 100 7/18 15 IV 0 3.86 40 7/23 20 I 0 5.70 100 7/26 23 II 0 0 -- 7/30 27 IV 0 6.15 100 7/31 28 III 0 0 — 8/1 29 I 0 6.00 100 8/5 33 I 0 4.49 62 46
Table 28. P. syringae populations on ERSHABET, 1986, Bozeman.
Date Day Repetition Symptoms log cfu/leaf % INA+
7/3 0 II I 3.11 100 7/3 0 I • 0 5.00 100 7/3 0 IV 0 4.86 100 7/3 0 III 0 0.74 100 7/8 5 II 0 3.45 80 7/9 6 III 0 4.54 63 7/11 8 IV 0 3.72 . 100 7/15 12 I 0 3.08 0 7/16 13 II 0 5.52 50 7/17 14 III 0 5.26 57 7/18 15 IV 0 4.04 60 7/23 20 I 0 5.88 80 7/26 23 II 0 6.30 0 7/30 27 IV 0 6.78 0 7/31 28 III 0 5.65 40 8/1 29 I 0 5.11 67 8/5 33 I 0 5.08 86
entries were studied in Arizona. 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 iue . . yigeppltoso R, oea, 1986. Bozeman, AR2, on populations syringae P. 4. Figure
log cfu/l eaf log ofu/leaf . syringae 1986.populationson Bozeman, ARl, 47 48
Figure 5. P. syringae populations on AR3, Bozeman, 1986.
Figure 6. P. syringae populations on AR4, Bozeman, 1986. iue8 P srna ouain o R, oea, 1986. Bozeman, AR6, on populations syringae P. 8. Figure
log ofu/leaf , log ofu/leaf . syringae 1986.populationson Bozeman, AR5, 49 50
Figure 9. P. syringae populations on AR7, Bozeman, 1986.
Figure 10 P. syringae populations on AR8, Bozeman, 1986. iue 2 P srna ouain o RO Bzmn 1986. Bozeman, ARlO, on populations syringae P. 12. Figure
log ofu/1 eof log cfu/1 eof I P syringae 1986. P.populationson Bozeman, AR9, LI. 51 iue 14. Figure
log cfu/l eof log cfu/l oaf srna ouain o R2 Bzmn 1986 Bozeman, AR12, on populations . syringae P 52 iue 6 P srna ouain nA1, oea, 1986 Bozeman, AR14, on populations syringae P, 16. Figure
Icsg cfu/leof log ofu/leof syringae 1986populationson Bozeman, AR13, 53 iue 8 P srna ouain o R6 Bzmn 1986. Bozeman, AR16, on populations syringae P. 18. Figure
log cfu/loof log cfu/leaf . syringaeP. 1986.populationson Bozeman, AR15, 54 iue2. . yigeppltoso 2-, oea, 1986. Bozeman, 222-1, on populations syringae P. 20. Figure
log cfu/Ieaf log cfu/leaf . syrlngaeP.populations 1986.on Bozeman,ARl7, 55 iue 22. Figure
log CfuZIeof ' log cfuZleof . yigeppltos nBL, oea, 1986 Bozeman, BOLD, on populations syringae P. . syringaeP. 1986.populations222-9,on Bozeman, 56 iue 4 P srna ouain o LGS Bzmn 1986. Bozeman, KLAGES, on populations syringae P. 24. Figure
log cfu/l eaf log cfu/l eof . syringaeP. 1986.populationsSTEPTOE,on Bozeman, 57 Figure 26. Figure
log cfu/leaf log ofu/leaf P . syringaeP. 1986.populationsonCLARK, Bozeman, yigeppltos nESAE, oea, 1986. Bozeman, ERSHABET, on populations syringae 58 59
Table 29. Percentages of INA+ bacteria in the population of P. syririgae, Bozeman, 1986.
ENTRY 100 99-80 79-60 59-40 39-20 19-i
ARl 12 I ; I AR2 12 I 2 I AR3 14 'I AR4 8 I I 2 AR5 3 4 I I 2 AR6 11 2 AR? 9 I 2 I AR8 6 2 . 3 I I AR9 7 I 2 I ARlO 10 I ARll 6 I 2 I I ARl 2 9 IIII ARl 3 16 I ARl 4 9 III AR15 12 I 2 ARl 6 9 2 I I AR17 9 2 2 222-1 8 I I 2 222-1 11 I 2 I BOLD 9 II 2 STEPTOE I 3 . 4 8 KLAGES 12 2 2 I CLARK 5 I 2 3 2 ERSHABET 5 3 3 ■ 3 3
TOTAL 212 26 33 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 alpha- ketoglutarate, but not D (-)tartrate. This places them in the P. syringae group. 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.
Date Day Repetition Symptoms log cfu/leaf % INA+
2/28 0 I 0 1.00 100 2/28 0 II 0 2.81 100 3/1 I I 0 0.70 100 3/1 I III 0 2.00 100 3/1 I IV 0 0 — 3/2 2 II 0 2.65 89 3/2 2 III 0 2.51 0 3/3 3 IV 0 1.30 75
Table 31. P l syringae populations on AR5, 1987, Arizona.
Date Day Repetition Symptoms log cfu/leaf % INA+
2/28 0 I 0 0 —— 2/28 0 II 0 2.18 90 3/1 I I 0 0.70 0 3/1 I III 0 0.70 100 3/1 I IV I 0 — 3/2 2 ' II 0 1.70 100 3/2 2 III 0 0 — 3/3 3 IV I 1.74 100
Table 32. Pl syringae populations on AR6, 1987, Arizona.
Date Day Repetition Symptoms log cfu/leaf % INA+
2/28 0 I I 4.26 100 2/28 0 II I 3.36 100 3/1 I I I 4.20 100 3/1 I III 0 4.15 95 3/1 I IV 0 3.81 100 3/2 2 II I 5.08 100 3/2 2 III 0 4.08 94 3/3 3 IV 0 4.26 100 61
Table 33. syringae populations on AR13, 1987, Arizona.
Date Day Repetition Symptoms log cfu/leaf % INA+
2/28 O I 1.90 100 2/28 O II. 2 0.70 100 3/1 I I 2 3.04 100 3/1 I III . . 3 2.81 100 3/1 I IV 2 1.90 100 3/2 2 TI .2 3.43 100 3/2 2 III 3 ■ NT* ' — 3/3 3 . IV 2 3.43 100
NT=Not tested ■:
Table 34. syringae populations on AR15, 1987, Arizona.
Date Day Repetition Symptoms log ofu/Teaf, % INA+
2/28 0 II 1.54 86 . 2/28 0 II 2 1.00 100 3/1 I II 2.00 100 3/1 I IIT 2 0 — 3/2 2 TI 2 1.48 67 3/2 • 2 III 2 2.08 0
A fourth repetition of AR15 was not planted.
Table 35. syringae populations on CLARK, 1987, Arizona.
Date Day Repetition Symptoms log cfu/leaf % INA+
2/28 0 ‘ I 0 0.70 0 2/28 0 II 0 0 — 3/1 I I Q 0 — 3/1 I III 0 0 — 3/1 I IV 0 0.70 100 3/2 .2 II 0 2.74 91 3/2 2 III 0 1.18 100 3/3 3 IV 0 3.32 100 6.2
Table 36. Percentages of'INA+ bacteria -in the population of P1 syringae, Arizona 1987.
ENTRY 100 9S-80 : 79-60 59-40 39-20 19-0
ARA 4 . I I :. - • : 1 AR5 3 I 0 AR6 6 2 AR13 7 AR15 2 I I I CLARK 3 I I.
TOTAL 25 6 . 2 - 3
• • •
Table 37. P. syringae populations on AR4, 1987, Bozeman.
Date Day Repetition Symptom's log cfu/leaf • % INA+ • Mean/SE
7/6 ■ 0 i . o . 0 .. ■ ■ 0 II 0 0 — III 0 0 -T IV 0 0 — —
7/13 . 7 I 0 ■ 0 -- ,0.57+0.80 II ■ . 0 NT — . H I 0 0 -; IV 0 1.70 ibq .
7/20 14 ■ I. . '0 3.00 50 1.41+1.42 II b ■ 2.65 0 III o . ' 0 — IV - 0 0 / —.
7/27 21 .I b • 4.85 o 5.04+0.21 II • 0 \ NT ■ — . . Ill : 0 V 5.33 .. 56, ; IV 0 4.95 , 0
8/10 35 • ’ I , 0 , 6.59 80 6.32+0.42 ", - II ' : o • 5.95 • ' .40. III • i 6.88 . 60 IV 6 ' 5 :. 88 14
NT=Not Tested - • 63
Table CO CO P. syringae populations on AR5, 1987, :Bozeman.
Date Day Repetition Symptoms log cfu/leaf % INA+ Mean/SE
7/6 . 0 I . 0 0 0 II ' 0 0 — III 0 0 — IV 0 0, —
7/13 7 I 0 0 — — 1.32+1.33 II 0 2.48 0 . III 0 0 — IV 0 2.81 0
7/20 14 I 0 2.70 0 2.26+0.26 II 0 2.18 100 III 0 2.18 0 IV 0 2.00 0
7/21 21 I 0 4.74 0 4.45+0.20 II 0 NT — III 0 4.34 0 IV 0 4.28 0
8/10 35 I " 0 0 — — 4.35+2.59 II 0 5.95 100 III 0 6.58 78 IV 0 4.88 71
NT=Not Tested 64
Table 39. P. syringae populations on AR6, 1987, Bozeman.
Date Day Repetition Symptoms log cfu/leaf % INA+ Mean/SE
7/6 0 I 0 . 2.70 0 2.08+1.20 II 0 0 — III 0 2.70 0 IV 0 2.90 0
7/13 7 I 0 NT — 0.85+0.85 II 0 NT — III 0 1.70 0 IV 0 0 —
7/20 14 I 0 3.18 33 2.27+1.32 II 0 . 3.18 0 III ' 0 0. — IV 0 2.70 0
7/27 21 I 0 5.88 0 5.55+0.41 ‘‘ II 0 5.00 0 III 0 6.00 0 IV 0 5.30 0
8/10 35 I 0 4.70 100 4.03+0.47 II I NT —- III 0 3.70 0 IV 0 3.70 0
NT=Not Tested 65
Table 40. P. syringae populations on AR13, 1987, Bozeman.
Date ' Day Repetition, Symptoms log cfu/leaf % INA+ Mean/SE
7/6 0 I 0 2.00 33 1.64+1.87 II 0 4.54 57 III 0 0 — IV 0 0 —
7/13 7 I 0 0 — 1.23+1.74 II 0 NT — III 0 0 — IV 0 3.70 100
7/20 14 I 0 NT —— 3.30+0.30 II 0 3.60 0 III 0 3.00 0 IV b NT ——
7/27 21 I I 7.84 0 7.58+0.27 II . I 7.28 38 Ill I 7.85 41 IV 0 7.34 67
8/10 35 I I 5.88 60 5.42+0.36 II I NT — III I 5.00 100 IV I 5.40 100
NT=Not Tested 66
Table 41. P. syringae populations on ARl5, 1987, Bozeman.
Date Day Repetition Symptoms. log cfu/leaf % INA+. Mean/SE
7/6 0 I 0 0 '- 0 II 0 0 — IIJ 0 0 — IV 0 0
7/13 7 I 0 0 — 1.46+1.46 II ■ 0 3.00 29 ' III 0 0 " IV 0 2.83 78
7/20 14 I .0 0 —— 1.52+1.54 II 0 3.40 0 III 0 0 — IV 0 2.70 0
7/27 21 I I 5.23 14 5.57+0.30 II 0 6.08 0 III 0 NT — IV 0 5.41 9
8/10 35 II 7.11 17 6.89+0.35 II 2 7.20 71 III I 6.93 18 IV 2 6.30 10
NT=Not Tested 67
Table 42. P. syringae populations on CLARK, 1987, Bozeman.
Date Day Repetition Symptoms . log cfu/leaf % INA+ Mean/SE
7/6 0 I 0 1.70 0 0.49+0.74 II • 0 0 —— III 0 o . — IV 0 . 0 ——
7/13 7 I 0 0 —— 0.50+0.87 II 0 2.00 P III 0 0 — IV 0 0 —
7/20 14 I 0 0 — 0.42+0.74 II 0 0 — III 0 0 — IV 0 1.70 0
7/27 21 I 0 4.60 75 4.75+0.22 II 0 4.48 0 III 0 4.95 0 IV 0 4.98 40
8/10 35 I ' 0 4.95 50 5.41+0.81 II 0 5.54 29 III 0 4.48 17 IV 0 6.65 0 iue 28. Figure
log cfu/l«»f ' log cfu/taaf . syringaeP 1987.populationson Bozeman, AR4, . yigeppltoso R, oea, 1987. Bozeman, AR5, on populations syringae P. 68 69
Figure 29. P. syringae populations on AR6, Bozeman, 1987.
Figure 30. P . syringae populations on AR13, Bozeman, 1987. iue 2 P srna ouain o LR, oea, 1987. Bozeman, CLARK, on populations syringae P. 32. Figure
log cfuZI eaf log ofu/leaf 70 71
8
7 • . : . , • ! • 6 • , i : • : : . • ! • • • : : • : o 5 • OJ ■ I * " i . : " . * s ' X • • • • . I ; • 5 4 . ! i I : u . ; :::: . • • : • . . ‘ ’ I CD I 1 2 i • £ 3 - • ’ . : 5 • : • , • S • • . ; . . • • • • : 2 : • • I . • :
- • I :
. 0 ------L_ ------0 10 20 30 40 days Figure 33. P. syringae populations on 24 barley cultivars and lines, Bozeman, 1986.
8 •
- 7 •
• 6
o 5 . _Q) • 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.
ENTRY 100 99-80 79-60 59-40 39-20 19-0
AR4 I I I ■, 3 4 AR5 2 2 8 AR6 I I 12 AR13 3 2 2 2 3 AR15 2 I 8 CLARK I • 2 I 7
TOTAL 7 I 8 7 5 42
Table 44. Comparison of the epiphytic populations of P. syringae on the six selected entries during 1986 and 1987 in Arizona and Bozeman.
Bozeman 1987 Arizona 1987 Bozeman 1986 Entry • ■ Mean Cultivar Mean Cultivar Mean
CLARK 2.64 A AR5 0.88 A AR5 2.18 A AR4 2.93 AB CLARK 1.08 A CLARK 2.92 AB AR5 2.94 AB . AR15 1.35 A AR4 3.76 ABC AR15 3.09 AB AR4 1.62 A AR15 4.40 BC AR6 3.29 AB AR13 2.73 A AR6 4.46 . BC AR13 4.53 B AR6 4.15 B ARl 3 5.53 C
Sign, level 5% 73
Table 45. Biochemical characteristics of the P. syringae isolates collection from Bozeman, 1986.
Utilization of O x i d a s e Fluor. A r g i n . H y p e r - a l p h a - D (-) I s o l a t e R e a c t i o n P i g m e n t D e h y d . S e n s i t . INA ketogl. tar. D G l O O - + - + + + - D G l O l - + - + + + - D G 1 0 2 - + - + + + - D G l 03 - + - + + + - D G 1 0 4 - + - + + + - D G 105 - + - + + + - D G 1 0 9 + - + + + - D G 1 1 2 - + - + + + - D G 1 1 4 - + - + + + - D G l 15 - + -- + + - D G 1 1 6 - + - + + 4 - D G l 17 - + - + + + - D G 1 1 8 - + - + + + - D G l 19 - + - + + + - D G 1 2 0 - + - + + + - D G 1 2 1 - + - + + + - D G 1 2 2 - + - + + + - D G 123 - + - + + + - D G 1 2 4 + - + + + - D G 125 - + - + + + - D G 1 2 6 - •f - + + + - D G 127 - + - + + + - D G 1 2 8 - + - + + + - D G 129 - + - + + + - D G l 20 - + - + + + - D G 1 3 1 - + - + + + - D G 1 3 2 - + - + + + - D G 1 3 3 - + - + + + - D G 1 3 4 - + - + + + - D G 1 3 5 - + - + + + - D G 1 3 6 - + - + + + - D G l 3 8 - + - + + + - D G 1 3 9 - + - + + + - D G l 40 - + - + + + - D G 1 4 1 - + - + + + - D G 142 - + - + + + - D G 143 - + - + + + - D G 144 - + - + + + - D G 145 - + - + + + - D G 146 - + --- + - D G l 47 - + - + + + - D G 148 - + - + - + - D G 149 - + - + + + - D G 1 5 0 - + - + + + - DGl 51 - + - + + + - D G 1 5 2 - + - + + + - D G 1 5 3 - + - + + + - 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).
Time Mean + S.E. (log cfu/leaf)
8 AM 5.88 + 0.13 12 PM 7.01 + 0.31 4 PM 7.06 + 0.30 8 PM 6.99 + 0.73 12 PM 7.25 + 0.58 4 AM 7.48 + 0.48 8 AM 7.11 + 0.22
V- 8 I 0
0 4 8 12 16 20 24 hours Figure 35. 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. Their establishment on the leaf surface was rather poor. 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). iue3. ouain fmre . yigeo LR, oea, 1986. Populationsof syringaemarkedP.onCLARK, Bozeman, Figure39.
log ofu/leaf * log ofu/leaf Populationsof syringaemarkedP.on Bozeman, KLAGES, 78 1986. iue4. ouain fmre . yigeo 2-, oea, 1986 Populationsof syringaeon222-9,markedP. Bozeman, Figure41.
log cfu/leaf ’ log cfu/leaf ouain fmre . yigeo 2-, oea, 1986Populationsof syringaeon222-1,markedP. Bozeman, 79 •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 rifampicln-
streptomycin (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 wild- type 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.
I 21°C 28°C Strain I I Klett # log cfu/ml Klett # log cfu/ml
DG104 I 157 9.35 • 212 . 9.49 I 49 8.65 . 96 8.80 9 7.95 . 22 8.10 I 0 7.25 4 7.40 I DG148 I 58 8.69 I 13 7.99 NT ' NT I 3 7.29 I DGl46 I 211 9.46 I 62 8.76 NT NT I 14 8.06 .0 7.36 I DG103 I 90 8.70 76 8.89 6 8.00 15 8.19 I 2 7.31 3 7.49 I DG105 I 40 8.85 209 ■ . 9.45 I 9 8.16 76 8.75 I 4 7.46 15 8.05 I 5 7.35 I DGlOl I 113 9.68 76 8.81 I 59 8.99 14 8.11 I 10 8.29 4 7.42 , 2 7.59 I DGlOO I 104 9.57 86 9.39 I 61 8.87 39 8.69 10 8.17 7 8.00 I 4 7.47 2 7.30 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 # Population (log fcfu/ml)
173-AKS ' 81 8.90 58 9.17 40 9.04 4 7.24
15I-JKS 48 9.00 . 2 8 8.57 10 8.28 ■5 : 7.66
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 = Rifampicin- Streptomycin resistant, KS = Kanamycin-Streptomycin resistant, RK = Rifampicin-Kanamycin resistant.
Strain Doubling Time (hrs) Strain Doubling Time (hrs)
DG151 1.11 + 0.08 151-2RK 1.29 + 0.36 151- IRS 0.97 + 0.29 151-3RK 1.04 + 0.25 151- 2RS 1.11 + 0.12 151-4RK 1.11 + 0.21 151- 3RS 1.43 + 0.44 .151-5RK 1.17 + 0.26 151- 4RS 1.19 + 0.05* DG126 1.70 + 0.06 151- 5RS 1.18 + 0.22 126-RK 1.38 + 0.14 151- 5KS 1.30 + 0.21 DG102 1.36 + 0.16 151- 6K(20)S 1.18 + 0.30 102-RD 1.20 + 0.12 151- 7K(20)S 1.13 + 0.25 DGliS 1.61 + 0.16 151-IOKS 1.39 + 0.30 118-IRK 1.63 + 0.09 151-12KS 0.97 + 0.11 118-2RK 1.83 + 0.32 151-13KS 1.02 .+ 0.10** 118-3RK 1.30 + 0.18 151-14KS 1.34 + 0.26 II8-4RK 1.70 + 0.07 151-15KS 1.29 + 0.04 DGl 17 . 1.68 + 0.15 151-16KS 1.12 + 0.10 117-RK 1.59 + 0.20 151-17KS 1.10 + 0.16 DG129 1.36 + 0.16 15I-DKS 1.42 + 0.36 129-RK 1.58 + 0.18 151-JKS 1.64 + 0.14 DG173 1.98 + 0.20 15I-KKS 1.62 + 0.11 173-KS 1.58 + 0.31 51-1RK 1.41 + 0.38 *Selected INA+ **Selected INA- 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. Both strains survived on
AR15 (Table 50, Figure 42). This is in accordance with previous research showing that exclusion of an INA+ pseudomonad from an INA- one, 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). Strain 151-4RS survived equally well on AR13 (Table 51, Figure
43). 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 plant- to-plant dissemination experiment..
Air Dissemination of P. syringae
Attempts to isolate the INA+ marked strain of F\_ syringae (151-
4RS) 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 !NA- Pseudomonas syringae on AR15, Bozeman, 1987.
Mean/SE of log cfu/leaf Date Day Total Marked INA+ • Marked INA-
7/7 0 4.30+0.78 4.25+0.60 4.10+0.48 7/8 I 3.09+0.97 3.37+0.62 2.16+0.46 7/9 2 3.02+0.12 2.92+0.74 1.69+1.06 7/10 3 3.70+1.00 3.70+0.63 3.51+0.65 7/12 5 0 3.33+0.66 2.69+1.75 7/14 7 3.55+1.09 3.58+0.28 . 2.64+1.56 7/16 9 2.73+0.72 2.50+1.61 2.28+1.37 7/21 14 3.44+0.28 3.03+0.51 1.35+0.93 7/22 15 4.37+0.77 3.65+0.36 • 1.78+1.41 8/7 31 5.83+0.63 4.28+0.41 1.97+1.52 8/11 35 5.98+0.22 3.94+0.41 2.43+0.20 9/4 59 NT . 3.03+0.82* NT
*0n dry leaves NT=Not Tested
Table 51. Populations of total and marked INA+ Pseudomonas syringae on ARl3, Bozeman, 1987.
Mean/SE of log cfu/leaf Date Day Total Marked INA+
7/8 0 4.83+0.77 3.73+0.49 7/9 I 4.30+0.70 3.07+0.64 7/10 2 5.21+0.37 3.17+0.22 7/12 4 5.56+0.25 2.88+1.67 7/14 6 5.97+0.79 3.92+0.88 7/16 8 3.79+1.09 4.26+0.51 7/21 13 5.18+0.00 3.99+0.47 7/22 14 4.81+0.19 4.22+0.61 8/7 30 6.26+0.15 3.89+0.63 8/11 34 5.91+0.37 3.27+1.56 9/4 58 NT 3.95+0.99*
*0n dry leaves NT=Not Tested 86
o total P.8 D INfi- P.8
Figure 42. Populations of total, marked INA+, and marked INA- P, 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: both fungi and non—fluorescent bacteria grew on the medium. 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 syringae moved as far as
70 m from a field inoculated with it. 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: P^ syringae can be disseminated under certain conditions. 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. The nature of these differences is unknown. 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 sprinkler- irrigated 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
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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 II Proteose Peptone #3 20 S K2HPO4 I,.5 g MgSO4 . 3 g Glycerol 17 ml Bacto-Agar 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 10 mg Vancomycin 6 mg Rifampicin 0 .5 mg Cycloheximide 100 mg Benomyl . 500 mg
BCRS (Modified King's medium B used in the 1986,, 1987 dissemination studies with marked strains).
1986 Study: King's medium B II
Antibiotics added as previously:
Rifampicin 1000 mg Streptomycin 1000 mg Cycloheximide 100 mg
1987 Study: Rifampicin 100 mg Streptomycin 500 mg Cycloheximide 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 10 mg Streptomycin 500 mg Cycloheximide 100 mg
Thornley' s medium 2A (for arginine dehydrolase, activity tests).
Water 1 1 . Peptone 1.0 g NaCl 5.0 g R2HPO4 6.3 g Bacto-Agar 3.0 g Phenol red 0.01 g Arginine HCl .10.0 g Adjusted to pH 7.2
Ayers et al. medium (for utilization of.carbohydrates).
Water I l' NH4H2PO4 I-Os KCl 0.2 g MgSO4 .TH2O 0.2 g Bromothymol blue Cl.6% ale. sol.)' . ' 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