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64 7662
LIB R A R Y

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“548‘

       
   

This is to certify that the

thesis entitled

Interaction Between Ozone and Xanthomonas phaseoli on

 

Navy (Pea) Bean Cu1tivars 'Seafarer' and 'NEP-2'

presented by

Brian Olson

has been accepted towards fulfillment
of the requirements for

M.S. Plant PathoIogy

degree in

W

Major professor

 

 

Date \ S \RWq

0-7639

 

 

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charge from circulation records

 

 

. INTERACTION BETWEEN OZONE AND XANTHOMONAS PHASEOLI ON
NAVY (PEA) BEAN CULTIVARS 'SEAFARER' AND 'NEP-Z'

BY

Brian Olson

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Botany and Plant Pathology

1979

ABSTRACT

INTERACTION BETWEEN OZONE AND XANTHOMONAS PHASEOLI ON
NAVY (PEA) BEAN CULTIVARS 'SEAFARER' AND 'NEP-Z'

BY

Brian Olson

Frequently in Michigan both ozone and common
bacterial blight (Xanthomonas phaseoli, Xp) damage occur
on dry Navy beans.‘ The research concerns interactions
between ozone and Xp (rifampin resistant mutant, Ra)
on Phaseolue vulgarie cultivars 'Seafarer' (ozone-
sensitive) and 'NEP-Z' (ozone-tolerant). Primary leaves
of ten-day-old plants were inoculated with Ra bacteria
and sometimes treated with an antioxidant N-(20(2-oxo-l-
imidozolidinyl)ethyl)-N-pheny1urea (EDU) before an eight
hour fumigation with ozone (470-544 ug/m3). A small
sometimes significant synergistic interaction between
ozone and blight occurred on both cultivars. Ozone
injury on both cultivars was significantly reduced when
Sprayed with EDU. Field experiments were inoculated with
Ra bacteria sprayed with EDU. No significant synergistic

interaction occurred between ozone and blight damage on

Brian Olson
either cultivar. 'Seafarer' plants sprayed with EDU were
significantly protected from ozone-injury compared to
non-sprayed plants, while 'NEP-Z' plants were not.
Significant differences of total yield only occurred

on 'Seafarer' plants inoculated with Ra.

To a good friend

George S. Lee

ii

ACKNOWLEDGMENTS

I would like to thank Dr. Saettler for his guidance
and encouragement throughout the research and preparation
of this thesis. -

I would also like to thank my committee members
Dr. Hooker and Dr. Smucker for their suggestions
throughout my research and evaluation of this manuscript.
I would like to thank, Dave Weller for his valuable
technical assistance and Dr. Jones for his evaluation of
this manuscript.

I especially thank my wife, Val, for her understand-

ing and patience.

111311

TABLE OF CONTENTS

Page
LIST OF FIGURESOOOOOOOOOOOOOCOOOOOOOOOOCOOOOOOOOOOC vi

LIST OF TABLESOOOOOOOOOOOOOOOOOOOOOOOCOOOOOOOOOOOOO vii
INTRODUCTIONOOO...IOOOOOOOOOOOOOOOOOOOOO0.0.00.0...

LITERATURE REVIWOOOOOOOOOOOOOOOOIO000......0......
comon Blight...’OOOOCOOOOOOOOOOOCOO00.0.0.0...

ozoneOO00......OOOOOOOOOOOCOOOOOCO0.000.000...

Ozone and Pathogen Interactions...............

@1519 N H

MATERIALS MDMETHODS.O0.00......OOOOOOOOOOOOOOOOOO 13

Greenhouse and Field Bean Plants.............. 13
Maintenance of Bacterial Cultures............. 14
Inoculum Preparation and Inoculation.......... 16
Application of Antioxidant Compound EDU....... 16
Bacterial Populations......................... 1?
Ozone Fumigation.............................. 18
Ozone and Blight Symptom Ratings and Yield
Determinations.............................. 21
Statistical Tests........................... 22

RESULTS.0.0.0....00.00.000.000....0.000COOOOOOOOOOO 24

Greenhouse Experiments........................ 24

Effects of inoculum concentration and

ozone fumigation on blight and ozone

injury and bacterial population of

'Seafarer' plants.......................... 24
Effects ofiinoculum concentration and

ozone fumigation on blight and ozone

injury and bacterial populations of

’NEP-Z' plants............................. 26
Effect of Ra bacteria, inoculation time

and ozone fumigation on ozone blight

symptoms and bacterial populations of
'Seafarer' plants.......................... 26
Effect of Ra bacteria, inoculation time

and ozone fumigation on ozone and blight
symptoms and bacterial populations of

'NEP-Z' plants............................. 28

iv

Effect of EDU, Ra bacteria and ozone fumi-
gation on ozone and blight symptoms and

Ba bacterial populations of 'Seafarer'
plants......... ............................
Effect of EDU, Ra bacteria and ozone fumi-
gation on ozone and blight symptoms and

Ba bacterial populations of 'NEP-Z'
plants.....................................
Ozone injury on 'Seafarer' and 'NEP-Z'......
Ra cultured bacteria affected by EDU........

Field Experiment..............................

DISCUSSIONOOOOOIOOOOOOOOO..0...00.0.0000... ..... .0.

LITEMTURE CITEDOOOOOOOOOOOOOOOOOOOOOOO ..... 0......-

31

31
32
36

36
48
S3

LIST OF FIGURES
Page
Figure 1. Diagram Of Field Plot................... 15

Figure 2. Ozone Fumigation Chamber................ 20

vi

LIST OF TABLES

Table Page

1 Effects of Ra Inoculum Concentration and
Ozone Fumigation on Ozone and Blight Symptoms
and Bacterial POpulations of 'Seafarer'
Plants........................................ 25

2 Effects of Inoculum Concentrations and Ozone
Fumigation on Ozone and Blight Symptoms and
Bacterial Populations of 'NEP-Z' Plants....... 27

3 Effect of Ra Bacteria, Inoculation Time and
Ozone Fumigation on Ozone and Blight
Symptoms and Bacterial Populations of
'Seafarer' Plants............................. 29

4 Effect of Ra Bacteria, Inoculation Time and
Ozone Fumigation on Ozone and Blight
Symptoms and Bacterial Populations of
'NEP-Z' Plants................................ 30

5 Effect of EDU, Ra Bacteria and Ozone
Fumigation on Ozone and Blight Symptoms
and Bacterial Populations of 'Seafarer'
Plants........................................ 33

6 Effect of EDU, Ra Bacteria and Ozone
Fumigation on Ozone and Blight Symptoms
and Bacterial Populations of 'NEP-Z'
P1ants........................................ 34

7 Ozone Injury on 'Seafarer' and 'NEP-Z'
Plants in the GreenhouseOOIOOOOOOOOOOOOOOOO... 35

8 Cultured Ra Bacteria Affected by EDU.......... 35

Blight Symptoms on 'Seafarer' Plants in
the FieldOOOOOOOOO0......OOOOOIOOIOOOOOOOOO0.. 37

10 Ozone Symptoms on 'Seafarer' Plants in
the FieldOOOOOOOOOOOO0.00000000000000000000000 39

ll Bacterial Blight Symptoms on 'NEP-Z' Plants
in the FieldOO0.0...ODD...OOOOOOOOOOOOOOOOCOOO 42

12 Ozone Symptoms on 'NEP-Z' Plants in the
FieldOOOOO0.0......00.0.0.0...IOOOOOOOOOOOOOOO ‘4

vii

13

14

15

Plant, Seed, and Pod Weight from Field
'Seafarer' and 'NEP-Z' Plants Treated with
Ra Bacteria and EDUOOOOOIOOOOOOOOOOOOOOIOOOOOO

Effect of Ra Bacteria and EDU on Bacterial
Blight Lesions on Pods of 'Seafarer' and
'NEP-Z' Plants...OOOOOOOOOOOOOOOOO0.0.0.000...

Presence of Ra Bacteria on 'Seafarer' and
'NEP-Z' Leaves with Blight Symptoms in
the FieldOOO0.000IOOOOOIOOOOOOOOO0.0.0....O...

viii

46‘

47

51

INTRODUCTION

Michigan annually produces approximately 90 percent
of the dry Navy (pea) beans in the United States (30).
Common and fuscous bacterial blights caused by
Xanthomonas phaseoli E.F. Smith Dowson (Xp) and
Xanthomonas phaseoli var. fuscans (Burkh.) (pr),
respectively, are major disease problems of Michigan
Navy (pea) beans (26). Both diseases are seed borne
and occasionally cause significant bean yield re-
ductions (1). In recent years, ozone injury has been
observed with increasing frequency in Michigan dry
bean fields (15).

Both ozone injury and blight damage are frequently
observed in the same bean field. Previous researchers
have demonstrated a cross protection phenomenon be-
tween ozone and obligate plant pathogens (16, 21). In
this study we investigated the possible interaction
between ozone injury and bean common blight (Xp) damage
in Navy bean (Phaseolus vulgaris L.) cultivars

'Seafarer' and 'NEP-2'.

LITERATURE REVEIW

Common Blight

 

Xp was first described by Beach in 1892 (2). In
1924, Burkholder reported pr, as having identical
symptoms as Xp, but the pr bacteria were a different
color (5).

Xp and pr bacteria both have a single polar
flagellum and are gram negative, straight rods,
obligately aerobic and produce non-diffusible yellow
pigments. Both bacteria produce hydrogen sulfide,
proteolize milk, hydrolize gelatin, starch and Tween
80, and produce an alkaline reaction with phenol red
dextrose agar. Results of biochemical tests for Xp
and pr are indistinguishable from those of Xanthomonas
campestris: Xp and pr are considered X. campestris
nomenspecies in Bergy's Manual 8th Ed. (4). The only
difference between Xp and pr is a brown diffusable
pigment produced by pr in certain culture media (5).

Xp and pr produce identical symptoms on leaves,
stems, pods and seed. Leaf symptoms are most

frequently observed, and begin with leaf cells becoming

plasmolyzed causing a water-soaked appearance on the
abaxial surface. The water soaked tissue becomes
chlorotic and characteristically bright yellow. The
chlorotic leaf tissue becomes necrotic and often lesions
coalesce, forming large portions of diseased tissue.
Heavily infected leaves may prematurely begin

senescence (31). Stem and pod infections also begin
with water-soaked lesions. The stem lesions become
sunken and turn reddish brown in color. Lesions on
mature dry pods turn dark brown.

Common and fuscous blights have traditionally been
considered late season diseases on dry beans. The
first observable symptoms normally occur in late July
and early August just after blossom. Recently Weller
(27) has detected common and fuscous blight symptoms
and the causal bacterial throughout the growing season.
Symptoms and bacterial populations begin in the early
seedling stage. Weller noted that leaves with blight
symptoms were usually located under younger symptomless
leaves, causing the entire plant to appear healthy.

At blossom, leaves in the outer canopy showed symptoms.

Xp snd pr are seed borne diseases. Seed
internally-infected with chn:pr, are usually yellow
or show a darkened hilum (27) and are considered the

primary source of inoculum. Bacteria on infected

seedlings are spread as secondary inoculum by blowing
or splashing rain.

Weller monitored bacterial populations on leaves,
stems, roots, pods and seed with Xp and pr isolates
resistant to 5.0 ppm rifampin (27). Bacterial
populations were determined by homogenizing plant
material in .01 M phosphate buffer (pH 7.2) and plating
the homogenate on nutrient agar media containing

rifampin and cycloheximide.

Ozone

Man has known about ozone, the molecule composed of
three oxygen atoms since the 1800's. In the late
1860's R.C. Kedzie, chemistry professor at Michigan
Agricultural College (M.S.U.), researched ozone
detection and suggested the possible health hazard of
ozone (18).

Awareness of air pollutants and ozone began in the
early 1950's. The modern world had become dependent
on the combustion engine and large industries were
located in most cities. At this time peOple in cities
such as Los Angeles became aware of smog and its danger
to human health.

In 1950 Middleton, Kendrick and Schwain attributed
injury on many herbaceous plants in the Los Angeles area

to smog or air pollution (25). Haagen-Smit et al., in

1952 experimentally demonstrated air pollution damagecni
spinach, beets, endive, oats and alfalfa.(8)t ant the
present time the Environmental Protection Agency (EPA)
determines smog levels by measuring ozone
concentration.

In nature ozone is found in both the upper and
lower atmospheres. The ozone responsible for plant
damage is found in the lower atmosphere, and is formed
by the photolytic reaction of NO2 from gasoline
combustion engines with 02. Ozone is a strong oxidiz-
ing agent and is short-lived. Because ozone is formed
with ultraviolet light and is a strong oxidizing agent,
ozone levels are generally diurnal, being high in the
day and low at night. Ozone also is formed during
electrical discharges, such as thunderstorm lightning
bolts, but such ozone does not significantly increase
ozone levels. For research purposes ozone is usually
produced by passing air over a high intensity ultra-
violet lamp.

Ozone is continually formed in our lower
atmosphere, but is a threat, only when conditions allow
ozone formation to exceed ozone decomposition. These
conditions include, clear skies and a thermal inversion
causing nitrous oxides to be trapped in the lower

atmosphere. In Michigan and other temperate regions

this may occur several times a year during the summer
months. Ozone episodes are not restricted to metro-
politan areas. Drifting air masses can cause damage to
agricultural crops in rural areas (9).

Ozone often causes a necrotic stippling or flecking
of leaves on affected plants. Bean leaves damaged by
ozone are termed bronzed due to the presence of small
pin-size necrotic regions on the adaxial leaf surface.
The necrotic regions are composed of dead palisade
cells; similar symptoms may also occur on bean pods.

In laboratory experiments researchers have exposed
plants to high levels of ozone without observable
damage (11). Subsequent studies and reviews have
shown that ozone is transported into the plant through
the stomata and that plants are not injured when the
stomata are closed (11). Several important factors
regulate stomatal function.

Juhren, Hull, and Went reported light intensities
of 3.21x>4.3 le were necessary to obtain traces of
oxidant (air pollution) injury on speargrass (Poa
annua L.) (17). Oxidant injury at 9.7to»12.9 le and
32t0v43 le were similar but significantly greater than
oxidant damage at 3.2tor4.3 le. Thus sufficient light
intensity is necessary to open stomata for ozone

injury.

High levels of relative humidity are necessary for
maximum stomatal opening. Some researchers have
recorded increases in air pollution injury on plants
when the relative humidity was increased (11).

In closed chamber fumigation studies, sufficient
air must pass through the chamber to prevent carbon
dioxide buildup. High CO2 levels stimulate stomatal
closure. -Heck and Dunning reported, that the ozone
sensitivity of pinto bean and tobacco plants decreased
when CO2 levels were increased (12).

The same authors also demonstrated that soil
conditions affect ozone sensitivity (12). Plants
growing in clay-loam mixture were least sensitive to
ozone, while plants growing in vermiculite and a peat-
perlite mixture were the most susceptible to ozone
injury. Lack of adequate soil moisture decreases plant's
sensitivity to ozone. Inadequate water supply causes
stomatal closure, and prevents ozone penetration into
the leaves.

Plant age is an important parameter in fumigation
studies. Beck and Dunning reported that fully
expanded mature pinto bean primary leaves were most
sensitive to ozone (12). In other cases, field grown
beans are most sensitive to ozone after the blossom

stage of plant development (9).

Air pollution studies under field conditions
involve some difficulties. Experiments using open-top
chambers are limited to space and therefore are
inadequate for large scale yield studies. Antioxidant
chemicals may also be used to determine ozone injury
affects on yield. Several compounds including benomyl
have shown protection against ozone damage (13). In
this research we have used N-(20(2—oxo-l-imidazolidinylr-
ethyl)-N-phenylurea (EDU), a protective compound
recorded as effective against ozone injury on bean

plants (6).

Ozone and Pathogen Interactions

Beagle and Manning have separately reviewed the
interaction of air pollutants and pathogens, primarily
fungal pathogens (ll, 24). Generally the reviews
noted, "ozone-injured plants appear to be more
susceptible to invasion by facultative parasitic and
facultative saprophytic fungi. Obligate parasitism
by fungi appears to be retarded by ozone and ozone-
injured host tissues? (24). More recently researchers
have studied the interactions between ozone and
bacterial and viral plant pathogens.

Brennan and Leone demonstrated protection against
ozone damage on tobacco plants (Nicotiana sylvestris)

inoculated with Tobacco Mosaic Virus six to 12 days

prior to a three to six hour ozone fumigation (588
ug/m3) (3). One day after ozone fumigation, non-
inoculated plants were ozone damaged and TMV-inoculated
plants were not. Davis and Smith noted protection
against ozone damage on pinto beans (Phaseolus vulgaris
L. Pinto) inoculated with Bean Common Mosaic Virus six
to 12 days prior to ozone fumigation (7). Both
researchers observed less protection against ozone-
injury when the time between viral inoculation and
ozone fumigation was decreased.

The first ozone-bacterial pathogen study
demonstrated little of no interaction or cross
protection. Kerr and Reinert inoculated red kidney
beans (Phaseolus vulgaris L.) with Pseudomonas phaseoli-
cola (halo bright) and one week later exposed the
plants to ozone (1176 ug/m3) for one hour (20). Ozone
fleck symptoms were observed on all leaf areas except
areas exhibiting typical necrotic and chlorotic halo
bright symptoms.

An interaction between ozone and bacterial leaf-
spot of alfalfa (Xanthomonas alfalfae) was observed
by Howell and Graham (16). Three alfalfa (Medicago
sativa) cultivars were used, one resistant to ozone
and two ozone-sensitive. Plants were split in two

groups, one group was inoculated with X. alfalfae

10

24 hrs before ozone fumigation (346 ug/m3 for four hrs)
and the other group was inoculated 24 hrs after fumiga-
tion. Plants inoculated before fumigation had less
severe ozone injury than non-inoculated plants and
plants inoculated after fumigation. Plants inoculated
after fumigation developed less bacterial leafspot
injury than non-fumigated plants and plants inoculated
before fumigation.

Laurence and Wood observed symptom differences on
soybean plants (Glycine max) fumigated with ozone and
inoculated with Pseudomonas glycinea (halo bright)

(21). Plants were fumigated with ozone 21 days after
planting with primary leaves almost fully expanded.
Sets of plants were inoculated with P. glycinea

between two days before and 16 days after ozone
fumigation. Plants were fumigated with 400 ug/m3 ozone
for four hours producing light to moderate ozone damage
on non-inoculated plants. Bacterial symptoms were only
less severe (on plants inoculated one day before and

two days after fumigation than on non-fumigated plants.
The authors suggested that reduced bacterial injury
may have been due to the production of bacteriostatic
or bactericida1.compounds in the plant caused bY
ozone-injury. Ozone injury on soybean was not affected
by bacterial inoculations before and after ozone

fumigation.

11

Laurence and Wood also observed an interaction
between ozone and Xanthomonas fragariae on wild
strawberry (22). Reduced bacterial symptoms were
recorded in all experiments when plants were exposed
to ozone (392 ug/m3) for three hours before and after
bacterial inoculations.

Several workers have reported that soybean and
bean plants injured by ozone produce phytolexin-type
compounds, which might account for differences in
disease severity of other pathogens (19, 25). One
theory suggests, the pathogen or ozone stimulates the
production of a general protective type compound which
then protects the plant from subsequent biotic or
abiotic attack. Howell observed protection against
ozone damage when alfalfa plants were first inoculated
with Xanthomonas alfalfae. He also reported protection
against X. alfalfae damage when plants were exposed to
ozone (16). This protection phenomenon is termed cross
protection. Saettler and Rubin (unpublished data)
reported the accumulation of coumestrol, a phytolexin-
type compound, in navy bean leaves damaged by ozone.
Recognizing that coumestrol may be bactericida1.or.
bacteriostatic and that common blight (Xp) and ozone-
injury occur on Michigan navy beans we decided to study

the interaction of these two diseases on navy beans.

12

In this research we studied the possible inter-
action of ozone and common bacterial blight (Xp) on two
navy bean (Phaseolus vulgaris L.) cultivars, ozone
susceptible 'Seafarer' and ozone resistant 'NEP-Z',

grown in the greenhouse and field.

MATERIALS AND METHODS

Greenhouse and Field Bean Plants

Ozone susceptible 'Seafarer' and ozone tolerant
'NEP-Z' navy bean cultivars were used in all studies.
'Seafarer' is a commercial variety extensively grown in
Michigan and 'NEP-Z' is a white bean developed through
seed mutation of the black bean cultivar ‘San Fernando'.

In greenhouse studies seed were germinated in
moist vermiculite for two days in the dark. One hund-
red and twenty germinated seedlings of uniform size
were transplanted at 1.5 cm depth in individual 7.5 cm
diameter sterile clay pots containing a soil mixture
of equal parts (volume) of sterilized peat, vermiculite
and sterilized sandy loam soil. Eight days after
germination 72 uniform plants were chosen for the
pertaining experiment. The plants were fumigated with
ozone lltx>13 days after germination. Plants were
watered alternately with deionized water and modified
Hoaglands solution (14). The plants were grown in a
greenhouse cooled with an evaporative cooler and

entering ambient air was drawn through charcoal filters.

13

14

Ozone levels in the greenhouse when monitored ranged
from zero to 78 ug/m3, while outdoor ambient ozone
levels ranged from 78 to 196 ug/m3. Greenhouse
temperatures ranged from 22 to 37 C and the relative
humidity ranged from 60 to 90%. No supplemental lighting
was used, because the experiments were performed from
6/1/78 to 8/26/78.

In field studies land was cultivated and treated
with herbicides and fertilizers using conventional
practices. Seed were planted on 6/15/78 and the plants
were harvested 9/15/78 and 9/17/78 (Fig. 1). Each
individual plot consisted of three rows, each 5.4 meters

in length.

Maintenance of Bacterial Cultures

Xanthomonas phaseoli mutant Ra, resistant to 50 ppm
rifampin, was obtained from D.M. Weller (28). Stock
cultures were prepared by growing bacteria in liquid
buffered-yeast extract (10 g yeast extract per 1000 ml
0.01 M phosphate buffer, pH -7.2) placed on a shaker.
After 48 hrs bacteria were transferred to 40% v/v aqueous
glyverol and stored at ~10 C. To prepare inoculum
Ra bacteria were transferred onto yeast extract
calcium carbonate agar plates (YCA: 10 g yeast extract,
15 g agar and l g CaCO3 per 1000 ml glass distilled

water). After 96 hrs growth, bacteria were transferred

15

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

'NEP-Z' 'NEP-Z'
Ra++ Ra+ Ra- Ra“. Ra+ Ra-
' *3.
E- 2+ 3+ E- E+ E- E- 8+ 3- 8+ E- 8+
*A? *A?
'8‘ age
'SEAFARER' 'SEAFARER'
Ra- Ra++ Ra+ Ra++ Ra+ Ra-
*3.
3+ E- 3- 8+ 8+ 3- E- E+ E_ E- E+ E-
*AF *3;
'SEAFARER'
Ra+ - Ra- Ra” '
E- E+ E- E+ E+ E-
*C* ‘3'
hi 'NEP-Z’
E Ra+ Ra++ Ra-
E+ E- 3- 3+ E- E+
KEY
Ra: Non-inoculated 8
Ra++ Inoculated with Ra 10 CPU/ml on 7/6/78.
Ra Inoculated with Ra 103 CFO/ml on 7/20/78.
3- Sprayed with tap water containing 0.1‘ v/v Tween 80.
2+ Sprayed with EDD (855 g/ml) containing 0.1\ v/v Tween 80.
‘A' Unplanted area one meter wide.
'3' Four border rows of 'Seafarer'.
‘C‘ Bulk 'Seafarer' planting.
*' Each individual treatment contains 3 rows and 5 meters in length. The rows are arranged
east to west.
Figure 1. Diagram of Field Plot

 

16

to fresh YCA plates for 48 hrs.

Inoculum Preparation and Inoculation

Bacteria were rinsed from YCA plates with phosphate
buffer. Bacterial concentrations were adjusted to 108
colony forming units (CFU)/ml using standard turbini-
metric and dilution plate techniques. Plants were
inoculated by one of two methods: 1) The abaxial surface
of primary leaves were Sprayed until runoff with a
Devilbiss atomizer operated at 1.4 kg/cm2 and held 15 to
20 cm from the leaf surface; 2) The abaxial surface of
primary leaves were sprayed to a water-soaked appearance

2 and

with a Devilbiss atomizer operated at 1.4 kg/cm
held 2 to 3 cm from the leaf surface. Several different
bacterial concentrations were used.

Inoculum for field experiments was prepared with
deionized water instead of phosphate buffer and
bacterial concentrations were adjusted to 108 CFU/ml.
Bacterial suspensions were directed upwards underneath

the plants using a knapsack sprayer Operated at 1.5 to

2.0 kg/cm2 delivering 99 ml(inoculum)/1ineal meter.

Application of Antioxidant Compound EDU

 

Aqueous solutions of EDU were prepared to contain
855 ug/ml (active ingredients) EDU and 0.1% v/v

Tween 80. In the greenhouse EDU solutions were

17

prepared in deionized water and were sprayed until
runoff on adaxial primary leaf surfaces using a
Devilbiss atomizer operated at 1.4 kg/cm2 and held

15 to 20 cm from the leaf surface. Check plants were
sprayed with deionized water containing 0.1% v/v Tween
80. Approximately 0.9 m1 of spray solution was applied
to each primary leaf.

In the field EDU solutions prepared in tap water
were sprayed onto adaxial leaf surfaces with a knapsack
sprayer operated at 2.8 to 4.2 kg/cm2 and delivering
33.46 m1(solution)/lineal meter or 57.25 mg(EDU)/1inea1
meter. Check plots were sprayed with tap water
containing 0.1% v/v Tween 80. All sprays were applied

weekly between 1200 and 1300 hours.

Bacterial'Populations

 

Populations of Xp Ra bacteria were determined by.
sampling six primary leaves in the greenhouse studies.
Leaf areas were measured with a Li Cor area meter
(Model 3000, Lambda Instruments Corp.) using either
leaf tracings on paper, giving a 1 five to ten percent
error, or by direct leaf measurements. Leaves were then
homogenized in a 75 ml of .01 M phosphate buffer pH 7.2
for 2.5 minutes. Homogenates were serially diluted and
aliquots plated on rifampin agar media,RAM (50 mg rif-

ampin and 25 mg cycloheximide/ 1000 m1 YCA) . Duplicate plates

18

were prepared for each dilution. After 96 hrs, dilution
plates containing 30 -— 300 colonies per plate were
counted. Final bacterial pOpulations were expressed as
the number of CFU/50 cm2 leaf tissue.

The presence of Ra bacteria in field studies was
confirmed by separately pressing three leaves per plot
exhibiting typical blight symptoms onto RAM plates
containing 75 ug/ml rifampin and 50 ug/ml cycloheximide.
Blighted leaf areas were outlined on the plates;

characteristic Xp bacterial growth after 96 hrs was

considered a positive indication of Ra bacteria.

Ozone Fumigation

Plants were fumigated with ozone in a 76.2 cm
cubical chamber constructed on all sides except the top
with .635 cm clear plexiglass lined with aluminum foil
on the outside. The chamber top was made of .635 cm
glass. Ten 91.44 cm length fluorescent tubes (four
30 watt Cool White and six 30 watt Gro Lux tubes) over
the chamber top provided 9.7 le at the leaf surface.

Air was forced through the exposure system at
265.0 l/min with a fan (Model 4C443 Dayton Mfg. Co.,
Chicago, IL) regulated by a rheostat (Cenco). Air
initially passed through a 6.35 cm diameter plastic
pipe into a 30.5 cm cubical humidifying chamber (Fig.

2) containing 5 -— 10 cm of standing distilled water

Figure 2.

l9

Ozone Fumigation Chamber

*A*
age
40*
spa
tEs
apt
sGa
*3:

Exposure chamber

Humidifying chamber

Fan

Ultraviolet lamp (ozone generator)
Flow meter

Cotton filter

Two ozone monitoring inlets
Exposure chamber exhaust

20.

Figure 2

 

#AJI'_

21

and six sheets of cheese-cloth hung top to bottom
perpendicular to the air flow. The air exited the
humidifying chamber through a 6.35 cm diameter plastic
pipe and was mixed with ozonated air just prior to
entering the exposure chamber.

Ozonated air was generated by passing 6.5 l/min
(Lab Crest Flowmeter, Fischer 7 Porter Co., Warminister,
PA) of cotton filtered compressed air over an ultra-
violet lamp (Model SCT4, Ultraviolet Products, Inc.,
San Gabriel, CA). Ozone in the exposure chamber was
monitored with a Dasibi ozone monitor (Model 1003-AH,
“Environmental Corp., Glendale, CA).

Thirty-six plants were randomly placed in the
chamber on six 0.635 cm x 6.35 cm x 71.12 cm glass
plates elevated 22.86 cm from the chamber floor.
Temperature was maintained at 22 C t 2 C and relative
humidity was maintained at 70% t 10%. The plants were
placed in the chamber at 0800 hours and a eight hour
Ozone fumigation with 470 to 549 ug/m3 was initiated at
0900 hours.

Ozone and Blight Symptom Ratings'and Yield Determinations
Ozone and bacterial blight symptoms were recorded

on each individual plant in the greenhouse experiments.

Ozone injury was recorded as the percentage of damaged

22

primary leaf tissue and was recorded at the following
times: 1) two days after ozone fumigation; 2)‘the day
of bacterial sampling; 3) sometime between fumigation
and sampling. Blight symptoms were recorded on a scale
of 0 to 100 (0 to 15, no symptoms to light water-soaking;
15 to 35, moderate to heavy water-soaking; 35 to 65,
light to severe chlorosis; 65 to 85, light to moderate
necrosis; 85 to 100, severe to complete necrosis).
Blight symptoms were always recorded at the time of
bacterial population sampling and sometimes between
ozone fumigation and bacterial sampling.

Ozone injury and blight symptoms were individually
recorded in the field as percent of damaged leaf tissue.
Symptoms were recorded every five days from 7/10/78 to
8/9/78 and every two days from 8/9/78 to 9/8/78.

For each plot a three meter length of the middle
row was harvested for yield data. The plants were
dired for two weeks in the greenhouse before weight
determinations were measured on total plants, seed plus
pods and seed. Pod blight symptoms were recorded as
the number of lesions per pod and 100 pods were examined
per plot.

Statistical Tests. In this research we analyzed
all of the data using the Northwestern University's
Statistical Package for the Social Sciences (SPSS)

program on Michigan State University's Control Data

23

6500 computer. Ozone and bacterial symptom data was
always transformed with the following equation before
analysis, (180/3.l4):c{arcsin [sqrt(value)]}. All of
the experiments were analyzed using the multi-variable
analysis of variance subprogram (MANOVA). In each case
one independent variable was used and a suitable
statistical design was formulated. The experiment
designed to analyze the affect of EDU on Ra colony
forming units was analyzed using SPSS's Student-Newman-

Keuls test.

RESULTS

Greenhouse Experiments

Effects of inoculum concentration and ozone fumi-
gation on blight and ozone injury and bacterial
population of 'Seafarer' plants. 'Seafarer' plants

4 6 and 108 Ra

were inoculated until runoff with 10 , 10
CFU/ml one day before ozone fumigation. This experi-
ment was designed to determine the effects of ozone
injury on different bacterial populations and the
effect of different inoculum concentrations on the
severity of ozone injury. Bacterial symptoms were
recorded ten days after ozone fumigation just prior to
sampling for bacterial populations. Ozone injury was
recorded two and ten days after fumigation. Ra
bacterial symptoms and populations were not significant-
ly different between ozone fumigated and non-fumigated.
plants (Table l). The severity of ozone injury was

not significantly different between plants inoculated

with Ra bacteria and those plants that were non—inocu-

lated (Table l).

24

25

TABLE 1. Effects of Ra Inoculum Concentration and Ozone Fumigation on Ozone and
Blight Symptoms and Bacterial Populations of 'Seafarer' Plants.

 

 

 

 

 

 

 

 

Experimegt Inoculum Ozone Ozone Injuay Blight Log Bacterialf
Number Concentragion Fumigation Symptoms Symptoms Pepulations
CPU/m1 2 day 10 day 10 day Ra Ciro/so c1112 leaf area
One 104 - 0.0 0.0 0.0 5.817
6 + 15.0 14.0 0.0 6.596
10 - 0.0 0.0 0.8 8.459
8 + 8.8 7.0 0.6 8.416
10 ~ 0.0 0.0 3.2 9.818
+ 9.7 7.0 2.7 9.821
Check - 0.0 0.0 0.0
+ 7.6 7.6 0.0
Two 10‘ - 0.0 0.0 0.0 -—
6 + 38.6 38.6 0.0 2.302
10 - 0.0 0.0 5.0 7.899
8 + 30.5 36.6 2.2 7.859
10 - 0.0 0.0 2.1 9.639
+ 37.3 44.3 2.1 9.686
Check - 0.0 0.0 0.0
+ 24.1 27.7 0.0
Three 10‘ - 0.0 0.0 0.0 5.229
6 + 2.3 3.0 0.0 4.975
10 - 0.0 0.0 0.0 7.117
8 + 6.5 4.0 0.0 6.896
10 - 0.0 0.0 1.6 8.476
+ 5.5 5.6 1.6 8.518
Check - 0.0 0.0 0.0
+ 2.9 2.2 0.0
Mean 104 - 0.0 0.0 0.0 3.682
6 + 18.7 18.5 0.0 4.624
10 — 0.0 0.0 1.9 7.825
8 + 15.3 15.9 0.9 7.724
10 - 0.0 0.0 2.3 9.311
+ 17.5 18.9 2.2 9.342
Check - 0.0 0.0 0.0
+ 11.5 12.5 0.0
Levels of Significance
Ozone Symptoms g
2 day 10 day
Experiments .00001* .00001*
Inoculum Concentration .10662 .08839
Blight Symptoms g
10 day
Experiments .00001'
Inoculum Concentration .00009'
Ozone Fumigation .20135
Inoculum Concentration by Ozone Fumigation .34774
Log of Bacterial Populations
Experiments .00002‘
Inoculum Concentration .00001‘
Ozone Fumigation .41676
Inoculum Concentration by Ozone Fumigation .43080

a Values for each of the three experiments and their means.

b Bacterial concentrations were sprayed until runoff on the abaxial primary leaf surface 1 day before
ozone fumigation.

0 Plants were ozone fumigated (+) and not fumigated (-) 10 days after seed germination.

d Ozone symptoms were recorded 2 and 10 days after ozone fumigation. Symptoms were recorded as the
percentage of ozone damaged primary leaf tissue.

a Blight symptoms were recorded 10 days after fumigation. Blight symptoms were recorded on a scale of
0-100 (0-15, no symptoms to light water-soaking; 15-35, moderate to heavy water-soaking; 35-65,
light to severe chlorsis; 65-85, light to moderate necrosis; 85-100, severe to complete necrosis).

f Bacterial populations were sampled 10 days after fumigation.

9 Ozone and blight symptom values were transformed (180/3.l4) x {arcsinlsqrt(value)l} and analyzed.

* Significant at the five percent level.

26

Effects of inoculum concentration and ozone fumi-
gation on blight and ozone injury and bacterial
populations of 'NEP-Z' plants. The same experimental
procedure as above was performed on the cultivar 'NEP-2'
with the following exceptions. Ozone injury was recorde
ed at two, six and nine days after ozone fumigation and
bacterial symptoms were recorded six and nine days after
fumigation. Ra bacterial populations were sampled nine
days after ozone fumigation. There were no significant
differences between the three replicate experiments
with respect to bacterial symptoms and populations
(Table 2). Ozone fumigation had no significant effect
on bacterial symptOms or populations (Table 2). Nine
days after fumigation ozone injury was significantly
more severe on Ra bacteria inoculated plants than non-

inoculated plants (Table 2).

Effect of Ra bacteria, inoculation time and ozone
fumigation on ozone blight symptoms and bacterial
populations of 'Seafarer' plants. Primary leaves of

'Seafarer"plants were inoculated (106

Ra CPU/ml) to a
water-soaked appearance, four and two days prior to
fumigation with ozone. The experiment was designed to
determine the effect of inoculation time on ozone
injury and the effect of ozone fumigation on Ra

bacteria populations and symptoms. Ozone injury was

recorded two, six and ten days after ozone fumigation.

27

TABLE 2. Effects of Inoculum Concentrations and Ozone Fumigation on Ozone and Blight
Symptoms and Bacterial Populations of 'NEP-Z' Plants.

 

 

 

 

 

 

Experiment Inoculum Ozone Ozone Injury Blight Log Bacterialf
Number“ Concentration Fumigationc Symptomsd Symptoms“ Populations
CPU/mlb 2 day 6 day 9 day 6 day 9 day Ra cw/so an? leaf area
One 104 - .o .o .o .o .o 5.225
6 + 2.0 3.1 3.9 .0 .0 5.679
10 - .0 .0 .0 .0 .0 7.444
8 + 4.1 3.9 4.3 .0 .0 7.698
10 - .0 .0 .0 .0 2.9 8.907
+ 1.4 3.3 3.3 .0 3.7 9.128
Check - .0 .0 .0 .0 .0
+ 2.6 3.3 3.6 .0 .0
Two 104 - .o .o .o .o .o 4.395
6 + 3.8 4.3 4.5 .0 .0 5.870
10 - .0 .0 .0 .0 .0 7.877
8 + 7.6 7.6 7.2 .0 .0 7.899
10 - .0 .0 .0 1.0 1.4 8.989
+ 7.4 6.2 8.4 .5 6.7 8.993
Check - .0 .0 .0 .0 .0
+ 4.6 3.9 3.5 .0 .0
Three 104 - .0 .0 .0 .0 .0 5.507
6 + 3.6 4.5 5.4 .0 .0 4.180
10 - .0 .0 .0 .0 .0 7.749
8 + 4.1 7.0 8.9 .0 .0 7.252
10 - .0 .0 .0 1.0 7.6 9.229
+ 4.6 7.6 7.3 1.0 5.7 9.221
Check - .0 .0 .0 .0 .0
+ 3.9 4.1 4.9 .0 .0
Mean 104 - .o .o .o .o .o 5.042
6 + 3.1 3.9 4.5 .0 .0 5.576
10 - .0 .0 .0 .0 .0 7.690
8 + 5.3 6.2 6.8 .0 .0 7.616
10 - .0 .0 .0 .7 3.9 9.042
+ 4.5 5.7 6.3 .5 5.3 9.114
Check - .0 .0 .0 .0 .0
+ 3.8 3.7 4.0 .0 .0

 

Levels of Significance
Ozone Symptoms g
2 da 6 da 9 da
Experiments _001Z§' .0168§* .02175‘
Inoculum Concentration .23616 .05691 .00620'
Blight Symptoms g

9 da
Experiments 05456

Ozone Fumigation .14051
Log of Bacterial Populations
Experiments .99374
Inoculum Concentration .00001‘
Ozone Fumigation .20747

Inoculum concentration by Ozone Fumigation v.18652

a Values presented are for each of the three experiments and their means.

b Bacterial suspensions were sprayed until runoff on the abaxial primary leaf surfaces 1 day before ozone
fumigation.

0 Plants were ozone fumigated (+) and non-fumigated (-) 10 days after seed germination.

d Ozone symptoms were recorded 2, 6, and 9 days after ozone fumigation. Symptoms were recorded as the
percentage of ozone damaged primary leaf tissue. Values presented are the means from 3 individual
ratings.

e Blight symptoms were recorded 6 and 9 days after ozone fumigation. Blight symptoms were recorded on a
scale of 0-100 (0-15, no symptoms to light water-soaking; 15-35, moderate to heavy water-soaking:
35-65, light to severe chlorsis: 65-85, light to moderate necrosis; 85-100, severe to complete
necrosis). Values presented are the means of 3 individual ratings.

f Bacterial populations were sampled 9 days after ozone fumigation. Values presented are the means of 3
individual samples each containing 3 plants.

9 Ozone and blight symptom values were transformed (ISO/3.14) x {arcsinlsqrt(value)]} and analyzed.

* Significant at the five percent level.

28

Ra bacterial populations were sampled ten days after ozone
fumigation. Two and six days after ozone fumigation,
ozone injury was significantly more severe on bacterial
inoculated plants than non-inoculated plants. Ozone
injury was not affected by the different inoculation
times. Blight symptoms and Ra bacterial populations

were not significantly different between ozone-fumigated

and non-fumigated plants (Table 3).

Effect of Ra bacteria, inoculation time and ozone
fumigation on ozone and blight symptoms and bacterial
populations of 'NEP-Z' plants. This experiment was
performed the same as the experiment above except that
ozone and blight symptoms were recorded two and six days
after ozone fumigation and Ra bacterial populations were
sampled six days after fumigation. Six days after
fumigation blight symptoms and bacterial populations
were significantly more severe and greater, respectively,
on ozone-fumigated than non-fumigated plants (Table 4).
Ozone injury recorded six days after fumigation was
significantly more severe on Ra bacteria inoculated
plants than non-inoculated plants. Note, ozone injury
was observed in the zone of bacterial inoculation for

both 'Sefarer' and 'NEP-2' plants.

29

TABLE 3. Effect of Ra Bacteria, Inoculation Time and Ozone Fumigation on Ozone and
Blight Symptoms and Bacterial Populations of 'Seafarer' Plants.

 

 

 

 

 

 

Experiment Inoculation c Ozone Ozone Inju y Blight Bacterial
Numbera Time” Bacteria Fumigation Symptoms Symptomsf Population
days 2 day 6 day 10 day 6 day 10 day Ra era/50 c1112 leaf area
One 4 - - .0 .0 .0 .0 .0
4 — + .7 1.3 1.4 .0 .0
4 + — .0 .0 0 2.1 99.0 9.739
4 + + 5.8 4.9 4.5 2.0 99.0 9.693
2 — - .0 .0 .0 .0 .0
2 - + 1.4 1.3 1.4 .0 .0
2 + - .0 .0 .0 8.4 22.1 9.414
2 + + 1.7 3.7 2.9 9.6 20.0 9.419
Two 4 - - .0 .0 .0 .0 .0
4 - + 5.1 4.7 3.1 .0 .0
4 + - .0 .0 .0 1.3 99.0 9.697
4 + + 11.0 6.6 3.4 2.1 99.0 - 9.730
2 - - .0 .0 .0 .0 .0
2 - + 7.6 5.8 4.3 .0 .0
2 + - .0 .0 .0 1.6 99.0 9.544
2 + + 8.8 10.5 5.0 4.8 99.0 9.672
Three 4 - - .0 .07 .0 .0 .0
4 - + 37.5 38.7 39.7 .0 .0
4 + b .0 .0 .0 99.0 99.0 9.829
4 + + 49.0 49.0 46.2 99.0 99.0 9.943
2 - - .0 .0 .0 .0 .0
2 - + 53.3 51.1 52.2 .0 .0
2 + - .0 .0 .0 18.3 99.0 9.932
2 + + 43.4 47.9 54.0 6.2 99.0 10.124
Mean 4 - - .0 .0 .0 .0 .0
4 - + 14.4 14.9 14.7 .0 .0
4 + - .0 .0 .0 34.1 99.0 9.755
4 + + 21.9 20.2 18.0 34.4 99.0 9.789
2 - - .0 .0 .0 .0 .0
2 - + 20.8 19.4 19.3 .0 .0
2 + - .0 .0 .0 9.4 99.0 9.630
2 + + 18.0 20.7 20.6 6.9 99.0 9.738

 

Levels of Significance
Ozone Symptomsh

2 d 6 d 10 d
Experiments .00fi1‘ 000% 300%

Inoculation Time .78610 .24348 .08712
Bacteria Inoculation .04908'.01148‘ .06242
Inoculation Time by Bacteria Inoculation .00633‘.42466 .54782

Blight Symptoms}:

6 da 10 da
Experiments .00001' 7000019

Inoculation Time ~ .00001' .00001'
Ozone Fumigation .90165 .92656
Inoculation Time by Ozone Fumigation .77589 .92656
Log of Bacterial Populations
Experiments .00001'
Inoculation Time .11844
Ozone Fumigation .21545
Inoculation Time by Ozone Fumigation .55978

a Values presented are for each of the three replicate experiments and their means.

b Primary leaves were inoculated 4 and 2 days before ozone fumigation.

a Abaxial leaf surfaces were inoculated until a water—soaked appearance with (-) phosphate buffer or (+)
106 Ra CPU/ml.

d Plants were (+) ozone fumigated 12 days after fumigation or (-) not fumigated.

0 Ozone symptoms were recorded 2, 6 and 10 days after fumigation. Symptoms were recorded as the percent-
age of ozone damaged primary leaf tissue. Values presented are the means from 3 individual plant
ratings.

f Bacterial blight symptoms were recorded 6 and 10 days after fumigation. Blight symptoms were recorded
on a scale of 0-100 (0-15, no symptoms to light water-soaking; 15-35, moderate to heavy water-soaking
35-65, light to severe chlorsis; 65-85, light to moderate necrosis; 85—100. severe to complete
necrosis). Values presented are the means of 3 individual ratings. .

3 Bacterial populations were sampled 10 day after ozone fumigation. Values presented are the means of 3
individual samples each containing 3 plants.

h Ozone and blight symptom values were transformed (180/3.4) x {arcsinlsgrt(value)]} and analyzed.

9 Significant at the five percent level.

30

TABLE 4. Effect of Ra Bacteria, Inoculation Time and Ozone Fumigation on Ozone and
Blight Symptoms and Bacterial Populations of 'NEP-2' Plants.

 

 

 

 

 

 

 

Ozone Injury Blight Log Bacterial
Experiment Inoculation Bacteriac Ozone. d Symptomse Symptoms Populations?
ra Time Fumigation 2
days 2 day, 6 day, 2 day 6 day Ra CFO/50 cm leaf area
One 4 - - 0.0 0.0 0.0 0.0-
4 - + 7.3 8.9 0.0 0.0
4 + - 0.0 0.0 20.0 80.0 9.561
4 + + 7.2 8.0 20.0 80.0 9.755
2 - - 0.0 0.0 0.0 0.0
2 - + 10.0 8.9 0.0 0.0
2 + - 0.0 0.0 20.0 21.0 9.490
2 + + 6.8 7.5 20.0 35.2 9.712
Two 4 - - 0.047 0.0 0.0 0.0
4 - + 3.2 3.9 0.0 0.0
4 + — 0.0 0.0 2.0 80.0 9.654
4 + + 4.2 4.9 2.0 80.0 9.862
2 - - 0.0 0.0 0.0 0.0
2 - + 4.1 3.6 0.0 0.0
2 + - 0.0 0.0 0.0 20.0 9.555
2 + + 2.1 3.8 0.0 26.5 9.709
Three 4 - - 0.0 0.0 0.0 0.0
4 — + 12.7 11.6 0.0 0.0
4 + - 0.0 0.0 20.0 80.0 9.527
4 + + 18.1 18.7 20.0 80.0 9.680
2 - - 0.0 0.0 0.0 0.0
2 - + 14.4 11.1 0.0 0.0
2 + - 0.0 0.0 5.0 15.6 9.192
2 + + 18.7 20.8 5.0 15.6 9.283
Mean 4 - - 0.0 0.0 0.0 0.0
4 - + 7.7 8.1 0.0 0.0
4 + - 0.0 0.0 14.0 80.0 9.581
4 + + 9.8 10.5 14.0 80.0 9.765
2 - - 0.0 0.0 0.0 0.0
2 - + 9.5 7.8 0.0 0.0
2 + - 0.0 0.0 8.3 18.9 9.412
2 + + 9.2 10.7 8.3 25.8 9.568

 

Levels of Significance

Ozone Symptoms h

2 da 6 da
Experiments .0000!‘ .00001'

Inoculation Time .65364 .77677
Bacteria Inoculation .58576 .01760'
Inoculation Time by Bacterial Fumigation .05137 .91306

Blight Symptoms h

2 da 6 da
Experiments .00001’ .00011'

Inoculation Time .00001' .00001'
Ozone Fumigation 1.00000 .00570'
Inoculation Time by Ozone Fumigation 1.00000 .00570'
Bacterial Populations
Experiments .00001'
Inoculation Time .00005'
Ozone Fumigation .00013'
Inoculation Time by Ozone Fumigation .70689

a Values presented are for each of three replicat experiments and their means.

b Primary leaves were inoculated 4 and 2 days before ozone fumigation.

c Abaxial leaf surfaces were inoculated until a water-soaked appearance with (-) phosphate buffer or (+)
105 Ra CPU/ml.

d Plants were ozone fumigated (+) or non-fumigated (-) 12 days after seed germination.

e Ozone symptoms were recorded 2 and 6 days after ozone fumigation. Symptoms were recorded as the
percentage of ozone damaged primary leaf tissue. Values presented are the means from 3 individual
plant ratings.

f Bacterial blight symptoms were recorded 2 and 6 days after ozone fumigation. Blight symptoms were
recorded on a scale of 0-100 (0-15, no symptoms to light water-soaking; 15-35, moderate to heavy
water-soaking: 35—65, light to severe chlorsis; 65-85, light to moderate necrosis; 85-100, severe to
complete necrosis). Values presented are the means of 3 individual ratings.

g Bacterial pepulations were sampled 6 days after ozone fumigation. Values presented are the means of 3
individual samples each containing 3 plants.

h Ozone and blight symptom values were transformed (lac/3.14) x {arcsinlsqrt(value)l} and analyzed.

' Significant at the five percent level.

31

Effect of EDU, Ra bacteria and ozone fumigation on
ozone and blight symptoms and Ra bacterial populations
of 'Seafarer' plants. Primary leaves of 'Seafarer'

6 Ra CFU/ml

plants were inoculated until runoff with 10
and sprayed with EDU, two and one day, respectively,
before fumigation with ozone. The experiment was
designed to determine the effect of EDU and ozone
fumigation on Ra bacterial populations and blight
symptoms. The effects of bacterial inoculation on
ozone injury was also observed. Ozone injury was
recorded two, six and ten days after fumigation and
bacterial symptoms were recorded six and ten days after
fumigation. Bacterial populations were sampled ten
days after fumigation. Ozone symptoms in each
replicate experiment were not significantly different
(Table 5). Blight symptoms were significantly less
severe on ozone fumigated plants than non-fumigated
plants. There were no significant differences in Ra
bacterial populations between ozone-fumigated and non-
fumigated plants. Blight symptoms were also signifi-
cantly less severe on EDU sprayed plants than check
sprayed plants. Ozone injury was not affected by

bacteria inoculation.

Effect of EDU, Ra bacteria and ozone fumigation on
ozone and blight symptoms and Ba bacterial populations

of 'NEP-Z' plants. The same experiment described above

32

was performed on 'NEP-Z' with the following exceptions.
Ozone injury was recorded two, five and eight days after
ozone fumigation and blight symptoms were recorded five
and eight days after fumigation. Bacterial populations
were sampled eight days after fumigation. Ozone-
fumigated plants had significantly higher bacterial
populations than non-fumigated plants. Blight symptoms
recorded eight days after fumigation were significantly
more severe on ozone fumigated plants than non-fumigated
plants. There were no significant differences in Ra
bacterial populations or blight symptoms between EDU
sprayed and check plants (Table 6). Bacterial inocu-
lated plants exhibited significantly greater ozone
injury than non-inoculated plants at two, five and

eight days after ozone fumigation. 'Seafarer' and
'NEP-Z' plants were totally protected from ozone injury
when sprayed with EDU (Table 5 and 6). EDU sprayed
plants were always a darker green than the non-sprayed

plants.

Ozone injury on 'Seafarer' and 'NEP-Z'. The
cultivar ‘NEP-2' is classified as field tolerant to
ozone injury. We simultaneously ozone-fumigated ten-
day-old 'Seafarer' and 'NEP-Z' plants to determine
their differences in ozone sensitivity (Table 7). The
primary leaves of both cultivars were equally sensitive

to ozone fumigation.

3C3

TABLE 5. Effect of EDU, Ra Bacteria and Ozone Fumigation on Ozone and Blight Symptoms
and Bacterial Populations of 'Seafarer' Plants.

 

 

 

 

 

 

 

 

 

Ozone Inju Blight Log Bacteria
Experimegt Bacteriab EDU dc 92°": d Symptoms5y Symptomsf Populationsé
"W“ 59"” ““9“ °" 2 day 6 day 10 day 6 day 10 day Ra CPU/50 cm‘ leaf area
One + - - 0.0 0.0 0.0 10.0 10.0 8.728
+ - + 11.1 13.4 9.6 10.0 10.0 8.779
+ + - 0.0 0.0 0.0 10.0 11.0 8.907
+ + + 0.0 0.0 0.0 10.0 10.0 8.860
- - - 0.0 0.0 0.0 0.0 0.0
- - + 9.8 9.8 7.6 0.0 0.0
- - 0.0 0.0 0.0 0.0 0.0
- I + 0.0 0.0 0.0 0.0 0.0
Two + - — 0.0 0.0 0.0 0.0 15.2 8.399
+ - + 9.4 6.8 7.4 0.0 3.1 8.640
+ + - 0.0 0.0 0.0 0.6 2.7 8.813
+ + + 0.0 0.0 0.0 0.0 2.9 8.832
- - - 0.0 0.0 0.0 0.0 0.0
- - + 11.1 6.7 7.7 0.0 0.0
- + - 0.0 0.0 0.0 0.0 0.0
- + + 0.0 0.0 0.0 0.0 0.0
Three + - - 0.07 0.0 0.0 0.0 0.5 8.176
+ - + 15.6 10.2 8.3 0.0 0.8 8.198
+ + - 0.0 0.0 0.0 0.0 0.8 8.154
+ + + 0.0 0.0 0.0 0.0 0.6 8.157
- - - 0.0 0.0 0.0 0.0 0.0
- - + 10.3 10.7 8.7 0.0 0.0
- + - 0.0 0.0 0.0 0.0 0.0
- + + 0.0 0.0 0.0 0.0 0.0
Mean + - - 0.0 0.0 0.0 3.3 8.6 8.434
+ - + 12.0 10.1 8.4 3.3 4.6 8.539
+ + - 0.0 0.0 0.0 3.4 5.0 8.625
+ + + 0.0 0.0 0.0 3.3 4.5 8.616
- - - 0.0 0.0 0.0 0.0 0.0
- - + 10.4 9.0 8.0 0.0 0.0
- + - 0.0 0.0 0.0 0.0 0.0
- + + 0.0 0.0 0.0 0.0 0.0

 

Levels of Significance

Ozone Symptoms h

Experiments .60gg5 .22239 %%6%%¥

Bacteria Inoculations .49656 .61223 .81097
Blight Symptoms h
10 da
Experiments .00001'
EDU Sprayed .01274'
Ozone Fumigation .00769'
Ozone Fumigation by EDU Sprayed .03191‘
Bacterial Populations
Experiment .00001'
EDU Sprayed .05552
Ozone Fumigation .48013
Ozone Fumigation by EDU Sprayed .40609

a Values presented are for each of the three experiments and their means.

b Abaxial primary leaf surfaces were sprayed until runoff with (+) 106 Ra CPU/ml or (-) phosphate buffer
2 days before ozone fumigation.

c Adaxial primary leaf surfaces were sprayed with (+) EDU or (-) deionized water.

d Plants were (+) ozone fumigated and (-) non-fumigated 10 days after seed germination.

0 Ozone symptoms were recorded 2, 6 and 10 days after ozone fumigation. Symptoms were recorded as the
percentage of ozone damaged primary leaf tissue. Values presented are the means from 3 individual
plant ratings.

f Blight symptoms were recorded 6 and 10 days after ozone fumigation. Blight symptoms were recorded on a
scale of 0-100 (0-15, no symptoms to light water-soaking: 15-35, moderate to heavy water-soaking; 35-
65, light to severe chlorsis; 65-85, light to moderate necrosis; 85-100, severe to complete necrosis).
Values presented are the means of 3 individual ratings.

g Bacterial populations were sampled 10 days after ozone fumigation. Values presented are the means of 3
individual samples each containing 3 plants.

h Ozone and blight symptom values were transformed (180/3.l4) x {arcsintsgrt(value)l} and analyzed.

' Significant. at the five percent level.

34

TABLE 6. Effect of EDU, Ra Bacteria and Ozone Fumigation on Ozone and Blight Symptoms
and Bacterial Populations of 'NEP-Z' Plants.

 

 

 

 

 

 

 

 

. Ozone injury Blight Log Bacterial
Ex::;;::2t Bacteriab S EDU dc P 05°“:i d Symptomse Symptoms Populationsg
praye ‘ umiga on 2 day 5 day 8 day, 5 day 8 day Ra CPU/50 cm2 leaf area
One + - - 0.0 0.0 0.0 0.0 0.9 8.486
+ - + 2.2 2.3 2.2 0.0 1.4 8.454
+ + - 0.0 0.0 0.0 0.0 0.6 8.540
+ + + 0.0 0.0 0.0 0.0 1.4 8.755
- - - 0.0 0.0 0.0 0.0 0.0
- - + 1.9 1.9 1.8 0.0 0.0
- + - 0.0 0.0 0.0 0.0 0.0
- + + 0.0 0.0 0.0 0.0 0.0
Two + - - 0.0 0.0 0.0 0.0 1.0 8.647 .
+ - + 1.0 1.9 2.0 0.0 1.3 8.732
+ + - 0.0 0.0 0.0 0.0 1.3 8.819
+ + + 0.0 0.0 0.0 0.0 1.6 8.841
- - - 0.0 0.0 0.0 0.0 0.0
- - + 1.1 1.5 2.2 0.0 0.0
- + - 0.0 0.0 0.0 0.0 0.0
- + + 0.0 0.0 0.0 0.0 0.0
Three + - - 0.0 0.0 0.0 0.0 0.5 8.442
+ - _+ 4.1 5.8 5.9 0.0 2.0 8.793
+ + - 0.0 0.0 0.0 0.0 0.2 8.468
+ + + 0.0 0.0 0.0 0.0 0.3 8.709
- - - 0.0 0.0 0.0 0.0 0.0
- - + 1.0 1.7 1.8 0.0 0.0
- + - 0.0 0.0 0.0 0.0 0.0
- + + 0.0 0.0 0.0 0.0 0.0
Mean + — - 0.0 0.0 0.0 0.0 0.8 8.525
+ - + 2.4 3.3 3.4 0.0 1.6 8.660
+ + - 0.0 0.0 0.0 0.0 0.7 8.609
+ + + 0.0 0.0 0.0 0.0 1.1 8.768
- - - 0.0 0.0 0.0 0.0 0.0
- - + 1.3 1.7 2.0 0.0 0.0
- + - 0.0 0.0 0.0 0.0 0.0
- + +_ 0.0 0.0 0.0 0.0 0.0

 

Levels of Significance
Ozone Symptoms h

8 d
Experiments .655%* .52877' .021%¥'

Bacteria Inoculations .03530' .00950' .01781‘
Blight Symptoms h
8 da

Experiments .04105‘

EDU Sprayed .14035

Ozone Fumigation .01953’

EDU Sprayed by Ozone Fumigation .48125
Bacterial Populations

Experiments .00857‘

EDU Sprayed .07104

Ozone Fumigation .00773'

EDU Sprayed By Ozone Fumigation .81588

a Values presented are for each of the three experiments and their means.

b Abaxial primary leaf surfaces were sprayed until runoff with 106 Ra CFU/ml (+) or phosphate buffer (-)
2 days before ozone fumigation.

c Adaxial primary leaf surfaces were sprayed with EDU (+) or deionized water (-).

d Plants were ozone fumigated (+) and non-fumigated (-) 10 days after seed germination.

e Ozone symptoms were recorded 2, 5 and 8 days after ozone fumigation. Symptoms were recorded as the
percentage of ozone damaged primary leaf tissue. Values presented are the means from 3 individual
plant ratings.

f Blight symptoms were recorded 5 and 8 days after ozone fumigation. Blight symptoms were recorded on a
scale of 0-100 (0-15, no symptoms to light water-soaking: 15-35, moderate to heavy water-soaking:
35-65, light to severe chlorsis; 65-85, light to moderate necrosis: 85-100, severe to complete
necrosis). Values presented are the means of 3 individual ratings.

g Bacterial populations were sampled 10 days after ozone fumigation. Values presented are the means of
3 individual samples each containing 3 plants.

h Ozone and blight symptom values were transformed (180/3.l4) x {arcsinlsqrt(value)l} and analyzed.

9 Significant at the five percent level.

35

TABLE 7. Ozone Injury on 'Seafarer' and 'NEP-Z' Plants
in the Greenhouse.

 

Percent Leaf Tissue Damaged with Ozone Injury

 

 

 

 

Experiment
'Seafarer'1 'NEP-Z'1
1 30 32
2 33 28
3 14 8
4 10 10
5 8 7
Mean l9 17
1

Each value presented is the mean of 15 individual plant ratings.
The plants were fumigated with ozone (470-549 ug/m3) for eight
hours. All the non-fumigated check plants had no ozone injury.
The analysis of variance levels of significance of ozone injury
between cultivars for all five experiments was .80055.

TABLE 8. Cultured Ra Bacteria Affected by EDU.1

 

Colony Forming Units

 

 

Experiment EDU (ug/ml)
0.0 3.4 7.9 28 111 433
1 183a 127b 130b 121b ll3b 122b
2 245a 231ab 210bc 200c 182c 188c

 

1Each value presented is the CPU mean from five plates.
Values without the same letters are significantly different at the ’
0.05 level.

36

Ra cultured bacteria affected by EDU. An experi-
ment was designed to determine the effect of EDU on Ra
bacterial growth (Table 8). RAM plants containing EDU
were prepared by separately sterilizing the RAM and EDU
stock solution (886 ug/ml) and combining the two solu-
tions to make five different EDU dilutions (443, 111,
28, 7.9 and 3.4 ug/ml). Ra bacteria were plated on the
RAM-EDU plates and after 96 hours there was a signifi-
cant 33% reduction of CFU on plates containing 443 ug/ml

EDU in one out of two experiments performed.

Field Experiment

 

The field experiment was designed as a split plot
experiment, with three blocks each containing plots of
'Seafarer' and "HEP-2' plants. Each cultivar plot in
each block was split into non-inoculated and Ra
inoculated (Time 1, 7/6/78; Time 2, 7/20/78) subplots.
Each subplot was split into EDU sprayed and non-sprayed
plots. The experiment was designed to determine the
effect of EDU (plants protected against ozone injury)
on blight symptoms and the effect of Ra bacteria on
ozone injury.

Blight symptoms were not significantly different
in either cultivar between EDU sprayed plants (protected
against ozone injury) and non-sprayed plants (Tables 9

and 11). In nine out of ten cases EDU significantly

37

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38

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39

 

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40

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41

reduced ozone injury on 'Seafarer' plants (Table 10).
EDU did not significantly affect the total plant weight,
pod and seed weight, or seed weight of 'Seafarer' plots
(Table 13). Ra bacteria inoculations did significantly
affect the total plant weight, pod and seed weight and
seed weight of 'Seafarer'. In all three weight categor-
ies the greatest weight was the bacterial inoculation on
7/6/78 and the least was bacterial inoculation on
7/20/78. Neither EDUrunrRa bacterial inoculation
affected the number of blight lesions per 100 pods
(Table 14).

In one out of ten cases EDU significantly reduced
ozone injury on 'NEP-Z' plants (Table 12). Neither
EDU nor Ra inoculation significantly affected, total
plant weight, pod and seed weight, seed weight and
blight lesions per 100 pods of 'NEP-Z' plants
(Tables 13 and 14).

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42

 

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43

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44

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45

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46

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47

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DISCUSSION

The data presented shows a small trend for a
synergistic interaction between ozone and Ra bacteria in
Navy (pea) bean plants. The interaction was so small
that it was only observed in greenhouse experiments
and not the field experiments.

Greenhouse experiments were arranged so that
bacterial inoculations always occurred before ozone
fumigation to simulate the occurrence of bacterial
blight and ozone injury in the field. Blight injury
begins in the early seedling stage and ozone injury
begins after blossom (9, 27). In seven out of sixteen
observations ozone injury was significantly more severe
on plants inoculated with bacteria than on plants not
inoculated with bacteria. Not all of the ozone injury
observations showed significant differences. This
inconsistency was probably caused by the inability to
detect such small ozone injury differences between
inoculated and non-inoculated plants. Bacterial
papulations were significantly different in two of six
experiments and always the bacterial populations were
higher in ozone fumigated plants than non-fumigated

48

49

plants and in another experiment bacterial symptoms were
significantly less severe on ozone fumigated plants than
non-fumigated plants. But in the latter case increased
bacterial populations did not correlate with increased
blight symptoms. This inconsistency was probably do to
the experimental error in detecting small differences of
blight symptoms and bacterial populations.

In one experiment blight symptoms on non-EDU sprayed
plants were significantly higher than EDU sprayed
plants. Greater bacterial populations were observed on
the EDU sprayed plants but the differences were not
significant. EDU (433 ug/ml) in rifampin agar media
(RAM) did significantly reduce Ra CEO by 33 percent.
This EDU concentration is half the concentration at
which EDU was Sprayed onto the plants. The high EDU
concentration in the culture plate probably does not
represent the EDU concentration found in the leaf
tissue. Probably the EDU concentration in the leaf
tissue is much lower than the concentration at which it
.was applied, therefore the bacterial concentrations
would not be affected.

In the field experiment no cross protection or
synergistic interactions were observed. The only
significant differences in yield data were with
'Seafarer' plants. Plants inoculated at 7/6/78 had the

greatest yield and plants inoculated at 7/20/78 had the

50

lowest yield. There are a few reasons to help explain
why check plots were not the highest yielding plots. 'In
observing the blight symptoms data (Table 9) it is
apparent that Xp bacteria had infected the check plots
and some of the bacteria were Xp rifampin resistant
mutant Ra (Table 15). Additionally, there may have been
some naturally Xp infected seed which was planted which
also may have contributed to the spread of volunteer Xp
bacteria (Table 15).

In the field experiment ozone injury was signifi-
cantly reduced by EDU on 'Seafarer' plants but not on
iNEP-Z' plants. This suggests that'Seafarer' is ozone
sensitive and 'NEP42' is ozone tolerant in the field.
However 'Seafarer' and 'NEP-Z' were equally susceptible
to ozone injury and were significantly protected from
Ozone injury with EDU in the greenhouse experiments. In
the field perhaps 'NEP-Z' plants are as sensitive to
ozone as 'Seafarer' plants, but because 'NEP-Z' plants
mature later than 'Seafarer' plants, the ozone sensitiv—
ity of 'NEP-2' plants does not coincide with the ozone
episodes in August. 'Or maybe, primary leaves are more
sensitive to ozone than trifoliolate leaves. When
we began these experiments we made the assumption that
primary leaves were similar to trifoliolate leaves in
their response to ozone and Xp bacteria. Weller has

demonstrated using the Xp rifampin resistant mutant

51

“on 625 a? eH.o ufifinuaoo Saxon One new 55 >335 sesame one: nufiHn x628

 

 

 

 

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52

(Ra) that primary and trifoliate leaves have similar
patterns of blight symptoms and bacterial papulations.
In the case of 'Seafarer' there was no contradiction of
ozone sensitivity between field and greenhouse
experiments. To determine the cause of ozone sensitiv-
ity differences between the field and greenhouse
experiments on 'NEP-Z', both youn (primary leaves) and
old (trifoliolate leaves) should be fumigated with ozone
under controlled conditions.

Contrary to our initial hypothesis and unlike the
other studies concerning the interaction between
bacterial plant pathogens and ozone we found no cross
protection reaction occurring between Xp and ozone
injury. Instead we found a small synergistic reaction
occurring between Xp and ozone injury. If there was a
cross protection interaction, coumestrol probably
wouldnot be responsible. Wyman and VanEtten showed
that coumestrol is neither bacteriostatic nor

bacteriocidal against the Xanthomonads (29).

10.

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