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This is to certify that the
thesis entitled
Systemic acquired resistance of bean to xcp and partial
characterization of bacteria isolated from Azuki bean plants

presented by

Seriba Katile

has been accepted towards fulfillment
of the requirements for

Master's degree in Botany & Plant Pathology

 

 

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Major professor

 

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SYSTEMIC ACQUIRED RESISTANCE OF COMMON BEAN TO XCP
AND PARTIAL CHARACTERIZATION OF BACTERIA ISOLATED
FROM DISEASED AZUKI BEAN PLANTS.
By

Seriba Ousmane Katile

A THESIS
Submitted to
Michigan State University
In partial fulfilment of the requirements
for the degree of
MASTER OF SCIENCE

Department of Botany and Plant Pathology

1996

ABSTRACT

SYSTEMIC ACQUIRED RESISTANCE OF COMMON BEAN TO XCP AND
PARTIALCHARACTERIZATION OF BACTERIA ISOLATED FROM AZUKI BEAN
PLANTS

By Seriba Ousmane Katile

Two cultivars (Mayflower and Midland) of common bean (Phaseolus vulgaris),
when treated with 2,6 dichloroisonicotinic acid (INA) or inoculated with a low
concentration of bacteria (X. campestris pv. phaseoli) exibited a resistant reaction when
challenge inoculated with a high concentration ofXanthomonas campestris pv. phaseoli.
In greenhouse experiments, INA caused a reduction in disease infection and severity on
cultivars on both cultivars. The systemic resistance was accompanied by increased
peroxidase activity. In field experiments, there was reduced infection on both cultivars
and an increase of yield on the moderately resistant cultivar Mayflower.

Several bacteria were isolated from Azuki bean plants, seeds, and seedlings and
tested for their characteristics and pathogenicity on common bean in greenhouse
experiments. It appeared that some of the strains were pathogenic to common bean when
they were inoculated on several cultivars. The pathogenic strains were tentatively

identified as C urtobacterium flaccumfasciens and Pseudomonas syringae.

‘ To my children Moussa, Adama, Aissata,

and my late father and mother’

iii

ACKNOWLEDGMENTS

This is a good opportunity for me to thank all those which have been involved in
the success of this program. I would like to thank my professor Dr. L. Patrick Hart for
his patience, his support, and his encouragement during this program. I really appreciate
his kindness and all the efforts he made for the success of this program.

I thank also all the members of my committee, Dr. Ray Hammerschmidt who
provided the INA for the experiments and much information about SAR, and the Students
in his lab for their help with native gels; Dr. Dennis Fulbright and students in his lab for
their help with the PCR work, and Dr. Jim Kelly and his students who provided bean seed
for the experiments. I appreciate their advice for my guidance during this program.

I would like to thank my wife Djelika who shared with me all the hard moments
during my study; the coordinators of the SPARC training program at the Institute of
International Agriculture (MSU), and all other people I did not mention here who have
been involved in this program, especially students and technicians in professor L. Patrick
Hart’s laboratory.

Before closing this chapter, I thank Moussa Katile and Mariam for taking care of

my children in Mali.

TABLE OF CONTENTS

Pages
LIST OF TABLES ............................................................................ viii
LIST OF FIGURES ........................................................................... x
PART I: INDUCED SYSTEMIC RESISTANCE OF
BEAN TO BACTERIAL DISEASES
INTRODUCTION ............................................................................... 1
LITERATURE REVIEW ..................................................................... 6
Historical perspectives of Systemic Induced resistance ..................... 6
Induced resistance to bacteria .......................................................... 9
Proposed mechanisms of systemic induced resistance ..................... 9
Signals involved in systemic resistance ............................................ 10
Salicylic acid (2- hydroxybenzoic acid) ............................................ 11
Chemicals inducers of resistance ..................................................... 1 1
2-6, dichloroisonicotinic acid .......................................................... I I
Peroxidase and chitinase activities ................................................... 13
Pathogenesis-related (PR) proteins .................................................. 14
Common bean - pathogen interactions ........................ . ...................... 15
MATERIALS AND METHODS ........................................................... 17
Plant materials ................................................................................... 1?
Bacterial isolates ................................................................................ 17
Greenhouse experiments ..................................................................... 18
Methodology for inoculation ............................. ‘ ................................. 18
Primary inoculation resistance inducing treatments ............................ 16
Challenge inoculation ........................................................................ 20
Multiple applications of INA ............................................................. 21
Analysis of bacteria growth ............................................................... 21
Dry bean peroxidase induction ........................................................... 22
Field experiments ............................................................................... 23
RESULTS ............................................................................................... 26
Greenhouse experiments ..................................................................... 26
Effect of INA on common blight infection .......................................... 26
Multiple applications of INA on the control of ch ........................... 33

Pages

Analysis of bacterial growth ................................................................ 36
Peroxidase activity ............................................................................. 39
Results of field experiments ................................................................. 46
Effect of INA on bacteria infection ..................................................... 46
Effect of INA on bean production ...................................................... 48

DISCUSSION AND CONCLUSIONS ..................................................... 50

PART II. CHARACTERIZATION OF BACTERIA ISOLATED FROM
DISEASED AZUKI BEAN PLANTS

INTRODUCTION .................................................................................... 54
LITERATURE REVIEW .......................................................................... 55
Chemical tests and morphological characteristics on specific
media ................................................................................................... 56
Molecular test: PCR ............................................................................. S7
Pathogenicity tests ................................................................................ 58
MATERIAL AND METHODS ................................................................ 59
Isolation from field infected Azuki bean in 1994 ................................... 59
Isolation from infected seed and seedlings grown from
commercial seed obtained in 1995 ......................................................... 59
Plants materials ..................................................................................... 6S
Bacterial isolates ................................................................................... 66
Preparation of inoculum and procedure of inoculation ........................... 67
Molecular analysis of bacterial strains ..................................................... 68
RESULTS ................................................................................................. 70
Effect of seed surface sterilization on germination and
isolation of bacteria ............................................................................... 7O
Characteristics of bacterial strains .......................................................... 71
Pathogenicity tests ................................................................................. 72
Molecular analysis ................................................................................. 77
DISCUSSION AND CONCLUSIONS ...................................................... 80
LIST OF REFERENCES .......................................................................... 81

vi

Pages

APPENDICES .......................................................................................... 88
Appendix A ................................................................................... 88
Appendix B .................................................................................... 90
Appendix C .................................................................................... 92
Appendix D .................................................................................... 93

vii

LIST OF TABLES
Pages

Table 1. Effect of pre-treating the common bean cultivar Mayflower
with 2, 6-dichloroisonicotinic acid on infection and disease
severity after challenge inoculation by X. campestris pv phaseoli

eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee

Table 2. Effect of pre-treating the common bean cultivar Midland
with 2,6 dichloroisonicotinic acid on infection and disease
severity after challenge inoculation by Xcampestris pv.phaseoli.

eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee

Table 3. Effect of multiple applications of INA on infection and disease
severity on the cultivar Mayflower inoculated with X. campesm's pv.

phaseoli ................................................................................................

Table 4. Effect of multiple applications of INA on infection and disease
severity on the cultivar Midland inoculated with X. campestris pv.

phaseoli .............................................................................................. .

Table 5. Effect of 2,6 dichloroisonicotic acid treatment on colony forming
units of X. campestris pv. phaseoli from leaf discs of the cultivar

Mayflower afier challenge inoculation ...................................................

Table 6. Effect of 2,6-dichloroisonicotinic acid treatments on colony forming
units of X. campestris pv. phaseoli from leaf discs of the cultivar

Midland afier challenge inoculation .......................................................

Table 7. Effect of INA inoculation on infection of bean, cultivars Mayflower

and Midland by X. campesm's pv. phaseoli in field inoculations ............

Table 8. Effect of INA treatments and challenge inoculation on bean seed yield
of cultivars Mayflower and Midland .....................................................

Table 9. Effect of seed treatment on germination and seedling infection

of Azuki bean cultivar Erimo .................................................................

viii

32

34

35

.37

38

47

49

71

Pages

Table 10. Characteristics of bacteria used in inoculation studies
of edible bean, azuki bean and cowpea .................................................. 73

Table 11. Comparison of pathogenicity and symptoms on cultivars
of edible bean, Azuki bean and cowpea inoculated with strains of
bacteria isolated from Azuki bean or edible bean .................................... 75

Table 12: Grouping of different isolates of bacteria ............................................ 76

LIST OF FIGURES

Pages

Fig .1. Structure of salicylic acid and 2-6, dichloroisonicotinic acid ........... 13
Fig. 2. Effect of INA inoculation on common blight infection after

challenge inoculation with ch on cultivar Mayflower ................... 27

Fig 3: Effect of INA soil drench and bacteria inoculation after challenge
inoculation with ch on leaf cultivar Mayflower ........................... 29

Fig. 4. SDS-polyacrylamide gel electrophoresis Of peroxidase from
leaves on the common bean cultivar Mayflower .............................. 40

Fig. 5. SDS-polyacrylamide gel electrophoresis of peroxidase from leaves
Of the common bean cultivar Midland ............................................. 42

Fig. 6 Native gel electrophoresis of peroxidase from leaves of

bean cultivars Mayflower and Midland .......................................... 44
Fig. 7. Azuki bean seedlings infected by bacteria ........................................ 61
Fig 8. Azuki bean leaf infected by bacteria ................................................. 63

Fig .9. PCR amplification of bacterial strains isolated from common
bean and Azuki bean ...................................................................... 78

PART I : SYSTEMIC ACQUIRED RESISTANCE TO COMMON BEAN TO XCP

I. INTRODUCTION.

Dry beans are susceptible to several disease causing bacteria. The three major
bacterial diseases are common bacterial blight, halo blight and bacterial brown spot. The
bacterial pathogens are Xanthomonas campestris pv. phaseoli, Pseudomonas syringae
pv. phaseolicola and Pseudomonas syringae pv. syringae, respectively. Among these
diseases, common bacterial blight is the most important and difficult to control because
there is no commercially available resistance, and chemical control is not reliable.

Symptoms of common blight on leaves initially appear as water soaked spots that
enlarge gradually, become flaccid and then necrotic. Lesions can be found throughout
the leaf including the margins. In severe infections dead leaves may remain attached to
the plant at maturity. Bacteria exude through stomata, thus providing inoculum for
secondary infections. Pod lesions are generally circular, slightly sunken and dark red-
brown.

Lesions are more abundant and severe on older leaves, but younger leaves can be
affected. The disease affects plant vigor, reduces yield, and can affect seed quality. When
the seed is internally contaminated, it is not possible to eradicate bacteria by seed
treatment. During extended periods of warm, humid weather, the disease can be highly
destructive causing loss in both quantity and quality of seed. Infected seeds are shriveled
and exhibit poor germination and vigor. When plants are severely infected, the entire plant

can die (Hall, 1991).

2

The bacterial pathogens causing common blight are X. phaseoli (Smith) Dawson
and its firscous variant X. phaseoli var. firscans (Burkholder) Starr and Burkholder, which
produces a brown pigment in culture. Both are now recognized as Xanthomonas
campestris pv. phaseoli (Smith) Dye. It is a gram negative, straight rod, aerobic
bacterium that is motile by a polar flagellum. The bacterium produces a yellow non-water
soluble carotenoid pigment (xanthomonadin) and mucoid growth on nutrient glucose agar
and YDC (yeast extract dextrose CaCO3). Common blight of bean can develop from
several inoculum sources but external and internal seed contamination are by far the most
important means of survival for Xanthomonaspampestris pv. phaseoli. The pathogen
may overwinter in plant debris for at least a year and longer in infected seed (Saettler,
1989)

The primary methods of disease control include the use of pathogen-free seed
and crop rotation. Chemical controls are not effective and all commercial cultivars of
edible beans are susceptible to X. campestris pv. phaseoli. In the absence of traditional
chemical and resistance strategies, an alternate method to reduce the disease is needed.
Among the techniques developed recently, systemic induced resistance may be an option.

Some plants species may react to pathogen infection by the induction of a long-
lasting broad - spectrum systemic resistance to subsequent infections by the same
pathogen or even other pathogens. The phenomenon has been known for many years and
has several names such as physiological acquired immunity (Chester 1933), induced

resistance or systemic acquired resistance (Ross 1961b).

General overview of induced resistance:

Systemic acquired resistance is a broad, physiological immunity that result from
infection with a necrotic pathogen (Kessmann et al 1994). In addition, certain natural or
synthetic chemical compounds can trigger similar plant responses (Kessmann er al, 1994).
Many plants develop an increased resistance against subsequent pathogen infection in
uninfected tissues. This systemic acqured resistance can be effective against viruses,
bacteria and fungi and is accompanied by the systemic expression of a group of genes
called SAR genes (Kuc, 1982; Ward et a1, 1991).

Most of the studies on systemic resistance have concentrated on tobacco (Ross
1961; Tuzun and Kuc; 1989) and cucurbits ( Hammerschmidt et a! 1976, Kuc, 1975 ),
but some effort has focused on other crops including wheat (Ride, 1980), cabbage
(Cook et a] 1985), rice (Smith et al, 1991), millet ( Kumar et a! 1993), and sugar beet
(Nielsen et al 1994).

Systemic induced resistance in plants is distinct from preexisting resistance
mechanisms (i.e physiological barriers), protein cross- linking, and phytoalexin
biosynthesis, the hypersensitive response and ethylene - induced physiological changes
(Ryals et a! 1994). Wounding or osmotic stress responses are not related to the induced
systemic resistance (Ryals et al 1994).

The first step in the development of systemic acquired resistance (SAR) is the
recognition by the plant of the pathogen infection. Compatible and incompatible
interactions can lead to an induction of SAR; thus the pathogen needs not to induce a

gene-for-gene resistance reaction (Kuc, 1982).

4

The induction of resistance usually coincides with the accumulation of
pathogenesis-related (PR) proteins. PR proteins and salicylic acid (SA) accumulate in
plant tissues following resistance inducing treatments that include inoculation with
microorganism and treatment with some chemical compounds. Some examples of PR-
proteins are chitinases (PR-3 group) and [3-1, 3 glucanases ( PR-2 group) which possess
antifungal activity and may play an active role in disease resistance. Salicylic acid and 2,
6 - dichloroisonicotinic acid (INA) are two known chemical inducers of resistance
(Nielsen et a], 1994). SA has induced both local resistance to TMV and the accumulation
of PR proteins (White, 1979). There is evidence to suggest SA be an endogenous signal
mediating disease resistance and an exogenous inducer of PR protein accumulation
(White, 1979 ). 2,6 dichloroisonicotinic acid (INA) induces local and systemic resistance
to pathogens in a number of plants ( Kuc 1982).

Bean induced resistance:

The systemic induction of resistance on beans was first reported by Sutton (1979).
The pre-inoculation of unifoliate leaves with spore suspensions of Colletotrichum
Iindemuthianum resulted in less severe symptom development in the first trifoliate leaves
compared with control plants when those leaves were challenged with the same pathogen
7 or 12 days later (Sutton 1979). Green bean plants were protected against anthracnose
caused by Colletotrichum Iindemuthianum when the hypocotyls of bean cultivars resistant
to some but not all the races of the fungus were inoculated, with nonpathogenic races of
the organism (Kuc, 1982). Bean plants also developed resistance against pathogens after

a primary infection by the same pathogen or other pathogens, or exposure to certain

5

chemicals (INA) that helped them to build up their defense system against P.5yringae pv.
phaseolicola (Dann and Deverall 1995).

Because conventional disease management strategies to control common bacterial
blight of bean such as use of clean seed, rotation, burial of crop debris by plowing,
chemicals and resistance are either not very effective or available, the research in this
thesis addressed the possibility of using SAR as a disease management tool. The
Objectives of this study were:

1 - to study the effectiveness of systemic acquired resistance of dry beans to bacterial
blight caused by Xanthomonas campestris pv. phaseoli in field and greenhouse
experiments.

2 - to analyze the effect of inoculating plants with INA or bacteria on PR- proteins and

peroxidase activities.

II. LITERATURE REVIEW:

Historical perspectives of Systemic Induced Resistance.

Systemic induced resistance or acquired systemic resistance has been
recognized for many years by naturalists and scientists ( Ryals 1994). The natural
phenomenon of resistance development in response to pathogen infection was first
recognized in 1901 by Ray & Beauverie, who worked with Botryris cinera on Begonia.
Chester ( 1933 ) reviewed 200 publications describing the phenomenon he termed
physiological acquired immunity. During the 30 years following Chester's review, many
papers were published but most of them were descriptive studies extending the earlier
observations.

The first systematic study on systemic acquired resistance (SAR) was
published by Ross in 1961 where he used TNV (Tobacco Necrosis Virus ) which causes
local lesions on Nicotiana tabacum, and demonstrated that infections of TNV were
restricted by a prior infection of the same virus. Ross established the validity of plant
immunization against virus diseases on tobacco (Nicotiana tabacum), bean (Phaseolus
vulgaris), and cowpea (Vigna unguiculata) using viruses as the inducing agents ( Ross
1964, 1966). Plant immunization was subsequently expanded to include many hosts and
viruses, bacteria, firngi, cellular components of infectious agents and chemicals ( Kuc
1976, 1981, a, b; Kuc and Caruso, 1977; Kuc and Richmond, 1977; McIntyre er al, 1981).
The hypersensitive reaction (HR) leading to systemic acquired resistance was first
characterized in Sweet William plants infected with “carnation mosaic virus” (Gilpatrik

and Weintraub 1952). The accumulation of groups of extracellular proteins called PR-

7

proteins (Pathogenesis related proteins) was correlated with the onset of SAR (van Loon
1982). White (1979) demonstrated that salicylic acid (SA) and certain benzoic acid (BA)
derivatives induced both resistance and accumulation of PR- proteins. As a result, SA was
considered as a possible endogenous signal (Van Loon and Antoniw, 1982).

Studies on cucumber and tobacco indicated that the lignification of the cell walls
(Hammerschmidt et al, 1982) or the induction of hydrolytic enzymes (Boller, 1987) and
other PR-proteins (Van Loon, 1985) were components of the induced defense
mechanism. Gottstein et al( 1989) found that solutions of K3PO,, K2HP04, Na3PO, and
Na2PO4 sprayed on the underside of the first and second true leaves of cucumber induced
systemic resistance in leaves three and four against anthracnose caused by Colletotrichum
Iagenomm. The induction of systemic resistance by the use of chemicals was studied by
Metraux et a] in 1991. 2,6—dichloroisonicotinic acid or INA (CGA 41396) is a chemical
compound formulated by Ciba-Geigy Ltd. with 25% active ingredient (a. i) that acts
indirectly by simulating the mechanisms of resistance in the host plant (Metraux er al
1991). They hypothesized that after a rapid uptake and translocation to other parts of the
plants INA acted directly with the defense reaction and not through the intermediate
systemic signal.

In 1991, Ward et a1, using a tobacco ITMV model system showed that steady-
state mRNA levels from at least nine families of genes were coordinately induced in
uninfected plants and they referred to these families collectively as SAR genes ( Ward et
al 1991). SA has been proposed as one signal leading to SAR because its concentration

rises dramatically afier pathogen infection (Malamy et al, 1990, Metraux er a1. 1990).

8

However, experiments by Hammerschmidt et a1 (1982) suggests that SA may not be a
systemic Signal. According to Ryals et a1 (1994), SAR can be conceptually divided into
two phases: aninitiation and transient phase that includes all of the events leading to the
establishment of resistance; and the maintenance phase describing the quasi steady-state
resistance that results from initiation.

Nielsen et a1 ( 1994 ) induced resistance in sugar beet to Cercospora beticola only
after repeated foliar applications of INA with concentrations ranging from 10-100 ppm,
but higher concentrations caused toxicity (necrosis). Four treatments with 25 ppm of INA
induced complete local and systemic resistance with no signs of phytotoxicity but fewer
treatments caused a delay in symptom appearance. Injection of INA directly in soil or
plants, soil drenching or immersion of roots into solutions of INA did not affect firngal
growth (Nielsen et al 1994). Vemooij et al (1995 ) found that the synthetic chemical
INA acts via the SAR signal transduction pathway. They showed that INA does not
induce SA accumulation in Arabidopsis and that INA is effective in transgenic plants
unable to accumulate SA. This suggested that INA induced the SAR signal transduction
pathway by acting either at the same site or downstream of SA translation.

Hoffland et a! (1995) showed that induction of resistance was possible in plants
which did not accumulate PR-proteins suggesting that the accumulation of PR-proteins
was not a prerequisite for the induction of systemic resistance.

A single application of INA at low concentration was sufficient to induce
resistance against several fungal and bacterial pathogens on different species of plants

(Metraux et al 1991). Inoculating millet (Penm'semm glaucum) seedlings with a low

9

concentration of zoospores, Singh et a1 (1993 ) induced systemic resistance to downy
mildew of millet caused by Sclerospora gramim'cola after challenging with higher

concentration of the same pathogen.

Induced resistance to bacteria:

Resistance was induced in number of plant species to bacterial diseases. These
include Pseudomonas solanacearum on tobacco (Seiquira, 1984), Erwim'a carotovora
subsp. carotovora on tobacco (Palva et a1, 1994), Pseudomonas lacrymans on cucumber
(Kuc, 1982), Pseudomonas syringae pv. phaseolicola on bean (Dann and Deverall 1995),
after a primary inoculation with microbial or chemical compounds. Infection of
Arabidopsis thaliana with turnip crinkle virus (TCV) leaded to resistance to TCV or
Pseudomonas syringae ( Ukness et a1. 1992). Active resistance to black rot caused by
Xanthomonas campestris pv campestris was induced in cabbage by inoculation with

Xanthomonas campestris pv. carotae (Cook and Robeson, 1985).

Proposed mechanisms of systemic acquired resistance.

Systemic resistance results from the release of endogenous compounds that are
translocated fi'om slowly necrotizing cells to other locations in the plants (sequeira, 1983)
For a systemic signal to increase resistance to a pathogen or insect, it must be perceived
by plant cells ( Hammerschmidt et al, 1993). For chemical signals, this may involve
binding to a receptor molecule in the plasma membrane (Ryan, 1992). The penetration of

appresoria of C. lindemuthianum into immunized cucumber was markedly reduced

10

whereas the gemiination of conidia was unaffected (Hammerschmidt, 1980;
Hammerschmidt and Kuc 1982; Jenns and Kuc, 1977; Jenns and Kuc 1980; Richmond et
al 1979). Lignification that occurs rapidly afier penetration was localized to the invaded
cells and a few adjacent cells and was associated with an increase in peroxidase activity.
AS with immunization, a single lesion on the inducer leaf resulted in a statistically
Significant systemic increase in peroxidase activity (Hammerschmidt et al, 1980). The
systemic increase in peroxidase was associated with markedly increased activity of several
peroxidases (Hammerschmidt, 1980).

Several reports present strong evidence that lignification is a plant disease
resistance mechanism, ( Asada et al, 1979; Henderson and Friend, 1979; Pearce and Ride
1978, 1980; Ride 1980; Vance et al, 1980). Lignification can restrict development of
pathogens by several possible mechanisms: increasing the mechanical resistance of the host
cell wall, reducing the susceptibility of the host cell wall to degradation by extracellular
enzymes, restricting the diffusion of pathotoxins and nutrients, inhibiting the growth of

pathogens by the action of toxic lignin precursors (Ride, 1980)

Signals involved in systemic resistance:

A number of chemical and non-chemical signals are involved in the induction of
systemic defense response of plant against pathogens (Enyedi et al 1992, Malamy and
Klessig, (1992). These include salicylic acid (SA), oligogalacturonides, the lipids derived
signals, peptides, abscisic acid, and non chemical signals ( Hammerschmidt, 1993). 2-6

dichloroisonicotinic acid (INA) is a chemical compound that induces resistance in plants

11

(Metraux, 1991). These compounds may affect different points in the signal transduction

pathway.

Salicylic acid (2-hydroxybenzoic acid):

Salicylic acid plays a central role in SAR signal transduction after pathogen
infection and is an exogenous inducer of PR-proteins accumulations and resistance
(Kessmann et al, 1994). In higher plants, SA has been proposed to be synthesized from
trans-cinnamic acid to SA, via the intermediates ortho-coumaric acid or BA (Ward et a1
1991). Such pathways provide a link between pathogen induction of phenyl propanoid
biosynthesis and SAR signal production (Ward et a1 1991). SA is required for SAR but it
is not the translocated signal molcule (Kessmann et a1 1994)

SA plays a central role in SAR-signal transduction afier pathogen infection and is
as an exogenous inducer of PR-proteins accumulation and resistance. The mechanism by
which SA induced gene expression is unknown, but a study by Chen et al, (993)

speculated that SA may be mediated by catalase inhibition.

Chemical inducers of resistance:
2-6 dichloroisonicotinic acid.

2-6 dichloronicotinic acid (CGA 41396) ( fig. 1) and its methyl ester (CGA
41397), both referred to as INA, induce systemic resistance in plants (Metraux et a]
1991). These compounds provide good protection against fungal and bacterial pathogens

on cucumber, rice and other crops in greenhouse and field experiments (Kessman et a1

12

1994). The compounds have no significant in vitro activity and are not converted into
antimicrobial metabolites (Kessman et a! 1994). INA induces B —1, 3-glucanases,
chitinases and 6-phosphoglucanate - hydrogenase (6-PGD) in tobacco, whereas
phenylalanine ammonia-lyase (PAL), acidic proteases, peroxidases and
phenylphenoloxidase were not affected by this compound (Kessman et al 1994). The
enzymes not affected by INA are typically observed in a local defense response associated
with the hypersensitive response (HR). INA apparently mimics the biological induction of
systemic disease resistance without affecting responses linked with local necrosis. INA
and its analogs sensitized plant tissue to respond to attempted infection with additional
defense reactions faster than untreated plants, in addition to the induction of SAR genes
and enzymes (Kauss et al 1992).

Kauss et a] (1992) showed that parsley cell cultures pre-treated with INA
produced various phenolpropanoid-derived metabolites much more rapidly following
treatment with fungal elicitor than did the control. In rice, INA induced a pronounced
increase in lipoxygenase activity within two days Of application, whereas an inactive
analog had no inducing effect (Smith and Metraux, 1991). The isonicotinic acid derivative
N-cyanomethyl-Z-chloroisonicotinade induced a high level of activity against the rice blast
pathogens (Seguchi et al 1992). Levels of lipoxygenases increased as did general lipid
metabolisms and peroxidases in chemically treated and blast-inoculated rice leaves
compared to inoculated controls. These results may indicate that other defensive

mechanisms are involved in the induction of SAR, besides those already characterized.

13

COOH

/

 

 

 

 

 

 

' \ \coou °V\N/\ cr

0 INA [CGA 413961
sallcyllc acid

Fig. 1: Chemicals structure of Salicylic acid and INA (CGA 41996)

Peroxidase and chitinase activities.

Resistance in plants is associated with inducible compounds that may function in
defense against disease: phytoalexins, peroxidases, lignin, hydroxyproline-rich
glycoproteins (extensins) and pathogenesis related proteins (Hammerschmidt, 1982;
Boller, 1987; van Loon, 1985). Increased peroxidase activity, enhanced lignification and
extensin deposition are associated with induced systemic resistance in cucumber against
Colletotrichum Iagenarium ( Hammerschmidt er a1, 1982; Dalisay and Kuc ,1995). PR-
proteins accumulation follows the induction of the systemic resistance ( Smith and
Hammerschmidt, 1988). The induction of chitinase activity occurred in different plant
species in response to infection and treatment with fiingal cell wall preparations ( Dalisay,
and Kuc, 1995). The observations that chitinase has inhibitory activity in vitro and

apparently in viva against some chitin-containing fungi but not those lacking chitin,

14

suggested a direct role of chitinase in plant defense.

Pathogenesis-related (PR) proteins:

Systemic induced resistance by pathogens or chemical agents is associated with a
concomitant accumulation of pathogenesis-related (PR) proteins. Currently, five families
of PR proteins have been classified (Carr and Klessig 1989). Within each family,
members of the so-called class 1 proteins are generally located in the vacuole, whereas
classes 2 and 3 are acidic extracellular proteins. The PR-l family of acidic extracellular
proteins have unknown firnction, but were the most abundant PR proteins in tobacco
(Payne et al 1988b). They include PR-la, PR- 1b and PR-lc. The PR-2 family of
proteins includes class 1 basic vacuolar proteins, class 2 and 3 proteins, and acidic
extracellular proteins with B-1,3 endoglucanase activity ( Kaufi‘man et al ., 1987, Ward et
al 1991). The PR-3 family includes class 1 basic proteins and class 2 acidic extracellular
proteins and endochitinase activity (Legrand etal., 1987; Payne et a1 1990; Heitz et a1
1994). PR-4 family includes acidic extracellular proteins with unknown function
(Friedrich et a! 1991). PR-S family has class 1 basic proteins and class 2 acidic
extracellular protein homologous to thaumatin (Payne et al 1988). Evidence is
accumulating that PR proteins possess direct antifiingal activity in vitro but their biological
role in vivo is unclear. Working with Pseudomonasfluorescens as an inducer of resistance
in the radish against F usarium oxysporium f.s. rapham’, Hoffland et al (1995)
demonstrated that accumulation of PR proteins was not a prerequisite for the expression

of induced systemic resistance.

15

Common bean-pathogen interaction,

The invasion of plant tissue by foliar bacterial pathogens generally occurs through
natural openings, e.g., stomata, hydatodes or nectaries, when there is a water congestion,
such as following a rainfall or heavy dew or under high humidity (Panopoulos and Schroth
1974). The bacteria then invade intercellular spaces, causing a gradual dissolution of the
middle lamella (Panopoulos and Schroth !974). One of the most conspiscious effects of
microorganisms on plant cell walls is enzymatic degradation (Walton 1994). Number of
cell wall dergrading enzymes have been reported from plant pathogen; these include
cellulase, pectinase, cutinase , xylase and protease (Walton 1994).

The most common simple interpretation of gene for gene system involving bacteria
and fiingi and for which there is direct evidence in a bacteria system is that the gene for
avirulence controls the production of an elicitor, the recognition of which is controlled by
the gene for resistance in the plant (Heath, 1991)

Colletotrichum lindemuthianum penetrates common bean by exerxing mechanical
pressure but there is evidence that the action of cutinases enzymes is also required for
successful penetartion of this pathogen (O’Connell et a1 1985). Subsequent penetration of
the epidermal cell appears to be mediated by enzymatic degradation and mechanical
pressure (O’Connell et al 1985).

Common bean may respond to bacterial infection by different means and bacteria
may also use different strategies to penetrate bean plant tissues. The role of gene-for-

gene interaction may be determinant in some bacterial diseases especially halo blight.

16

Incompatible reactions lead to necrosis and death of the surrounding cells, and compatible
reactions lead to disease development.

As described in the introduction there is no commercially available resistance to
common bacterial blight (Xanthomonas campestris pv. phaseoli) of edible bean, and
chemical controls have not been effective. The previous research on SAR suggested that
this may be a practical approach to reduce the severe economic losses to this disease
expected in Michigan. The objectives were:

1- to study the effectiveness of systemic acquired resistance in dry beans to
bacterial blight caused by Xanthomonas campestris pv. phaseoli in field and greenhouse
experiments.

2- To analyze the effect of inoculating plants with INA or bacteria on PR-

proteins and peroxidase activities.

11]. MATERIALS AND METHODS
WIS:

Two commercial cultivars of common bean (Phaseolus vulgaris L.) were used in
this study: Mayflower and Midland. These cultivars are not as commonly grown in
Michigan as they were several years ago, but their reaction to X. campestris pv. phaseoli
is distinct: Mayflower is moderately susceptible, Midland is highly susceptible. Both
cultivars were used in field and greenhouse experiments. Seed was provided by Dr. J.
Kelly, Department of Crop and Soil Science (MSU).

Bacterialisclam:

The bacterial isolate used both in the field and greenhouse study was a strain of X.
campestris pv. phaseoli (Smith) Dye (DWF 151) isolated from infected leaf tissue and
obtained from Dr. D.W. Fulbright (Department of Botany and Plant pathology, MSU).
The bacterial cultures were grown on NBY (nutrient-broth yeast extract agar) medium.
The composition of this medium was the following: nutrient broth, 8 g/l; yeast extract, 2
g/l; KZI-IPO4, 2 g/l; KI-I2P04, 0.5 g/l; glucose, 2.5 g/l; Bacto-agar, 15 g/l. The medium
was autoclaved for 20 minutes and poured in 100 mm x 10 mm plastic petri dishes.
After cooling, a metal loop was sterilized with alcohol and flamed, and single colonies
were streaked on the solidified medium. Plates were kept under fluorescent light to
enhance growth. A day before inoculation of plants, single colonies were picked with a

sterile toothpick and dropped into 200 ml of nutrient broth / 500 ml Erlenmeyer flasks.

17

18

The flasks were placed in a controlled environment incubator shaker (New Brunswick
Scientific) incubator shaker at 30°C and the bacteria were grown overnight. The
bacterial solution was centrifugated at lxlO‘ g in an [EC Centra-4B centrifiige and the
pellet collected. The pellet was diluted in sterile water and a Beckman DB-G grating
spectrophotometer was used to measure the Optical density of the solution which was
adjusted to an OD. 600= 0.05 for the inducing inoculation and OD 600= 0.6 for the

challenge inoculation.

WM:

Commercial navy bean cultivars Midland and Mayflower were grown in 15 cm clay
pots filled with bacto-professional soil. For each treatment, 2 to 3 plants were used.
Plants were watered daily and the temperature was maintained at 20 to 25° C during the
day and 16 - 18° C during the night. Plants were fertilized once with a 10% water solution
of urea to enhance growth. Plants received the primary SAR inducing inoculation two
weeks afier planting. The experiments were performed five times and the data for the 3

best experiments were collected.

M II I I I} . I |° :
WW: Three primary SAR inducing traetments were
used to induce SAR in the dry bean cultivars: 1) injection of INA solution into the leaves;

2) soil drenches with a solution of INA; 3) inoculation of leaves with a low concentration

of X. campestris pv. phaseoli. Primary leaves were injected with a 50-ppm water solution

19

of INA. Preparation of INA solution consisted of diluting 200 mg of the INA powder (25
% a.i) in 1 liter of distilled water (Hammerchmidt, personal communication).
Approximatively 50 ul of INA solution were infiltrated in the lower surface at four to five
locations using a 1 ml syringe without a needle. Leaf intercellular spaces were infiltrated
by placing the leaf tissue between the syringe opening and the thumb and depressing the
syringe plunger. For the soil drenches, the soil surface of each pot containing three
plants was drenched with 100 ml of a 0.5 ppm solution of INA. For the bacterial primary
SAR inducing inoculation, plants were inoculated with Xcampestris pv. phaseoli strain
DWF 151. The strain was grown on NBY ( as described previously) for one week and
single colonies collected and grown in a nutrient broth solution for 24 hours at 30°C in a
controlled environment incubator shaker (New Brunswick Scientific). Bacterial pellets
were collected by centrifugation as described previously and diluted with sterile distilled
water. Using small centrifiIge tubes the pellet was diluted with distilled water and the
optical density was adjusted an OD. 600 = 0.05 using a spectrOphotometer. Preliminary
Studies showed that an OD. 600 = 0.05 corresponded to 10s cfu/ml". The bacteria were
infiltrated into the lower surface of the leaves using a 1 ml syringe, in the same manner as
the INA solution.

A day prior to inoculation, the plants were placed in an air-conditioned mist
chamber at 100% relative humidity and a temperature of 20° C. Afier inoculation, the
plants were placed in plastic bags and kept in the mist chamber for 24 hours. The plastic
bags were removed and plants then placed on benches in the greenhouse 5 to 7 days

before the challenge inoculation. A day before the challenge inoculation, plants were

20
again placed in plastic bags in the mist chamber.
C1 11 . l . .

Plants were challenge inoculated with the same strain used in the primary
inoculation X. campestris pv. phaseoli strain DWF 151 grown as described above,
however the concentration of bacteria for the challenge inoculation was higher. The
O.D.,,00 was 0.6 corresponding to (109 cfu/ml“). For the challenge inoculation the plants
were sprayed to run off with strain DWF 151 strain using a hand field 550 ml sprayer,
then re-bagged in plastic and kept in the mist chamber for an additional 2 to 3 days. The
treatments were the following:

To: Control (no treatments).

T1: INA inoculation only

T2: INA soil drenches only.

T3, Bacteria low concentration only - primary inoculation
T4: Control + challenge with bacteria

T5: INA inoculation + challenge with bacteria

T6: INA soil drench + challenge with bacteria

T7: Bacteria low cfu + challenge with bacteria.

Plants were evaluated lO-days after the challenge inoculation for typical lesions on
leaves. The data collected included the percentage of infected plants, number of infected
leaves, number of bacterial lesions per leaf or plant, disease severity, and the number of
colony forming units (cfu / per leaf disc) at l, 2, 4, 6, and 8, days after the challenge

inoculation.

21

Multiple applications of INA.

To study the effect of increasing the number of INA applications on common
blight infection, INA was sprayed one, two, or three times on both cultivars using a 250-
ml hand sprayer. The first application of INA was 10 days after planting, the second
application, 4 days after the first and the third application was made 4 days after the
second. The data recorded included the total number of plants, the number of infected
plant, the number of infected leaves, and disease severity. The severity was recorded fi'om
0 to 4 where 0 = no symptoms, 1 = weak infection, 2 = moderate infection, 3 = severe
infection, 4 = very severe infection. The results of multiple treatments were compared to
the drench inoculation and pre-inoculation with bacteria. Phytotoxicity was recorded when

it was observed.

Analysinthaflcflalammth:

After the challenge inoculation, the disc culture method was used for analysis of
bacterial growth (Fulbright personal communication). A one cm disc of the leaf was
removed using a sterile cork borer for each treatment at different times after the challenge
inoculation. For each treatment, discs were taken from a single leaf (one disc per
treatment for each day of collect). The discs were surface sterilized with 20% bleach and
homogenized in 0.9% saline solution. The homogenate was serially diluted and 0.1 ml of
each dilution from each treatment was pipetted on NBY medium, and the number of

colony forming units (cfu) determined. The experiments were performed three times.

22
Won:

The effect of resistance inducing treatments on peroxidase activity was tested by
infiltrating the lower surface of cotyledon leaves ( first unifoliate leaves) of 10 day old
plants of Mayflower and Midland in the greenhouse with 500 ul of INA or 500 ul of
bacterial suspension (O.D.600 = 0.6 corresponding to l x 109 cfu/ml) at five to ten points.
A l-ml disposable syringe was used for infiltration as described previously. For soil
drenches 100 ml of INA solution (0.5 ppm) was poured over the soil surface of each pot.
Control plants received no treatments. Six days afier treatments, the first and second
trifoliate leaves were collected for peroxidase assays.

Treatment leaves were collected from the treated and control plants by cutting
carefiIlly the petiole about 5-10 mm from the base of the leaves. Three leaflets collected
from different treatments were placed into separate 250 ml beakers and covered with
50 ml Of an ice— water mixture. The beakers were placed into a desiccant chamber and
vacuum applied for 15 to 20 minutes to infiltrate the leaves with water. The leaves were
removed, blotted dry on a paper towel and then carefiilly rolled into 50 ml centrifuge
tubes. The water in the intercellular space was collected by centrifugation at 1500 x g for
15 minutes in an IEC-Centra-4B Centrifuge. The intercellular fluid was transferred to 1.5
ml Eppendorf tubes and store tubes at -20°C until analyzed for peroxidase activity. Two
methods were used for this study, SDS-PAGE gel (Laemmli, 1970) and the native gel

Sample preparation for SDS-PAGE consisted of adding 10 ul of sample Buffer
(see the appendix B) to 30 ul of each sample of intercellular fluid collected as described

above. The samples were heated for five minute at 95° C and the entire 40 ul was loaded

23

into single wells of a 12% SDS-PAGE gel (see appendix B). Electrophoresis was carried
out according to Laemmli (1970) in a Bio-Rad Mini-Protean II system (Bio-Rad,
Hercules, CA) at the recommended power of 200 volts for optimal resolution with
minimal thermal band distortion. The current was approximatively 10 mA per gel and the
running time was 45 minutes. The gel was stained in Coomassie brilliant blue R250
overnight and then destained in a solution of 50% methanol, 10% acetic acid and 40%
distilled H20.

The native gel was run following the procedure described by Hammerschmidt
(1990). Approximatively 50 ul of each sample of intercellular fluid was collected as
described above, and mixed with 1 ul of blue dye (see appendix A). The sample was
loaded in a 7.5 % Acrylamide native gel (see appendix A). The current was set to run at
50 volts overnight, approximatively 16 hours. The gel was stained in a solution containing
120 mg Of4-dichlorO-l- naphthol, 40 ml methanol, 20 ml of 10 X-PBS pH 7.3, 0.38 ml of

hydrogen peroxide and 140 ml of dde.

W:

During the summer of 1995, Mayflower and Midland were grown in the field to
evaluate several INA treatments on the development of common blight. This experiment
was performed to confirm the greenhouse results on the effect of INA on disease
reduction on bean. The plants were grown on the Botany East Farm in a sandy soil in 75
cm rows. Each treatment plot consisted of four rows of plants, 3 m long. All of the

rows in the treatment plot were treated, but only the center 2 rows were rated for disease

24

severity and yield. There was a single border row of plants between every four rows of
treated plants. The treatments were replicated 3 times in a completely randomized design.

The date of planting was July 5, 1995 and a post emergence herbicide was applied
on July 24, 1995. INA was sprayed up to 3 times using a one - gallon hand sprayer
(GREENLAWN, model 010PEXG, Gilmour, PA). The INA solution was sprayed over
the plant until small drops of solution remained on the surface of the tOp leaves. The first
application of INA was July 27 (week 1), the second on August 4 (week 2) and the third
on August 11 (week 3).

The concentration of INA was 50 ppm which was applied in 20 gallons of water
per acre. Challenge inoculation with bacteria was on August 23, 1995. The concentration
of bacteria was 107 cfu/ml" corresponding to a spectrophometer reading O.D_600 = 0.1 on
the Beckman spectrophotometer. Challenge inoculation was four weeks after the first
application of INA. The treatments were as the follows:

1: Control, no treatment.
2: INA, 1 spray at week 1 + challenge inoculation with bacteria.
3: INA, 2 sprays at weeks 1 and 2 + challenge inoculations with bacteria.
4: INA, 3 sprays at weeks 1, 2 and 3 + challenge with bacteria.
5: INA, I spray, at week 2, no challenge inoculation.
6: bacteria inoculation only (no INA spray).
7: INA, 1 spray at week 3 + challenge with bacteria.
Disease incidence was measured by counting the number of individual lesions on

leaves from the inoculated plants during the 2 weeks following the challenge inoculation.

25

The plot was harvested for seed yield on a dry weight basis on October 12. The data were

analyzed by one way analysis of variance using MSTATC ( MSU, Crop and Soil Science).

III. RESULTS.
W5:

Effect of INA on common blight infection

Pre-treatment with 2-6 dichloroisonicotinoc acid (INA) reduced the rate of
infection on cultivars Mayflower and Midland challenge inoculated with Xanthomonas
campestris pv. phaseoli (Tables I and 2). Pre-treatment with INA by syringe injection
or soil drenches provided about equal levels of control and were better than a pre-
inoculation of cotyledons with X. campestris pv. phaseoli. Lesions were more prevalent
on control plants challenge inoculated with X. campestris pv. phaseoli than on plants pre-
treated with INA or bacteria (Fig 2A, 2B, and Fig 3C and 3D), and challenge inoculated
with X. campestris pv. phaseoli. A primary inoculation with a low concentration of X.
campestris pv.phaseoli was more effective on the cultivar Mayflower (Table 1) than the
cultivar Midland (Table 2). The results indicated that a primary treatment with INA or X.
campestris pv. phaseoli reduced the amount and severity of disease on both dry bean
cultivars (Table 1 and 2). The differences between the cultivars Mayflower and Midland
receiving a primary inoculation with X. campestris pv. phaseoli may have been due to the
higher susceptibility of the cultivar Midland to this pathogen. The cultivar Mayflower was
more resistant to common blight pathogen. These results tend to confirm field
Observations that Mayflower is moderately susceptible and Midland highly susceptible (Dr.

J. Kelly, personal communication).

26

27

Fig 2 : Resistant reaction on leaf of common bean cultivar Mayflower pre-
treated with 2,6-dichloroisonicotininc acid and challenge inoculated with
Xanthomonas campestris pv. phaseoli after 7 days.

A. Control challenge inoculated with X. campestris pv. phaseoli.

B. INA pre-treated and challenge inoculated with Xcampesm's pv. phaseoli.

28

 

29

Fig. 3: Resistant reaction on leaf of common bean cultivar Mayflower pre-treated
with 2,6 dichloroisonicotinic acid or bacteria and challenge inoculated with
Xanthomonas campestris pv. phaesoli afier 7 days.

C. INA drench and challenge inoculated with X. campestris pv. phaseoli

D. Primary and challenge inoculated with Xcampestris pv.phaseoli.

 

30

 

31

Iahlfl: Effect of pre-treating the common bean cultivar Mayflower with 2,6-
dichloroisonicotinic acid on infection and disease severity afler challenge inoculation with

Xanthomonas campestris pv. phaseoli.

Control+ch INA inoc + ch INA drench +ch ch + ch

     

Treatments

 

Total plants 2 3 2 2
Infected plants 2 3 l 2
Total leaves 32 47 30 39
Infected leaves 8 7 4 8
% infection 25 14.9 13.3 20.5
Total lesions 53 12 7 l I
Lesions/ infleaf 6.6 1.7 g 1.8 1.4
Severin 4 l 2 3

 

These results are the means of 3 greenhouse experiments in 1995. Control + ch =
control plants challenged with X. campestris pv. phaseoli; INA inoc + ch = plants
primarily inoculated by INA injection and challenged with X. campestris pv. phaseoli;
INA drenc + ch = Plants primarily inoculated with INA solution in soil and challenged
with X. campestris pv. phaseoli; ch + ch = plants primarily inoculated with X.
Campestris pv. phaseoli at low cfu / ml and challenged later with high cfiJ / ml of X
campestris pv. phaseoli. Lesion/inf. Leaf = average number of lesions per infected leaf.
Total lesion was the total number of lesions on all the leaves. Severity recorded from 0 to
4 where 0= no symptoms, l= weak infection, 2= moderate infection, 3=severe infection,

4= very severe infection. Data were recorded 10 days afier challenge inoculation.

32
Table}: Effect of pre-treating the common bean cultivar Midland with 2,6-
dichloroisonicotinic acid on infection and disease severity after challenge inoculation with

Xanthomonas campestris pv. phaseoli.

 

 

Treatments Control + ch INA inoc + ch INA drench + ch ch + ch
Total plants 2 2 2 3
Infected plants 2 l 2 2

Total leaves 54 45 48 54
Infected leaves I l 3 6 9

% infection 20.4 6.7 12.5 16.7
Total lesions 42 l 3 14 32
Lesion/ infleaf 3.8 4.3 2.3 3.5
Severity 4 1 l 4

 

The results are the means of 3 greenhouse experiments in 1995. Control + ch = control
plants challenged with X campestris pv phaseoli; INA inoc + ch = Plants primary
inoculated by INA injection and challenged with X. campestris pv. phaseoli; INA drench
+ ch = Plants primary inoculated with INA solution in soil and challenged with
Xcampestris pv. phaseoli; ch + ch = Plant primary inoculated with X. Campestris pv
phaseoli at low cfu/ml and challenged later with high cfu/ml of X. campesm's pv.phaseoli.
lesion/ infleaf = average number of lesions per infected leaf Total lesion are the total
number of lesions on all the leaves. Severity recorded from 0 to 4 where 0= no symptoms
1= weak infection; 2 = moderate infection; 3 = severe infection, 4= very severe

infection. Data were recorded 10 days after challenge inoculation

33

Multiple applications of INA on the control of ch.

INA was sprayed onto plants 1, 2 or 3 times to evaluate the effect of multiple
applications of INA on the control of common blight (Tables 3 and 4). It appeared that
increasing the number of treatments with INA influenced infection by X campestris pv.
phaseoli. On control plants inoculated with bacteria only, disease symptoms appeared
on all leaves. Symptoms were severe on the top leaves of the control plants with wilted
and dead leaves on the cultivar Mayflower. On the cultivar Midland, symptoms were
even more severe on control plants inoculated only with X. campestris pv. phaseoli. Top
leaves were dead and all the bottom leaves were necrotic. Both cultivars receiving a
single primary treatment of INA (INA I) and challenged with X. campestris pv. phaseoli
had disease symptoms on the second and third trifoliate leaves but the top leaves were
healthy. With two applications of INA, symptoms were visible only on the third trifoliate
leaves after challenge inoculation on Mayflower, but on Midland, almost all the top leaves
were severely wilted. With 3 applications of INA followed by a challenge with X.
campestris pv. phaseoli, symptoms were generally localized on the fourth trifoliate leaves
and all of the bottom leaves were healthy. Two and three applications of INA on Midland
resulted in short plants with small leaves and symptoms were present only on fourth
leaves. Curling of the top leaves was visible on all plants of the cultivar Midland receiving

three sprays of INA.

34

Iahlel, Effect of multiple applications of INA on infection and disease severity on

cultivar Mayflower challenge inoculated with Xanthomonas campestris pv. phaseoli.

 

 

Treatments Cont+ch Inal+ch Ina2+ch In33+ch InaD-I-ch ch+ch
Total plants 3 3 3 4 3 3
Infected plants 3 3 2 3 3 3
Total leaves 41 50 60 103 69 48
Infected leaves 20 9 l 0 6 14 17
%infection 48.8 18 16.7 5.8 20.3 35.4
Total lesions 47 l l 14 9 39 50
Lesion/leaf 2.3 1.2 1.4 1.5 2.8 2.9
Severity 4 2 l l 2 3

 

Note: These are the means of 4 greenhouse experiments in 1995 and 1996

cont+ ch = control inoculated with Xcampestris pv. phaseoli, lnal + ch = one Spray

of INA + Challenge with ch, Ina2 + ch = Two sprays of INA and challenge with ch,

Ina3 + ch = three sprays of INA + challenge with ch, Ina D + ch = INA in soil

drench and challenge with Xcampestris pv phaseoli. ch + ch : primary and challenge

inoculation with Xcampestris pv phaseoli. These data were recorded 10 days after

challenge inoculation. Total lesions the total number of lesions on all the leaves of plants.

Lesion/inf. leafi average number of lesion per infected leaf.

Disease severity was recorded on a scale from 0 to 4 where 0 = no symptoms, 1=

weak infection, 2 =moderate infection, 3 = severe infection, 4 = very severe infection.

35

jljable 4; Effect of multiple applications of INA on infection and disease severity on the

cultivar Midland challenge inoculated with Xanthomonas campeslris pv. phaseoli.

 

1

 

Treatments Cont+ch Ina1+ch InaZ+ch Ina3+ch lnaD+ch ch+ch
Total plants 3 2 3 4 3 3
infected plts 3 2 1 2 3 3

total leaves 53 56 44 72 69 58
infected leav. 25 9 4 7 23 22

% infection 47.2 16.0 9.0 9.7 33.3 37.9
Total lesions 58 1 8 9 8 45 6 l
lesion/ infplants 2.3 2 2.4 1.1 1.9 2.7
Severity. 4 1 l 1 2 3

 

These are the means of 4 greenhouse experiments in 1995 and 1996. Severity
Cont+ch = control plant challenged with Xcampestris pv. phaseoli. Ina 1 + ch = one
spray of INA + challenge with ch, Ina 2 + ch = Two sprays of INA + challenge with
ch, Ina 3 + ch = Three sprays of INA + challenge + ch; INA D + ch = INA in soil
drench and challenge with ch; ch + ch = Primary and challenge inoculation with ch.
These data were recorded 10 days after challenge inoculation. Total lesions are the total
number of lesions on all the leaves. Lesion/inf. leaf = average number of lesions per
infected leaf.

The disease severity was recorded from 0 to where O = no symptoms, 1 = weak infection,

2 = moderate infection, 3 = severe infection, 4 = very severe infection.

36

l I . [I | . I II

Afier the challenge inoculation, leaves of both cultivars Midland and Mayflower
were collected from each treatment at 1, 2, 4, 6 and 8 days afier the challenge inoculation
with X. campestris pv. phaseoli to analyze the effect of INA on bacterial growth

The bacterial population increased to high levels on control plants receiving only a
challenge inoculation compared to plants pre-treated with INA or X. campestris pv.
phaseoli and challenged inoculated with the same bacteria (Tables 5 and 6). On plants
treated with INA, the population per ml increased at a slower rate than on the control
plants (Tables 5 and 6). The population of ch from the cultivar Mayflower (Table 5) was
generally lower than from Midland (Table 6) at each sampling period, and the total

number of cfu’s afier 8 days was slightly less on Mayflower.

37

Table; The effect of 2,6 dichloroisonicotinic acid treatments on colony forming units of

X. campestris pv. phaseoli from leaf discs of cultivar Mayflower afier challenge

 

 

 

 

inoculation.

DAYS AFTER CHALLENGE INOCULATION
Treatments (cfu) 1 2 4 6 8

number Ofcfu/ml

control + ch 1x 10’ 2 x104 7 x105 5.5 x10° 7 x105
lNAl-l-ch 1.5x10‘ 9x 104 2x104 2x10‘ 2x104
INA2+ch 9x103 6x 103 1.2x 104 1.2x10‘ 1.2x10‘
INA3+ch 1x103 4x103 4x104 4x10’ 4x104
INAlD-I-ch 10 7x 103 1x 105 1x105 1x105
ch+ch 5x102 2x102 2x102 3.5x 10’ 5x105

Results are the means of 3 experiments.
Leaf discs were take on the same leaf for each treatment at different dates .

cfu = colony forming units

13121162 Effect of 2,6 dichloroisonicotinic acid treatments on colony forming units of X.

campestris pv. phaseoli from leaf discs of the cultivar Midland afier challenge

 

 

 

 

inoculation.

DAYS AFTER CHALLENGE INOCULATION

Treatments 1 2 4 6 8
Number of cfu / ml

control +ch 1.2 x10’ 1.1x10" 9 x105 1x 10° 7 x 10°
INA1+ch 2x104 8x 104 2x 105 2x105 2x10’
INA2+ch 4x103 1x104 0 1x104 1x104
INA3+ch 2x10" 0 o o 2:110s
INAD+ch 2x 10‘ 6x 10‘ 1x 104 1x104 1x105
ch + ch O 6 x103 5.5 x 10° 5.5 x 10° 5.5 x 10°

 

The results are the means of 3 experiments.

Leafdiscs were taken on the same leaf for each treatment at different dates.

For 2 and 3 applications of INA, some treatements have no cfu in the first day following

the challenge inoculation.

39

WI

The two cultivars of edible bean Mayflower and Midland were pre-treated with
INA solution by injection, soil drench or bacterial solution by injection to study their effect
on peroxidase activity. Extracts from intercellular fluid were used to analyze the effect of
INA inoculation on peroxidase. Plants receiving a primary treatment with INA by
injection and those inoculated with a low concentration of bacteria showed stronger
protein bands (Figs 4 and 5). There was an increase in peroxidase activity in both
cultivars Mayflower and Midland when treated plants were compared to control plants
(Fig. 6). Bands appeared stronger than on control plants which showed a weak
peroxidase activity in gel electrophoresis (Fig.6). These results suggested that increased
protein accumulation and peroxidase activity occurs in bean plants treated with INA or

bacteria solution.

40

Fig 4. SDS-polyacrylamide gel electrOphoresis for proteins

from the leaf of cultivar Mayflower (Coomassie blue stain).

Control = control plant (no pre-treatments)

(no pre-treatment); INA inoc = plant pre-treated with INA by injection;

INA drench = plant pre-treated with INA in soil drench; ch inoc = plant
pre-treated with Xcampestris pv. phaeoli in low concentration, INA + ch =
plant pre-treated with INA by injection and challenge inoculated with

X. campestris pv. phaseoli.

41

MAYF LOWE R

8x + <2.
85 aux
cocoa—O <2.
605 <2.
_o.=coo

.2856

Kd

 

42

Fig 5: SDS-polyacrylamide gel electrophoresis for protein
fi'om leaf of cultivar Midland (Coomassie blue stain).
Control = control plant (no pre-treatment);
INA inoc = plant pre-treated with INA by injection; INA drench = plant
pre-treated with INA in soil drench; ch inoc = plant pre-treated with
Xcampestris pv. phaeoli in low concentration, INA + ch = plant
pre—treated with INA by injection and challenge inoculated with

Xcampestris pv. phaseoli.

43

MIDLAND

.2855
35:00
8:. <2.
:22... <2.

00:. aux ,

8x + <2.

 

44

Fig 6: Native gel electrophoresis for peroxidase activity from the cultivars
Mayflower and Midland. Control = control plant (no pie-treatments);

INA inoc = plant treated with INA by injection; INA drench = plant treated
with INA by soil drench; ch = plant treated with Xantomonas campestris pv.

phaseoli in lowconcentration.

45

MIDLAND

MAYFLOWER

cox
cocci <2.

005 <2.
.2200

aux
cocoa“. <2.

so... <2. .

.2250

 

46

W.
Effect of INA on bacteria infection

The 1995 growing season was characterized by periods of drought during the
month of August. It appeared that challenge inoculation of bean plants was affected by
the drought since there was no supplemental irrigation. INA had different phytotoxic
effects on the two cultivars Mayflower and Midland . While Mayflower did not have a
noticeable response to INA, curling of leaves was clearly evident on the cultivar Midland
especially after 3 applications of INA.

On the cultivar Mayflower, the application of INA significantly reduced the
severity of infection, as determined by the average number of infected plants only afier
three primary treatments with INA and challenge inoculated with Xcampestris pv.
phaseoli (Table 7). Statistically, 1, 2 or 3 applications of INA significantly reduced the
percentage of infected plants in the variety Midland compared to the control plants also
challenged with Xcampestris pv. phaseoli. A single late application of INA also reduced
subsequent infection on Midland by X. campeslris pv. phaseoli, but not on the cultivar
Mayflower. Midland responded better than Mayflower to INA treatments when the
number of infected plants was compared. However, overall disease severity was low even
when the number control plants infected was as high as forty six percent (Midland table 7).
One, two and three applications of INA on Midland significantly reduced the number of
infected leaves but only three applications on Mayflower resulted in a significant reduction
(Table 7). Mayflower appeared to be less susceptible than Mildland to infection by X.

campesm's pv. phaseoli (Tables 6 and 7)

47

Iahlel. Effect of INA treatments on the number of infected plants on the bean cultivars

Mayflower and Midland challenge inoculated with X. campestris pv.phaseoli in field

 

 

experiments.
cultivars Mayflower Midland
Treatments % infection % of control % infection % of control

 

Control (no treats) 24.5 ab 92.03 35.10 b 76.00
INA 1 + ch 14.11 abc 53.00 20.75 cd 44.95
INA 2 + ch 21.97 abc 82.53 9.46 e 20.56
INA 3 + ch 11.24 c 42.22 13.29 de 28.89
INA 1 late + ch 18.08 abc 67.91 24.26 c 52.62
INA only(no chall) 13.52 be 50.78 21.18 cd 45.94
Control + ch 26.62 a 100.00 46.16 a 100
LSD 12.71 9.09

CV. 40.16 40.8

 

Note: Means followed by the same letters are not significantly different. Control plants

did not receive any INA or challenge inoculation, INA 1 = 1 application of INA in week
1, INA2 = application on INA in week 1 and 2, INA 3 = application of INA in week 1, 2
and 3, INA 1 late = 1 application in week 3. All treatments were challenge inoculated 1

week afier the last application of INA in treatment INA 3.

% of infection = percentage of plants infected by ch.

48

Effect of INA treatment on yield.

The field plot was harvested on October 12, 1995. Mayflower plots receiving 1,
2, and 3 applications of INA still had some plants with green leaves and stems while
control plants not treated with INA were completely dry. On the cultivar Midland, plants
from all treated plots were mature (i.e dry) but some curling of leaves was evident before
harvest. There were no major differences in maturation between the different treatments
in the cultivar Midland although 2 and 3 applications of INA did leave some green leaves
in some plots. All the other plants were dried.

On Mayflower, all of the INA treatments significantly increased seed yields
regardless of whether there was a challenge inoculation (Table 8), except for the single
late application of INA followed by challenge inoculation with Xcampestris pv. phaseoli.
This was true for both wet and dry weights. Statistical analysis indicated no significant
differences in wet or dry weight seed yields between different treatments on the cultivar

Midland (Table 8).

49

Table}: Effect of INA treatments on the bean cultivars Mayflower and Midland after

challenge inoculation with X. campestris pv. phaseoli on seed yields.

 

 

 

Cultivar Mayflower Midland
Treatments dry weight g/m2 dry weight g/m2
Control (no treats) 72.44 c 64.82 NS“

INA 1 + ch 85.11 a 56.64

INA 2 + ch 86.44 a 66.71

INA 3 + ch 76.44 b 55.57

INA 1 only (no chal) 88.77 a 70.53

INA (late) + ch 66.42 d 61.37

Control + ch 57.06 e 56.91

CV. 25.75 26.28

 

 

Average yields from the two central rows of each plot in three replications .
INA 1+ ch= INA application in the first week and challenged 3 weeks later with ch;
INA 2 + ch = INA application first and second weeks and challenged 2 weeks later with
ch; INA 3+ ch = INA application in first, second, and third weeks and challenged one
week later with ch; INAl only (no chal) = INA application in the first week and no
challenge with ch; INA (late) + ch= INA application in third week only and
challenged on a week later with ch. NS“: no significant difference between treatments.
These data are the results of one year experiment. Numbers followed by the same letter

are not significantly different according to the Fisher Anova analysis. The dry weight was

DISCUSSION AND CONCLUSIONS:

The results from this study confirmed previous work on systemic resistance in
edible beans. The observed reduction in disease severity caused by a foliar bacterial
pathogen of bean following pre-treatment with a pathogen or chemical is consistent with
the results of earlier work on bean by Sutton (1979); Cloud and Deverall (1987); (Dann
and Deverall, 1995), and on other plants (Kuc, I990; Hammerschmidt and Kuc , 1990;
Metraux et al, 1991). Bean plants have shown resistance to many foliar diseases
including anthracnose (Cloud and Deverall, 1987), rust (Dan and Deverall, 1995), and
soilborne diseases (Kuc, 1990) after they were systemically induced. This was the first
report on the induction of systemic acquired resistance in bean to the common blight
pathogen X. campestris pv phaseoli.

The two culivars of common bean responded differentially to the induction of the
systemic resistance. The cultivar Midland, highly susceptible to common blight was
sensitive to INA treatments when the number of applications was increased by some
visible signs on the leaves. The curling and reduced size of leaves were some of the signs
on this cultivar in the greenhouse as well as in field experiments. On the cultivar
Mayflower, there were no particular signs of phytotoxicity but maturity was delayed by
INA application in the field experiments. INA applied as a soil drench appeared not to
provide as good level of protection in bean against Xcampestris pv. phaseoli on either of
the two cultivars. Inoculation with a low concentration of bacterial suspension, although

more effective on the reduction of disease infection than the control when they were

50

51

challenged was also not as effective in protecting the two cultivars as injecting INA into
the leaves. The primary inoculation with bacteria lead to small lesions on the leaves that
might have been a source of inoculum for other parts of the plants by the same pathogen.

INA treatment directly into the leaves may delay the infection of bean by X.
campestris pv. phaseoli. The number of INA applications affected disease development
by reducing the number of infected leaves, the number of lesions on the leaves and the
severity of the disease. The first leaves adjacent to the INA treated leaves had better
protection than upper leaves. This was evident by the presence of more lesions on the top
leaves than on the bottom leaves of both cultivars when plants where inoculated two or
three times with INA and challenge inoculated with X. campestris pv. phaseoli.
Symptoms appeared earlier on control plants challenge inoculated with bacteria than on
plants receiving INA treatments. Therefore, INA application may delay the infection of
the bean plant by X. campestrr‘s pv. phaseoli. The method of pre-treatment can also affect
the induction of resistance. Injections provided better induction than spray.

The number of colony forming units was not affected by INA or bacteria
treatments during the first 3 days after the leaves were treated but by four days more
colony forming units were present on control plant leaves. Plants receiving INA treatment
had less cfu’s on NBY medium than control or plants treated with bacteria in both
cultivars. The cultivar Midland had a higher number of cfu’s on control plants than on
the cultivar Mayflower. The induction of resistance of bean plants was correlated with an
increase in peroxidase activity for both cultivars of common bean. Both the native and

polyacrylamide gels electrophoresis of plant intercellular space fluid showed that plants

52

treated with INA or bacteria had similar bands which were missing or weak on control
plants (fig. 4, 5, and 6)

In the field experiments, the early application of INA resulted in a reduced
percentage of infected leaves by Xcampestris pv.phaseoli after the challenge inoculation.
INA reduced infection of the plants challenge inoculated with bacteria below that of the
control plants naturally infected by Xcampestris pv. phaseoli. A late single application of
INA was not as effective as a single early application. Two or three applications of INA
reduced the amount of infection, but did cause some injury on the cultivar Midland.

The results of the field experiments confirmed those obtained from the greenhouse
about the role of INA in the systemic induction of resistance in bean plants. INA provided
good protection on both cultivars of bean against bacterial blight by reducing the number
of infected leaves, the percentage leaf infection, disease severity and the number of
bacterial cfiI’S on both cultivars in the greenhouse. In field experiments, INA reduced the
percentage of infected plants on both cultivars and increased the seed yield on the cultivar
Mayflower. Based on the results of percentage of leaf infection, disease severity, number
of cfu’s, and peroxidase activity in the greenhouse experiments and the percentage of
infected plants in field experiments, it appeared that the susceptible cultivar Midland
responded better to SAR than Mayflower. INA treatment may have contributed to the
increase in yield in Mayflower, but on Midland, the phytotoxic effect observed on this
cultivar (curling of leaves) could have reduced the yield because of its sensitivity to INA.

The potential impact of INA on bean production is not clear. Many diseases are

responsible for the reduction of yield in different species of plants. The cultivar

53

Mayflower showed an increase in seed yields on plants inoculated with INA over the
control, but on Midland, there was a decrease in yield in plants treated two to three times
with INA The results, although limited suggest a cultivar INA interaction that would
require an evaluation of INA on every cultivars to determine if SAR was of potential
benefit for specific cultivar. Future studies should include more cultivars of bean both in
greenhouse and in field evaluations. Cultivar specificity has been shown to be important in
the induction of SAR by bean plants and will have to be considered before INA can
become a component of disease management practices.

The increase in peroxidase activity in greehouse experiments was a good indication
of the systemic induction of resistance in bean plants. Increasing peroxidase activity
followed INA treatments indicated that the defense system of bean responded to further
infection of the plants by pathogens. In greehouse experiments, the decrease in lesion
number in INA treated plants indicated a slow bacterial growth. This was confirmed by
the low number of cfu’s on INA treated plants. INA treatments also reduced the disease
severity to a low level (generally less than 10 % of leaf surface area infected). INA in the
future might replace conventional fungides that pollute the environment but there is a
necessity to evaluate the costs and other impacts.

Common blight was of minor importance in the Michigan bean production in 1995
(Hart, personal communication). Long periods of drought might have affected the
infection of field grown plants afier challenge inoculation was. The experiments reported
here need to be conducted over several years to determine how the environment influences

the INA - bean interaction in the development of SAR.

PART II : PARTIAL CHARACTERIZATION OF BACTERIA ISOLATED FROM
AZUKI BEAN PLANTS

I. INTRODUCTION:

During the summer of 1994, bacterial lesions on leaves were observed in
commercial fields of Azuki bean grown in Michigan. These lesions were characterized by
necrotic areas surrounded by yellow halos and necrosis, and were similar in appearance to
halo blight caused by Pseudomonas syringae pv. phaseolicola and common blight caused
by Xanthomonas campestris pv. phaseoli on Michigan dry bean cultivars. We were
concerned that Michigan dry bean cultivars might be susceptible to pathogens causing
disease on Azuki bean. Therefore, a study was conducted to identify and characterize the
pathogens causing disease on Azuki bean and determine if the host range included
Michigan common beans.

To achieve this goal, a series of tests and experiments were conducted in the
laboratory and greenhouse. The overall goal of this research was to determine if bacterial
diseases of the Azuki bean observed in Michigan fields in 1994 were limited to Azuki
bean, or if the Michigan dry bean industry was at risk from potentially new bacterial
diseases. Specific objectives of this research were to:

1) isolate and characterize the bacteria associated with the disease, using morphological,
biochemical and molecular criteria;
2) complete Koch’s postulate and determine the host range of the isolates;

3) compare the isolates with known strains of the pathogens.

54

H. LITERATURE REVIEW:

Azuki bean ( Vigna angularis) is an important legume in most countries in the
world, especially in the East and Southeast Asia. It belongs to the family of Fabiaceae,
tribe Phaseoleae, in the genus of Vigna. In the United States, Azuki beans are grown
mainly for export, and much of the seed originates in foreign countries. Several species of
bacteria including Curtobacterium, Pseudomonas, and Xanthomonas have been reported
as pathogens of Azuki. In Nebraska, several highly virulent isolates of Curtobacterium
flaccumfasciens pvflaccumfasciens on Azuki bean were reported (Coyne et a1., 1963;
Bradbury, 1986). Another bacterial disease, bacteria stem rot, caused by Pseudomonas
adzudicola (Bradbury, 1986) was reported as a host-specific pathogen of Azuki. Bacterial
brown stem rot has characteristics closely resembling Pseudomonas syringae pv. glycinea,
Pseudomonas syringae pv phaseolicola and Pseudomonas syringae pv. mori ( Tanii and
Baba, 1971). Kennedy et al 1984 reported some new bacterial diseases of Azuki bean in
Minnesota that caused stem rot. Pathogenicity was Confirmed in greenhouse inoculations
and some cultivars of common bean were susceptible to this pathogen. The bacterial
strain resembles P. adzudicola, which was described for the first time in 1979 in Japan.

Identification of plant pathogenic bacteria involve different Steps including the
Gram-stain, the color of the colony on different types of media, the growth habit, the
form of the cell, presence of a spore, flagellation, the respiration and differential carbon

carbon source for growth. Other important tests include antibody testing, DNA typing, a

55

56

pathogenicity testusing tobacco to determine the hypersensitive reaction (HR), and host

range on other plant species.

Chemical test and morphological characteristics on specific media.

Bacteria are divided in two groups: the Gram - positive bacteria which retain the
primary violet dye after washing with alcohol, and the Gram - negative bacteria which lose
the violet coloration after alcohol washing and counter staining with safranin. The Gram
stain gives a purple to blue-black color for gram-positive bacteria and red color for Gram -
negative bacteria (Schaad, 1994). The KOI-I test (3% KOH) provides a rapid response to
determine the gram reaction but it is not always reliable.

Bacteria grown on different media often result in colonies of different color and
growth characteristics. For example, on nutrient glucose agar (NGA), yeast extract-
dextrose-CaCO3 (YDC), or nutrient-broth yeast extract agar (NBY), the Xamhomonas
group gives yellow colonies while Pseudomonas does not. Erwinia grows on Miller-
Schroth (MS) medium, but Xanthomonas does not; Pseudomonas can produce
fluorescent pigment on King’s B agar, while Agrobacterium cannot. Agrobacterium is the
only group which grows on D-l agar (Schaad, 1994). The presence of more than four
peritrichous flagella or not can help to define the different groups. Erwim'a has more than
four peritrichous flagella. The form or shape of the bacterial cell can also help to
determine the bacterial group. The shape can be rod, coccus, bacillus or coryneform. The
presence of a spore is a characteristic of the Bacillus group and Streptomyces have aerial

mycelia (Schaad, 1994). Other tests including the use of carbon sources, oxidase reaction

57

and arginine dehydrogenase are also important for determination of species or pathovars.

Molecular tests: Polymerase Chain Reaction

Chemical and morphological tests are generally sufficient to differentiate species.
The use of molecular tests can provide good separation among closely related bacterial
groups. Polymerase chain reaction or PCR is one of the most powerful tools because it
can provide a DNA fingerprint that can be strain or species specific. PCR is performed by
amplifying segments of genomic DNA for different species of bacteria. DNA polymerase
uses Single-stranded DNA as a template for the synthesis of a complementary new strand (
Watson et a1 1992). The PCR mixture contains buffer, dNTPs, primers, enzyme, T aq
polymerase and water at desirable concentrations (Watson et al 1992). The DNA template
contains the target segment to amplify. A drop of oil is added to the samples to prevent
evaporation during the process. The mixture is placed in well of a PCR machine called a
thennocycler. The reaction can be completed in 30 to 60 cycles. The starting material is
double - stranded DNA. The strands are separated by heating the reaction mixture and
then cooled so that the primers anneal to the two primer binding sites that flank the target
region. T aq polymerase synthesizes new strands of DNA, complementary to the
template. T aq polymerase improves the sensitivity and specificity of the PCR. The
reaction mixture goes through repeated cycles of primer annealing, DNA synthesis and

denaturation of DNA fragments. The target sequence doubles in concentration for each

cycle.

58

Pathogenicity tests.

Not all bacterial species are pathogenic to plants. Some are saprophytes even
though they are ofien isolated from infected tissue. The pathogenicity of bacterial isolates
can be determined by observing the hypersensitive reaction (HR) on tobacco, or by
inoculating different plants of the same family or species (Fulbright personal
communication). When plant pathogenic bacteria are injected into the leaf of tobacco, the
hypersensitive reaction (HR) occurs (necrosis or death of tissue surrounding the

inoculation point). There is no reaction to non pathogenic bacteria.

III. MATERIAL AND METHODS.

Il’fi fill'fi lll'l 'IE9I

Lesions were observed on Azuki bean leaves and pods during the summer of

1994. Samples of infected leaves and pods were collected by Dr. Patrick Hart for
examination. Lesions on leaves were characterized by necrosis and yellowing. The lower
surface of leaves exhibited water soaking lesions. Symptoms on pods were also
characterized by water soaking lesions. Samples of leaves and pods were surface
sterilized with 10% clorox bleach for five minutes. Diseased pieces of leaves were then
chopped with a razor blade in a drop of sterile water in a petri dish and small loops of
water were serially transferred to additional drops of sterile distilled water. The dilutions
were streaked onto nutrient agar plates and placed under a fluorescent light for three to
five days. Singles colonies of bacteria (white, yellow and orange) were observed on the
medium. These single colonies were restreaked on medium to obtain pure cultures of the
bacterial isolates

IOcOIIOIII' '0 "'-.I' "III! 2,0WI,OII Ollll‘ . "'OO<I"I
1225..
A supply of Azuki bean seed (cultivar Erimo) was obtained for pathogenicity tests.
Preliminary attempts to grow plants resulted in severe stem rotting and post emergence
damping off (Fig. 10 and 11). Therefore, the cause of early death of young seedlings of
Azuki bean was also investigated as a possible source of contamination. Seeds were

surface with sterilized with 5% clorox for 10 minutes to determine if the contamination

59

60

was internal or external. The seeds were crushed in sterile petri plates in sterile distilled
water and a drop of the liquid was streaked on nutrient glucose agar. From non- surface
sterilized seed, three types of bacteria colonies were obtained ( yellow, white and orange)
and fi'om surface sterilized seed two types of colonies were obtained ( yellow and white).
Treated (10% clorox ) and untreated seeds were grown in sterilized clay pots filled
with Bacto-Professional planting mix. A total of 10 pots for each treatment was planted
with three seeds per pot. Ten days after germination the seedlings were evaluated for

disease symptoms and percent of germination.

61

Fig. 7: Azuki bean seedlings showing symptoms of natural infection by
bacteria 10 days afier planting ( cultivar Erimo).

1= wilting of the seedling, 2 = necrotic lesions on leaves.

62

 

63

Fig 8: Azuki bean leaves showing necrotic spots and chlorosis from
naturally infected seed on cultivar Erimo 10 days after planting.

3 = Necrosis and curling on leaves; 4= Chlorosis on leaves.

 

65

W:

In greenhouse experiments, cultivars of several Michigan edible bean varieties,
Azuki beans and cowpea were grown in clays pots filled with Bacto - professional
planting mix. A total of six cultivars of common bean (Phaseolus vulgaris), two cultivars
of disease-flee Azuki bean (Vigna angularis), and three varieties of cowpea (Vigna
unguiculata) all obtained from Dr. J. Kelly's lab in Crop and Soil Sciences Department
were used in this study.

Plants were maintained in the greenhouse at the temperature of 20 to 25 ° C during
the day and 18 to 21 ° C during the night under continuous fluorescent lighting. A total of
ten pots per variety with three plants in each pot were grown for the experiments. The

characteristics of the different cultivars were the following:

Qcmmmjcamulflxam
Cultixauame CommerciaLclass
Mayflower : Navy
Aztec : Pinto
Alpine : GN(Great Northern)
Montcalm : DRK (Dark Red kidney)
Huron :
Taylor : Cranberry

I I . I II' .

Erimo UJBU (Japan)

Common 424 (Washington)

66

W
CB5 : California’s black eye
CB46 : California’s black eye
CB88 : California’s black eye
B | . I . I |

To evaluate the relative virulence and pathogenicity of bacteria isolated from
Azuki bean, known isolates of pathogenic bacteria were used to compare with the
isolates obtained from Azuki leaves, seedlings and seed. An isolate of Xanthomonas
campestris pv phaseoli strain DE 151 and Pseudomonas syringae pv. phaseolicola,
were obtained from Dr. D. Fulbright. C urtobacterium flaccumfasciens sp.
flaccumfasciens, strain NE 21 was obtained from the University of Nebraska (A.Vidaver).
This srain had the same characteristics as the other strains obtained from Nebraska
(SWM2 and var. aurantiacun 2A-Lt) In addition to these known pathogenic strains, six
isolates of bacteria from Azuki leaves, stem and seed were used in pathogenicity tests on
the different cultivars of common bean Azuki bean, and cowpea (Table 10). The medium
used to maintain the bacteria was nutrient-broth yeast extract agar: nutrient broth (8 g/l),
yeast extract (2.0g/l), KZI-IPO4 (2.g/1), KHZPO,( 0.5g/l), glucose ( 2.5/1), agar (ISg/l).
The morphological charactereistics used for differentiation included the colony
morphology, the Gram reaction, the hypersensitve reaction on tobacco and the fluorescent

test on King B medium.

67
Wmflnmlumndjnnculaflnnnmdum:

Common bean, Azuki bean and cowpea plants were grown in the greenhouse to the
2-3 trifoliate stage and then incubated in a mist chamber at about 20°C for 24 hours prior
to inoculation. Bacterial isolates from single colonies were grown overnight on a
nutrient broth medium in a rotary incubator at 27°C and at 150 rpm. Bacterial cells were
collected in the log phase. Bacteria were centrifuged at 1 x 10‘ rpm in a 50-ml tube in an
[EC-4B centrifiIge, and the pellets collected and diluted in sterile distilled water. The
OD. 600 was adjusted between 0.05 and 0.1 using a spectrophotometer.

Two methods of inoculation were used in these experiments. A scion was made at
the point the cotyledon was attached to the stem to allow inoculation by introducing
bacteria into the cut with a needle and syringe. A second method of inoculation was from
the lower side of the cotyledons leaves using a syringe without a needle and placing the
leaf between the syringe opening and the thumb to infiltrate the bacterial inoculum into the
mesophile of the leaves. Each pot contained 3 plants. The inoculated plants were
transferred to a mist chamber, temperature about 20°C for 24 to 48 hours. The plants were
then placed on greenhouse benches and watered daily. Each cultivar of common bean,
Azuki bean and cowpea was inoculated with the six strains of bacteria isolated from Azuki
bean plus the three control pathogens: X. campestris pv. phaseoli, P. syringae pv.
phaseolicola, and C. flaccumfasciens ssp. flaccumfasciens (isolate NE 21). Ten days
after inoculation, cultivars were evaluated for their reaction to the different strains. The
plants were evaluated according to the type of symptoms ranging from a general wilting to

leaf necrosis. The experiment was repeated three times and the results are the means for

68

the three experiments.

“11 I’EII‘I'

All of the bacteria isolates tested for pathogenicity were evaluated by PCR for
differences in DNA fingerprints. The PCR box primer was used in this analysis ( from
Fulbright Laboratory). The preparation of the sample was the first step in the analysis.
The primer used in this study was BOX lAR. The preparation of the master mix (BO X
lAR) (Carmen personal communication) was as follows:

For each sample:

5.0 ul Gitschier Buffer,

0.2 ul BSA,

2.5 ul 100% DMSO

13.5 ul dHZO

1.0 ul Tween 20 (1%)

1.25 ul 25 mM Mix odeTP’s (l:l:1:l)

1.0 ul primer (0.3 ug/ul = 50 umol/ul)

0.4 ul Taq polymerase 2 U Perkin and Elmer.

The total volume of the sample was 24.85 ul. The correct volume of master mixture was
distributed to 0.5 m1 Eppendorf tubes. For each tube, the master mix was 23.8 ul and 1 ul
of sample DNA ( 50 ng /ul), 25 ul of mineral oil covered the top of the sample. The
samples were centrifuged for five seconds and loaded in the therrnocycler. The box

program was 007-008-009 and there were 35 cycles (see appendix C). After

69

amplification, 6 ul of the amplified product was mixed with 1 drop (approximatively 1 ul)
of tracking dye and the DNA bands separated on agarose gel (Sambrook et al). The

standard was 1 ul of a 100bp ladder, 1 ul of dye and 5 ul of deO. The gel was run at 81
volts for 18 hours. The gel was stained in Ethidium bromide and a picture taken using /

57-Polaroid film, (light exposure at 8).

IV. RESULTS

Effects of seed surface sterilization on germination and isolation of bacteria:

The surface sterilized seeds of the cultivar Erimo had a higher percentage
germination compared to the non-surface sterilized seed. Of 30 surface sterilized seeds
planted 29 germinated (96%), but only 24 out of 30 non- sterilized seeds germinated
(80%). Seed treatment slightly improved germination. Seedlings were evaluated for
disease symptoms 10 days afier planting. Early symptoms on seedlings were characterized
by stem rotting and leaf puckering (Figs. 10 and 11). Treated and untreated plants
exhibited similar symptoms with 75% (22) and 62% (15) of seedlings affected respectively
(Table 9). These results suggested that the seed infection was internal as well as external

for this paricular lot of seeds.

70

 

71

1312119: Effect of seed treatment on germination and seedling infection on the Azuki bean

 

 

cultivar Erimo.

Treatments treated seeds untreated seeds
Total seed planted 30 30

germination 29 24

% of germination 96 80

infected seedlings 22 15

% infection 75 62

 

Results are the means of two experiments. Symptoms appeared earlier on

untreated plants and most of the infected seedlings later died.

72

Characteristics of bacterial strains:

The different strains of bacteria isolated from Azuki bean were compared with
known bacterial pathogens of bean (Table 11) in a series of tests. The strains were
designated according to their origin and the year of their collection. Two isolates
obtained from Azuki bean leaves in Michigan in 1994 from the field were provided by Dr.
Hart ( KS-94-1 and KS-94-2). KS-94-l was a yellow Gram - positive strain and KS-94-2
was an orange Gram - positive strain. KS-94-2 did not cause a HR on tobacco. Two
isolates from the seed of Azuki bean cultiva Erimo were designated KS-95- and KS-95-2.
KS-95-1 was Gram - positive with a yellow colony appearance and KS-95-2 was gram -
negative with a white colony appearance. The isolates obtained from the stem of Azuki
bean seedlings cultivar Erimo in the greenhouse were designated as KS-95-3 and KS-95-4.
These were a white, gram negative, and a yellow gram positive respectively.

The known strain of X. campestris pv. phaseoli was Gram - negative and
produced yellow colonies on YDC or NBY medium (Table 11). This bacteria caused
common blight on bean. The strain C. flaccumfasciens sp. flaccumfasciens was Gram
positive bacteria and caused wilting in a number of bean varieties (Tables 11 and 12). It
produced yellow to orange colonies on NBY or YDC medium. The strain P. syringae pv.
phaseolicola was gram negative and produced white colonies on different media. It

caused halo blight in some edible bean cultivars (table 10).

73

mm: characteristics of bacterial isolates used in inoculation studies of common bean,

 

 

Azuki bean, and cowpea.

bacteria origin colony Gram reaction on fluorescens
isolates morphology reaction tobacco test
X.c.phaseoli Fulbright yellow negative HR NO
C.f.f. NE 21 Nebraska yellow positive HR NO
P.s.phaseolic Fulbright white negative HR NO
KS- 94-1 Field Mich yellow positive HR NO
KS-94-2 Field Mich orange positive NO NO
KS-95-3 Greenhouse white negative HR YES
KS-95-4 Greenhouse yellow positive HR NO
KS-95-1 Seed MI yellow positive HR NO
KS-95-2 Seed MI white negative HR NO

 

Note: X.c.p =Xanthomonas campestris pv. phaseoli; C.f.f = Curlobaclerium
flaccumfasciens species flaccumfasciens (Nebraska), KS- 94-1: bacteria isolated from
Azuki leaf in 94- isolate #1; KS- 94-2: bacteria isolated from Azuki leaf in 94, isolate # 2;
KS-95-3 : bacteria isolate from Azuki stems in 95, isolate #1; KS-95-4 : bacteria isolate
from Azuki stems in 95, isolate # 2, KS- 95-1: bacteria isolate from Azuki seed in 95,
isolate # 1; KS-.95-2: bacteria isolate from Azuki seed in 95 isolate # 2; Ps.phaseolic:

Pseudomonas syringae pv phaseolicola. M1 = Michigan

74
Pathogenicity tests:

All the bacterial isolates tested except one were pathogenic. Symptoms
characterized by wilting and necrosis were more frequent than chlorosis. Both stems and
leaves were inoculated as described previously. On cowpea, only the strain C.
flaccumfasciens Sp. flaccumfasciens from Nebraska was able to cause infection, all other
isolates including X.campestris pv.phaseoli and Pseudomonas syringae pv.syringae were
not pathogenic. KS-94-1, a field isolate from Azuki bean infected all the cultivars of
common beans, including Azuki bean. KS-94-2 which was negative on the HR test on
tobacco caused minor wilting on Erimo only, and KS-95-2 caused minor necrosis also on

Erimo. The other strains had variable reaction on different cultivars (table 11).

75
Iahlell: Comparison of pathogenicity and symptoms on cultivars of common bean Azuki

bean, and cowpea inoculated with strains of bacteria isolated from Azuki bean or common

bean in greenhouse.

 

strains

 

  
 

 

KS-95-

 

C.fi‘.N Psp" KS-94- KS-94- KS-95- KS-95- KS-95-

E 21 l 2 3 4 1 2
Cowpea
CB5 wilt - - - - - - -
CB46 - wilt - - - - - - -
CB88 - - - - - - - wilt -
Azuki bean
Erimo necros. - - necros. wilt necros. wilt - necros.
commo dead wilt - necros. - wilt - - ~
common bean
Aztec wilt,nec wilt chloro necros. - wilt wilt wilt -
Huron necros. wilt necros. chloros - - wilt - -
Alpine wilt,nec wilt necros. necros. - wilt wilt necros. -
Montca necros wilt wilt necros. - - - - -
Mayflo wilt,nec wilt wilt necros. - - wilt wilt -
Taylor wilt,nec wilt wilt,ne necros. - - wilt - -

r

 

* isolated from edible beans.
wilt = wilting, nec or necros = necrosis, chloros = chlorosis; Mayflo = Mayflower;

Montca = Montcalm, (- )= no reaction.

76

In comparison to known strains, it appeared that none of the bacteria isolates from Azuki
were closely related to Xanthomonas. The two isolates KS-94-1 and KS-95-1 were
similar to Curtobacterium. The two isolates KS-95-3 and KS-92-2 were close to the
known group Pseudomonas, all Gram - negative with white colonies on NBY medium,
but only KS-95-3 was fluorescent on king’s B medium. The following table provides

grouping of the isolates.

W: grouping of different isolates of bacteria

 

Shaun fimunl Simian;
Xanthomonas C urtobacterium Pseudomonas
- KS-95-1 KS-94-3
- KS-95-l KS -95-2

- KS-95-4

 

Notejhe strain Lea-94-2 did not cause a HR on tobacco and did not belong to any of the

known groups.

77

Molecular analysis.

The DNA fingerprints of the different strains of bacteria used in this study were
analyzed to determine if the isolates from Azuki bean were related to the known
pathogens of edible bean in Michigan. KS-94-1 and KS-95-1 were very similar in colony
color, type of colony and both were gram positive, but showed large differences in bands
when their DNA was amplified. However, KS-95-1 shared 2 bands with C.
flaccumfasciens spflaccumfasciens, a gram positve bacteria that caused wilting in edible
bean plants. The two isolates with white colonies and gram negative characteristics
where similar to Pseudomonas syringae pv. phaseolicola but their DNA fingerprint was
quite different. (Fig 12). The DNA fingerprint showed some similarty in band between

KS-94-l, isolated from Azuki leaves and KS-95-4 isolated from Azuki stem .

78

 

Ejg_9: PCR amplifications of bacteria strain isolated from Azuki bean leaves,
seed and stems of seedlings.
PCR results of bacterial strains used in this study showed difference
between strains that had the same types of colonies, same color and morphology
and different from known strains that caused symptoms on common bean plants

M = marker, W = water (control), 1 = Cff, 2 = Cmm; 3 = KS-95-1, 4 = KS-94-1,

5 = PSp, 6 = KS-95-2, 7= KS-95-3, 8= ch; 9= KS-95-4.

 

kb

1.0-

 

9.0.0 .0
[0005 01
I I I

79

MW123456789

. u.‘

 

DISCUSSION AND CONCLUSIONS

The different strains of bacteria found on Azuki bean had morphological
characteristics close to some species of bacterial pathogens of common bean in Michigan.
The wilting, stem rotting and damping off were the most frequent symptoms on
seedlings of Azuki. The bacterial transmission was probably by seed externally and
internally contaminated. However, bacteria isolated from seed had different characteristics
from those obtained from seedlings or leaves. Symptoms described by Kennedy in 1984
on Azuki bean in Minnesota were Similar to those found in this study. Most of the strains
identified in this study in Michigan caused necrosis and wilting on other species of bean,
but not on cowpea. Two strains KS-94-l and KS-95-1, were very similar to one strain
Cflaccumfasciens sp. flaccumfasciens NE 21 from Nebraska. These were all gram
positive, yellow colonies on NBY, and caused HR on tobacco. Despite these similarities
only the strain KS-95-1 had a DNA fingerprint to close that of Curlobacterium. KS-95-4
strain appeared different from the subspecies obtained from Nebraska. KS-94-1 caused
necrosis on most of the bean species, while KS-95-1' caused wilting on most species. The
pathogenic species described as Pseudomonas adzudicola could be closely related to two
of the strains as determined by their colony color and their gram stains (KS-95-3 and KS
95-2). These strains were gram negative and had white colonies resembling those of our
strains of Pseudomonas syringae pv phaseolicola (DWF) which are also characteristics of
Pseudomonas adzudicola. A strain of Pseudomonas adzudicola was not availablefor

DNA fingerprinting. Therefore, the comparison was not complete. None of the strains

80

81

were similar to Xanthomonas species. The strain KS-94-2 appears to be a saprophytic
bacteria of bean.

The results obtained from the morphlogical, chemical, pathogenicity and
molecular tests showed that the strais responsible for the different symptoms on Azuki
bean could be the two strains KS-94-1 and KS-95-4. These two strain shared multiple
bands in their DNA fingerprint and were pathogenic to most of the cultivars of common
bean in greenhouse experiments.

Not all these bacterial strains were pathogenic to the common bean cultivar of
Phaseolus vulgaris grown in Michigan. The strain KS-94-2 which had orange colonies on
NBY did not cause any symptoms on common bean, but most other strains KS-95-1, KS-
95-3, KS-95-4 and KS- 94-1 caused symptoms on most bean species when different
cultivars were inoculated in the stem or on the leaf surface.

This study reports on the characterization of different trains of bacteria isolated
from Azuki bean. Tentative identification of the isolates as Curtobacterium and
Pseudomonas were made but additional tests should be made. Symptoms that developed
on common bean cultivars after inoculation with bacterial strains doesn’t necessarily mean
that common beans grown in Michigan are at risk from pathogenic bacteria of Azuki
bean. A State survey to determine if Curtobacterium or Pseudomonas adzudicola are

causing disease on Michigan’s dry bean should be considered for future studies.

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Boller, T., Metraux J-P., (1988). Extracellular localization of chitinase in cucumber.
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Bradbury, J. F., (1986). Guide to Plant Pathogenic Bacteria. CAB International
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Dalisay, RF, and Kuc, J ., (1995). Persistence of induced reistance and enhanced
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Dann, E. K., and Deverall, B. 1., (1995). Effectiveness of systemic resistance in bean
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Gottsetein, H.D., and Kuc, J ., (1989). Induction of systemic resistance to
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Hall, R., (1991). Compendium of Bean Diseases. APS Press. 73 pp.

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Hammerschmidt, R., Nuckles, E.M., and Kuc, J ., (1982). Association of enhanced
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Henderson, S.J., and Friend, J ., (1979). Increase in PAL and lignin-like compound as
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Hoflland, E., Pieterse, C. M. J., Bik, L., and Pelt Van, J. A., (1995). Induced
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Jenns, A and Kuc, J., (1980). Charactersitics of anthracnose resistance of cucumber to
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Kauffmann, S., Legrand , M., geoffroy, P., and Fritig, B. (1987). Biological
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Dichloroisonicotinic and salycilic acid, inducers of systemic acquired
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Kennedy, B. W., and Denny, R., (1984). Bacterial stem rot, a new disease of Adzuki
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Kennedy, B. W., (1983 a). Bacterial spot rot, a devasting disease of Azuki beans in
Minnesota. Annual Report of Bean Improvement Cooperative. 27 : 160 (Abstr).

Kennedy, B.W., (1984 b). Natural and artificial pathogenicity ofPseudomonas
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Kessmann, H., Staub, T., Hoffmann, C., Maetzke, T., Herzog, J ., Ward, E.,
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Kuc, J., (1982). Induced immunity to plant disease. Bioscience 32: 854-859.

Kuc, J., and Strobel, N., (1992). Induced resistance using pathogens and non-
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Kumar, V.U., Meera, M. S., Hindumathy, C. K., and Shetty, H. S., (1993).
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Legrand, M., Kauffmann, S.,~ Geoffroy, P., and Fritig, B., (1987). Biological fiinction of
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Lumpkin, T. A., and McClary, D. C., (1994). Azuki Bean, botany, production and
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85

Metraux, J-P., Signer, H., Ryals, J ., Ward, R, Wyss-Benz, M., Gaudin, J .,
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a;_

 

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W5

APPENDIX A

El 1 '1 . . H13 I. 1 li 1,119;
Lam-L221

(1) 30g acrylamide

0.8 g bis acrylamide

Water to 100 ml. Keep in fridge max lmonth.

(2)185 g tris base

2.1 ml concentrated HCl or 24 ml 1M HCl

0.24 ml TEMED

water to 100 ml

(3) 10% ammonium persulfate. Make fresh weekly, keep cooled

for 1.5 mm gel mix 20 ml water, 0.28 ml ammonium persulfate (3) 10 ml acrylamide mix,
10 ml Tris mix (2).

Pour overlay water. Set for 40 mn. Pour off water.

Stacking gel.

(5) 10 g acrylamide

0.8 g bis acrylamide

Dilute to 100 ml of water, keep in fridge for one month.

(6) 2.23 g tris base

12.8 ml 1M H3PO4

0.1 ml TEMED.

For 1.5 mm gel, mix 10 ml of water, 70 ml ammonium persulfate (3), 5 ml acrylamide
mix (5) 5 ml Tris mix (6).

Pour set in a comb approximately for 3 hours.

Upper buffer.

5.16 g Tris base

3.48 g glycine

water to 1000 ml pH = 8.9

Lower Buffer.

14.5g Tris base

5.2 g concentrated HCl or 60 ml 1N HCl
Water to 1000 ml.

Load. Run to 50 V.

89

90

For staining:

4-dichloro-1-naphtol ( 120 mg)

140 ml dde

40 ml methanol

20 ml 10 X PBS pH 7.3

40 ul H202 ( hydrogen peroxide), addition 0.36 ml of H202.
In few minutes bands start to appear.

Coomassie blue- stain preparation.

For 1 liter:

0.25 g Coomassie R-250 ( brilliant blue)

400ml methanol

stir until dissolved

Add 70 ml acetic acid (glacial)

Add 530 m1 H20

Filter through whatman # 1 filter paper using a funnel under gravity.

For overnight staining: 4 parts of stain (80%): 1 part of destain (20% solution I)

Destain solution I:
500 ml methanol.

100 ml acetic acid
400 ml H20

Destain solution H:
70 ml acetic acid glacial

50 ml methanol
880 ml H20.

91

APPENDIX B

Protocol\SDS-PAGE.

RECIPES FOR SDS-PAGE.

References: B.D Hames and D. Rickwood , editors, 1981, " Gel electrophoresis of
proteins : a practical approach", IRL Press.

W 6.5% 7% 7.5% 8% 9% 10% 12% 13% 15%
(10ml)

 

Acrylamide 2.17 2.3 2.5 2.7 3.0 3.3 4.0 4.3 5.0 ml

1.75MTris pH 2 2 2 2 2 2 2 2 2 ml
8.8

10%SDS 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 ml
Water 5.66 5.5 5.3 5.13 4.8 4.53 3.83 3.5 2.83 ml
TEMED10% 10 10 10 10 10 10 10 10 10 ul

 

ammonium 60 60 60 60 60 60 60 60 60 ul
persulfate

Stackinaaelt

(6 ml)

1. Acrylamide 1.0 ml

25 M Tris pH 6.8 0.6 ml

10% SDS 60 ul

Water 4.3 ml

TEMED10% 5 ul

Ammonium persulfate 50 ul

 

Prior to adding TEMED and ammonium persulfate, degas the combination for 15
minutes,
AW = 30 gm acrylamide, 0.8 gm bis-acrylamide per 100 ml.
Filter through whatman # 1.
LliMIIiS = 21.2 gm/ 100 m1 pH 8.8
W= 15.1 gm/ 100 ml. pH.6.8

W: (per liter)

Tris base 3.0 gm
glycine 14.4 gm
SDS 1.0 gm

SampleBuffer: (2 x) ( 4 X )
( To make 25 ml )

1.25 M Tris -Hcl, pH 6.8
mercaptoethanol

[or DDT

SDS

Sucrose

Bromophenol blue
Water

92

2.5 ml

2.5 ml

20 Mm]

10 ml 10%

5.0 gm
1.0 gm ( add until color looks right
to 25 ml ( 10 ml) ( 6 ml).

93

APPENDIX C

Primer: (BO X IAR)

Preparation of Master Mix:

5 ul Gitschier Buffer

0.2 ul BSA

2,5 ul 100% DMSO

13.5 ul H20

1.0 ul Tween 20 ( 1% )

1.25 u125 mM Mix odeTP’s( 1:1:121)
1.0 ul primer ( 0.3 ug/ul -- 50 umon)
0.4 ul Taq polymerase 2 U Perkin Elmer.

Prepare per tube:

Master mix 24 ul
DNA (50 ng/ul ) 1 ul
Mineral oil 25 ul

Spin for 5 seconds and load in PCR machine.

The program applies to thermocycler in room 34 ( D. Fulbright).

 

 

 

 

 

 

 

 

 

 

 

. program # for BOX
1 X 007 950 C 7 minutes
940 C 1 minutes
35 X 008 530 C 1 minutes
650 C 8 minutes
1 X 009 650 C 15 minutes
= 40C Soak

 

Load 6 ul of PCR preparation per sample onto gel.
Samples are mixed with 2 ul of blue dye before loading.
1 ul of Marker (ladder) is used for the size of the bands

WWW:

Agarose 3.75 gm

0.5 X TAE Buffer 250 ml

The mixture is placed in a small autolave with vapor pressure for 15 minutes and cooled
down using a stirrer (a magnetic bar) until ready to pour in a comb tray until it solidifies.

Appendix D:

94

Morphological and chemichal tests of bacteria isolated from Azuki bean leaf in 1994.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Strains/test ch P55 P. Fluores KS-94-1 KS-94-2
Gram test - - - + +
Motility + + + + +
aeration + + + + +
pigment yellow white white brown yellow orange
shape rod rod rod coryneform rod
growth 370C fast moderate very fast very fast slow
oxidase test - - + - -
ADH tets - + + -
PAF + (brown) + + + +
YDC + (brown) + + + +

no carbon - - - - -
sucrose + + + + +
Mannitol + + + + +

D. Tatrate - + - - +

L. Lactate + + - - -
Sorbitol - + + + -
Tobacco HR + + + + +

4 ‘0 clock - - - - -
Bunsi + + - + -
Midland + + - + -
Mayflower + + - + -
Azuki bean + + + + +
Fluorescent - + + - -

 

 

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