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THESIS

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This is to certify that the
dissertation entitled
Mode of action of the secondary
metabolite of Pseudomonas fluorescens Q2-87

in the Biological control of Phythium species.

presented by
Sahar Ali Gamal El-din Youssef

has been accepted towards fulfillment
of the requirements for

Ph . D . degree in Plant Lathology

W

Major professor

Date ({é ’10.!”

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6/01 cJCIRC/DateDuepGS-p. 1 5

MODE OF ACTION OF THE SECONDARY METABOLITE OF PSEUDOMONAS

FLUORESCENS Q2-87 IN THE BIOLOGICAL CONTROL OF PYT HI UM SPECIES

By
Sahar Ali Gamal El-din Youssef

A DISSERTATION

Submitted to
Michigan State University
In partial fulfillment of the requirements
For the degree of

DOCTOR OF PHILOSOPHY

Department of Plant Pathology

2002

ABSTRACT

MODE OF ACTION OF THE SECONDARY METABOLITE OF PSEUDOMONAS

FLUORESCENS Q2-87 IN THE BIOLOGICAL CONTROL OF PYT HI UM SPECIES

By

Sahar Ali Gamal Eldin Youssef

Pseudomonasfluorescens Q2-87 produces the antifungal compound 2,4-
diacetylphloroglucinol and it is effective as a biological control agent in reducing plant
diseases caused by Oomycetes. This dissertation is an investigation of the mechanism of
biological control using three members of genus Pythium: P. ultimum, P.
apham'dermatum and P. intermedium. The study by light microscopy of the antagonistic
reactions between the Pseudomonas and the Pythium showed that there were changes on
the hyphal morphology. Study by the scanning electron microscope revealed loss of the
rough hyphal surface, loss of turgor, collapse and gradual cell wall degradation.
Examination of cell-free supematants of bacterial growth showed absence of production
of cell wall degrading enzymes by the bacterium. An active compound that induced the
changes in hyphal morphology was isolated and identified as 2,4-DAPG which occurred
along with presence of 2-MAPG and phloroglucinol. A synthetic 2,4-DAPG was used as ‘
a positive control. Assaying cell wall degrading enzymes 13-1, 3-glucanase and B—1,4-
glucanase (cellulase) in P. ultimum mycelium after treating with a partially purified 2,4-
DAPG showed increase in enzyme activity several fold compared to the untreated

mycelium. Similarly, exposed mycelium to the compound expressed a higher activity of

cellulase than that of the unexposed mycelium. 2,4-DAPG induced cell wall degrading
enzymes, glucanase and cellulase, and caused them to be over expressed in the fimgal
tissue causing cell wall degradation and death. Studying the enzymes present in the
mycelimn using PAGE, revealed two enzymes in Pythium tissue, one isozyme of
glucanase and two isozymes of cellulase. Confirmation of glucanase activity was
achieved with PAGE. Effect of 2,4-DAPG on P. ultimum ultrastructure was also
examined with transmission electron microscopy. Examination showed changes occurred
on cell walls, mitochondria, nuclei, cytoplasm and number of vacuoles present in the
treated hyphae. The treated hyphae lost their cell wall while they remained turgid and
regular in the untreated samples. Mitochondria and nuclei were disrupted and the
cytoplasm aggregated. Presence of large numbers of vacuoles in the treated cells was an
indication of senescence. Production of lytic proteases was evident 30 min after treatment
of mycelium with 2,4-DAPG. The enzyme production continued to increase until it
reached 5 fold, 48 h after treatment. Assaying the RNase activity in P. ultimum tissue
after treatment with 2,4-DAPG revealed increase of its activity after 30 min. Increase of
RNase activity correlated with increase of incubation time with the antifungal compound
2,4-DAPG. Study of the expression of protein patterns was made on SDS-PAGE and

changes of protein expression recorded 30 min after the treatment.

Cepyright by
SAHAR ALI GAMAL ELDIN YOUSSEF

2002

I dedicate this thesis to my parents, husband and children for all the love and support

ACKNOWLEDGMENTS

I would like to express my gratitude and thanks to my major advisor Dr. Joe
Vargas for his guidance and support to my research, to Dr. Jerry Adams for his time and
help with my questions, to Dr. Frances Trail for her helpful advise and to Dr. Muralee
Nair for all his time guidance and useful discussions.

Special thanks to Dr. Kamel Tartoura for all his time, support and helpful
discussions. I extend my gratitude to Dr. Andrea Darocha for her help and time.

I also would like to thank Ron Detweiler for his help in whatever area I asked for it and
to my collegeous Phil Dwyer for his friendship and encouragement.

Finally, I am forever indebted to my mother and father who their love, support

and belief in my abilities helped me to reach my goal.

vi

TABLE or CONTENTS

INTRODUCTION

CHAPTER 1
LITERATURE REVIEW

CHAPTER 2 '
CHRONOLOICAL ULTRASTRUCTURAL ANALYSIS OF THE HYPHAE OF THREE

PYTHI UM SPECIES TREATED WITH THE BIOCONTROL PSEUDOMONAS FL UORESCENS
Q2-87 PRODUCING 2,4-DIACETYLPLOROGLUCINOL

Introduction

Materials and Methods
Results

Discussion

CHAPTER 3

INDUCTION OF CELL WALL DEGRADING ENZYMES IN PYT HI UM ULTIMUM BY
2,4-DIACETYLPHLOROGLUCINOL PRODUCED BY PSEUDOMONAS FLUORESCENS
Q2-87

Introduction

Materials and Methods
Results

Discussion

CHAPTER 4

ULTRASTRUCTURAL, HYDROLYTIC ENZYMES AND MOLECULAR ANALYSIS OF
THE EFFECT OF 2,4-DIACETYLPHLOROGLUCINOL PRODUCED BY PSEUDOMONAS
FLUORESCENS ON PYTHIUM ULTIMUM

Introduction

Materials and Methods
Results

Discussion

SUMMARY

LIST OF REFERENCES

vii

15

I6
17
I9
20

28

29
30
35
37

47

48
50
54
57

69

INTRODUCTION

Biological control using antagonists against plant pathogens is a promising field
for the control plant diseases. Several microorganisms including fimgi, bacteria and
viruses have been reported as potential biocontrol agents. The Pseudomonads are some of
the most promising microorganism for biocontrol. Many strains of Pseudomonas
fluorescens are capable of reducing the occurrence of plant diseases via production of
antibiotics (Cronin et al., 1997a; Cornin et al., 1997b; and Duffy and Défago, 1999).
Antibiotics produced by Pseudomonads include 2,4-diacetylphloroglucinol (Shanahan et
al., 1992), phenazines (Pierson and Weller, 1994), and pyrrolnitn'n (Howell and
Stipanovic, 1979). 2,4-DAPG has a broad spectrum of antibiosis (Broadbent et al., 1976;
Keel et al., 1992; Levy et al., 1992; Reedy and Borovkov, 1970), including antifungal
(Garagulya 1974, Keel et al., 1992, Levy et al., 1992, Nowak-Thompson et al., 1994,
Tomts-Lorente et al., 1989, and Vincent et al., 1991), phytotoxic (Kataryan and
Torgashova, 1976, Keel et al., 1992, and Reddy et al., 1969) and antiviral (Tada et al.,
1990) activity. 2,4-DAPG is considered to be the only antifimgal agent produced by P.
fluorescens strain F113, CHAO, and Q2-87 (Shanahan et al., 1992; Keel et al., 1992;
Fenton et al., 1992; and Bonsall et al., 1997).

Pseudomonasfluorescens Q2-87 produces the antifungal compound 2,4- DAPG
as a secondary metabolite. The compound 2,4-DAPG plays a major role as a biocontrol
compound and suppresses various soil-borne plant pathogens such as black root rot of

tobacco and F usarium wilt of tomato (Keel et al., 1996), Pythium damping-off of cotton

(Howell and Stipanovic 1980) and cucumber (Kraus and Loper 1992), Rhizoctom'a
damping-off of cotton (Howell and Stipanovic, 1979), take-all, Rhizoctom'a root rot, and
Pythium damping-off of wheat (Gurusiddaiah et al., 1986). 2,4-DAPG is a major factor in
controlling some diseases such as damping off in sugarbeet (F enton et al., 1992), black
rot of tobacco (Keel et al., 1992) and take-all of wheat (Raaijmakers and Weller, 1997).
Deletion of the biosynthetic locus of 2,4-DAPG from the genome resulted in loss of the
ability to inhibit the fungal pathogens (Keel et al., 1992; Fenton et al., 1992; and Carroll
etal., 1995).

Review of the literature revealed that most of the studies were done either on the
isolation, identification and characterization of the metabolite 2,4-diacetylphloroglucinol
(Shanahan et al., 1992; Keel et al., 1992; Gurusiddiah et al., 1986; Kraus and Loper
1994) or genetics analysis of the cluster genes regulating this metabolite (Vincent er al.,
1991; Carroll et al., 1995; Bangera and Thomashow 1999) and enhancement of its
production (Raaijmakers et al., 1999). Other studies included a study by Mazzola et al.,
(1995) screening the compound against Gaeumannomyces gramim's isolates, the causal
agent of Take-all of wheat, and some other pathogens such as F usarium wilt of tomato
(Keel et al., 1996), Pythium damping-off of cotton (Howell and Stipanovic 1980) and
cucumber (Kraus and Loper 1992), Rhizoctom'a damping-off of cotton (Howell and
Stipanovic, 1979), Take-all, Rhizoctonia root rot, and Pythium damping-off of wheat
(Gurusiddaiah et al., 1986).

Although the mechanism by which 2,4-DAPG inhibits fungal grth has not
received much attention, some hypotheses have been pr0posed. Certain plant species

produce phloroglucinol, which is externally deposited on the plant surfaces and may be

involved in defense against invasion (Tomas-Lorenete et al. 1989). 2,4-DAPG is a
member of the phloroglucinol class and may act by disrupting the fungal membrane, thus
blocking the pathogen’s ability to cause disease. Phloroglucinols can also be phytotoxic
and show greater activity against dicots than monocots (Keel et al. 1992). Kataryan and
Torgashova (1976) observed that 2,4-DAPG behaves similarly to the herbicide 2,4-
dichlorophenoxyacetate, and substituted phloroglucinol have been shown to act as potent
photosystem II inhibitors in plants (Keel et al. 1992, Yoneyama et a1. 1990). Production
of 2,4-DAPG by a bacterium may activate the host plant’s defense systems (Keel et al.
1992). This hypothesis is based on the observation that the presence of the compound in
the rhizosphere inhibits black root rot caused by T hielaviopsis basicola without
drastically reducing the fungal population.

By contrast, there is neither information on the mode of action of the compound
2,4-DAPG on any pathogen, whether true fungi or Oomycetes, nor any information on
the defense mechanism of the pathogen. This study was initiated to better understand the
effect of the secondary metabolite 2,4-DAPG produced by Pseudomonasfluorescens Q2-
87 on Pythium species. The objective was to clarify the mechanism by which 2,4—DAPG
controls Oomycetes. Specifically, SEM is used to determine the effect of 2,4-DAPG on
the hyphae. TLC is used to confirm the presence of the secondary metabolite, to
demonstrate that the bacterium does not produce enzymes as a second mode of action,
and for identification and quantification of hyphal cell wall degrading the enzymes. TEM
is used to study the effect on the Pythium ultrastructure, production of self-destructive
enzyme related to senesces, and to study the changes in hyphal protein patterns following

exposure to 2,4-DAPG.

Chapter 1

LITERATURE REVIEW

Pseudomonasfluorescens

Flourescent Pseudomonads are a large group of bacteria and some members have
been shown to inhibit plant pathogens, including fungi and bacteria. Howell and
Stipanovic (1979) isolated a strain of Pseudomonasfluorescens, called Pf-S that
controlled Rhizoctonia solam' Kuhn, the causal agent of damping off of cotton seedlings.
They found that this strain produced the antifungal substance pyrrolnitrin. Although this
strain was able to inhibit Rhizoctonia solani, it showed no inhibitory effect on Pythium
ultimum. Another strain of P. fluorescens was found to be effective against P. ultimum
and called Pf-S also. That led to a conclusion that another antibiotic had been produced.
That antibiotic was identified as pyoluteorin in 1980.

P. aureofaciens strains producing the antibiotic phenazine are found through out
the world (Pierson, 1997). The primary phenazine antibiotics, shown to be responsible for
fungal inhibition, are phenazine-l-carboxylic acid (PCA), 2-hydroxy-phenazine-1-
carboxylic acid (2OHPCA), and 2-hydroxy-phenazine (2PZ). There are two known
strains that produce these antibiotics; one is called P. aureofaciens 2-79 and the other is
called 30-84. Mutant strains of 30-84 that do not produce phenazines also have lost the
ability to inhibit plant pathogens. Restoration of phenazine production in the mutants

results in restoration of pathogen inhibition (Pierson and Thomashow, 1992). The

primary mode of actions of phenazines in inhibiting plant pathogens is by disruption of
cell membrane functions, inhibition of RNA synthesis, and DNA replication and
transcription processes (Pierson and Pierson, 1996).

Other important members of the Pseudomonads group are P. fluorescens Q2-87,
F113 and CHAO that produce the antifungal compound 2,4-diacetylphloroglucinol (2,4-
DAPG). 2,4-DAPG is considered to be the main antifungal compound produced by the
three strains (Shanahan, et al., 1992; Keel, et al., 1992; Fenton, et al., 1992; Harrison et
al., 1993; Bonsall et al., 1997). P. fluorescens CHAO produces also hydrogen cyanide,
and pyoluteorin that contribute to the inhibition of fungal growth and disease suppression
(V oisard et al., 1989; Maurhofer et al., l992and Keel et al., 1990). Keel, et al., (1992)
found that mutant strains, obtained by Tn5 insertion, that had lost production of 2,4-
DAPG also completely lost the ability to inhibit Thielavr'opsis basicola and
Gaeumannomyces graminis var. tritici. A quantitative relationship between antibiotic
production and disease suppressiveness is suggested by the enhancement of production of
2,4-DAPG and pyoluteorin accomplished by adding extra copies of '22kb fragment of
DNA to improve suppression of Pythium on cucumber by CHAO (Maurhofer et al.,
1992)

Examination of 2,4-DAPG production by strain F113 under different growth
conditions, in vitro, showed that temperature had a direct impact on the production of the
compound (Shanahan et al., 1992). The optimum temperature was found to be 12°C and a
low ratio of culture volume to surface area available to bacterial growth was critical for
optimum production. Iron concentration, pH and oxygen had no effect on 2,4-DAPG

productions. Carbon sources were found to have a direct impact on antifungal production,

whereas sucrose, fructose and mannitol enhanced the amount of 2,4-DAPG produced by
strain F113, glucose and sorbose reduced production. In another study of the effect of
growth conditions on 2,4-DAPG production in vitro by strain CHAO, Duffy and Défago
(1999) found that glucose and zinc enhanced 2,4-DAPG productions. Studying the effect
of biotic factors on 2,4-DAPG production by the same strain CHAO in the rhizosphere
was achieved by Notz et al., (2001). In this study a translational phLA’-‘LacZ fusion was
used to observe 2,4-DAPG production in vitro and in vivo. Using a clay and sand-based
artificial soil significantly enhanced 2,4-DAPG production in the rhizosphers of maize
and wheat (monocots) than that was produced in soil planted with bean and cucumber,
(dicots). This observation led to the examination of the effect of different plant cultivars
on 2,4-DAPG productions. Significant differences in production of 2,4-DAPG in
association with six different cultivars of maize Using transgenic maize plants versus
non-transgenic plants showed no significant differences of 2,4-DAPG productions (N otz
et al., 2001 ). Planting of pre-germinated maize enhanced phLA’-‘LacZ the reporter gene
expression at 24 h after planting and decreased expression to ‘/4 48 h, suggesting an effect
of plant age on 2,4-DAPG gene expression. When cucumber and maize roots were
infected with P. ultimum, infection enhanced gene expression and 2,4-DAPG productions
(Notz er al., 2001).

2,4-DAPG has broad antiviral (Tada etal., 1990), antibacterial (Keel et al., 1992;
Reddy and Borovkov, 1970), antifungal (Nowak-Thompson, etal., 1994; Keel et al.,
1992; Vincent et al., 1991) and antihelminthic (Bowden et al., 1965) properties.
Examining the synthetic 2,4-DAPG against Erwinia carotovora subsp. atroseptica

causing soft rot of potato showed inhibition under in vitro conditions. P. fluorescens

F113 producing 2,4-DAPG was examined and showed ability to control the soft rot
potato pathogen. A mutant non-producing strain and the wild type were examined liquid
medium and on wounded potato tuber. The wild type was able to inhibit bacterial grth
and protected potato tubers from rotting, whereas, the mutant negative showed no effect.
The same results were obtained when E. carotovora population size was examined in soil
and on diced potato tuber in an experiment carried out with unplanted soil and soil
planted with diced potato tuber. It has been concluded that P. fluorescens F113 is a
potential biocontol agent against E. carotovora (Cronin et al., 1997).

P. fluorescens F113 was examined also against the potato cyst narnatode
Globodera rostochiensis in vitro and in soil. The results obtained showed an increase of
the hatch eggs by two fold, and a decrease in the mobile juveniles by three fold, after
incubation with the bacteria either in vitro or in vivo. The same results were obtained
when the nematode cysts and juveniles were incubated with synthetic 2,4-DAPG. This
experiment confirmed the effect of 2- DAPG produced by bacteria strain F113.

P. fluorescens strain CHAO suppresses two fungal root pathogens Thielaviopsis
basicola causal agent of black root rot of tobacco and G. g. var. tritici causal agent of
take all of wheat. The role of 2,4-DAPG for in disease suppression was examined. A
mutant negative strain, lacking 2,4-DAPG production obtained by TnS insertion, was
used as a negative control. The two strains were tested in vitro against the two pathogens.
The mutant negative strain did not show inhibition of both pathogens compared with the
wild type. Restoration of 2,4-DAPG production restored the bacteria ability to protect

tobacco and wheat from black root rot and take-all respectively. Production of 2,4-DAPG

played an important role in the ability of strain CHAO to suppress plant pathogens (Keel
etaL,1992)

Conservation of the genomic locus of biosynthesis 2,4—DAPG among
Pseudomonad strains from different geographic regions was studied by Keel, et al.,
(1996). A genomic fragment 4.8kb from P. fluorescens Q2-87 was used as a probe to test
against all 2,4-DAPG producing strains from a worldwide collection. The probe
hybridized with all genomic DNA of strains from Europe, Ghana, Italy, Switzerland and
Washington despite of phenotype and genotype diversity. There were to different
phenotypes observed. The first phenotype was strains able to produce 2,4-DAPG,
pyoluteorin and hydrogen cyanide (HCN). These strains presented isolates from all
locations in USA, Europe, and Africa. A second phenotype of isolates able to produce
only 2,4-DAPG and HCN was found in USA and Europe. Analysis of genotypes revealed
that strains producing 2,4-DAPG and pyoluteorin were genetically similar, however,
strains that did not produce pyoluteorin expressed a greater diversity in genotypes.
Researchers concluded that highly similar strains could be found throughout the world

(Keel et al., 1996).

P. fluorescens Q2-87

P. fluorescens Q2-87, previously known as P. aureofaciens Q2-87 (Vincent et al.,
1991), was originally isolated from wheat rhizospheres in a take-all suppressive soil
(Shano silt loam, pH 5.7) near Quincy, Washington that inhibited G. g. var. tritici. When
applied to soil or wheat seeds, P. fluorescens Q2-87 protected wheat against Take-all

disease. Examination of the bacteria genome revealed that 6.5-kb fragment was

responsible for 2,4-DAPG production, and transfer of this genomic region to a 2,4-DAPG
non- producing strain resulted in production of the compound (Bangera and Thomashow,
1999). The 6.5 fragment was identified as having six open reading frames, in which four
of them (phLA, phLC, phLB and phLD) were expressed in Escherichia coli and resulted
in a polyketide biosynthesis. PhLD was responsible for production of
monoacetylphloroglucinol (MAPG). The other genomic regions phLA, PhLC and PhLB

were all essential in converting MAPG to 2,4-DAPG as well as in synthesis of MAPG.

Biocontrol Mode of Actions

The biocontrol agents differ in their modes of action. In general there are several
mechanisms that have been described: competition for substrate and niche exclusion
(Elad and Chet, 1987), antibiosis (Howie and Suslow, 1991; Benhamou et al., 1996) or
production of extracellular enzymes (F ridlender et al., 1993). Although antibiosis had
been studied extensively and considered one of the main modes of action of biocontrol
agents, other mechanism such as production of hydrolytic enzymes may play an
important role in biological control (Handelsman and Stabb, 1996).

Production of hydrolytic enzymes that act against soilborne pathogens are well
documented in plants (Kim and Hwang, 1997; and Kim and Hwang, 1997), in biocontrol
fungi such as Trichoderma spp. (Thrane et al., 2000, Elad and Kapat, 1999, Thrane et al.,
1997), in mycophylic fungi such as Sporotrichum mycophyllum (Pisarevskaya, 1997) and
in biocontrol bacteria (Fraidlender et al., 1993, Velazhahan et al., 1999, Meena et al.,

2001).

Studying the enzymatic activity by Phytophthora capsici on infected pepper stems
revealed that the cell wall degrading enzyme 1, 3-B-glucanase (a 34 kilodalton (kD)
produced by plants) was able to inhibit the fungal growth. Using a high amount of the
enzyme, 100 ug/ml, caused hyphal and zoospore lysis. A synergistic effect could be
obtained by combining glucanase and chitinase to inhibit F usarium oxysporum f. sp.
Cucumerinum (Kim and Hwang, 1997).

Lysis by hydrolytic enzymes is well known as a mode of action of some
biocontrol agents. Chitinase and B-1 , 3-glucanase are particularly essential fungus
controlling enzymes due to their capability to degrade the fungal cell wall of chitin and B-
1, 3-glucan (Bartnicki-Garcia and Lippman, 1973; Schroth and Hancock, 1981).

Studies by Fraidlender et al., (1993) have documented the ability of the bacterium
Pseudomonas cepacia to control several plant pathogens in the greenhouse including
Pythium ultimum, F usarium oxysporum, Rhizoctonia solani, and Sclerotium roljsz'i by
producing hydrolytic enzymes. Thrane et al., (1997) studied the extracellular enzymes
glucanase and cellulase produced by T. harzianum against Pythium spp. Both enzymes
were purified and glucanase was found to be a small protein with a molecular mass of 17
KD. The two cellulase isozymes had a molecular mass of 40 and 45 KD. Testing the
purified enzymes against Pythium species revealed that low concentration was enough to
inhibit fungal growth. Follows up with the pervious results, Thrane et al., (2000) used
two isolates of T richoderma spp. producing the hydrolytic enzymes, cellulase and endo-
l, 3-B-glucanase, but they differed in their ability to colonize and compete in peat moss
and wood chips against P. ultimum in vitro and in vivo. The two isolates were able to

inhibit the fungal growth and protect cucumber seedlings although there were significant

10

differences in the amount of enzymes they produced. Examining the culture filtrates of
the two isolates grown on media amended with different carbon sources revealed
differences in response. Enzyme production was affected in one isolate more than the
other. In a study by Downer et al., (2001) examining the effect of glucanase and cellulase
against different fungal structure of Phytophthora cinnamomi, fungal mycelium,
zoospores, zoospores cysts and infected roots were incubated with different enzyme
concentrations. They found that glucanase had a slight effect on zoosporangia and
chlamydospore formation when used in low concentration. However, at high
concentrations, glucanase was able to prevent encystment of fungal structures more so
than cellulase. The cellulase significantly inhibit the development of zoosporangia and
chlamydospores when used at concentration more than 10 units/ml. Whereas cellulase
reduced zoospore production, glucanase had no effect on it. The conclusion was that each
enzyme played a major role on Oomycetes structures depending'on differences of cell

wall components.

Enzyme effect on fungal cell

‘Picard et al., (2000) studied the cytological effect on Phytophthora parasitica of
cellulase produced by the biocontrol agent Pythium oligandrum. It was observed was that
P. oligandrum growth toward Phytophthora mycelium in dual culture resulted in plasma
membrane aggregation and cytoplasm disorganization. These changes were accompanied
with deposition of cellulose material in the cell wall. However, P. oligandrum was able to
penetrate that thick cell wall which suggested that degradation took place on the

Oomycetes hypha. Measuring cellulase activity of P. oligandrum in substrate-free

11

medium resulted in detection of active enzyme as active as those obtained from
Trichoderma viride on degrading Phytophthora cell wall.

Observation of the antagonistic process of Trichoderma harzianum against P.
ultimum was studied by transmission electron microscope (TEM). Abnormal cellulose
depositions on Pythium cell walls at the early stage of antagonism were observed.
Although deposition of cellulose represented a defense mechanism from Pythium cell, the
biocontrol agent was able to penetrate the cell wall, suggesting production of cellulase by
Trichoderma. Structural changes inside the cell were evident including a sharp increase
in vacuoles number and retraction of the plasma membrane. After 6'days of incubation,
Pythium hypha appeared free of cell components. A gold labeling of cellulose in the cell
wall of Pythium was present even though cell wall seemed severely damaged, suggested
presence of another cell wall degrading enzyme. Incubation of Pythium cells with gold-
complexed B-1,3-glucanas revealed a sharp decrease in the labeling as early as 2 days
after inoculation with T richoderma. At 6 days of the treatment, labeling of Pythium cell
wall with gold complex had totally disappeared, suggesting that glucan was dissolved.
Production of the hydrolytic enzymes glucanase and cellulase contributed equally to the
antagonistic ability of Trichoderma harzianum against P. ultimum (Benhamou and Chet,

1997i

Oomycetes Cell Wall
Oomycete cell wall components are mainly [3- 1,3- and 0-1, 6- glucans and
cellulose (B-l, 4-linked glucan) ( Wang and Bartnicki-Garcia, 1966; Bartnicki-Garcia,

1968; Bartnicki-Garcia and Lippman,]973). Bartnicki-Garcia (1968) stated that glucan

l2

was the major component of the Oomycete cell wall in the different genera he examined
for cell wall components, including the genus Pythium. Furthermore, removal of cellulose
from the cell wall of the Oomycete Phytophthora by cuprammonium extraction did not
change the hyphal morphology visibly. Thus, glucan was probably responsible for the

skeletal network of the cell wall.

Pythium ultimum and biological control

P. ultimum is a wide spread plant pathogen and its presence was confirmed in
USA, Canada, England and Egypt causing damping-off of most crops. The fungicide
metalaxyl was used world wide to control this pathogen. Because of development of
fungicide resistant strains, another method to control Pythium species needed. Roberts et
al., (1997) used Enterobacter cloacae strain 501 R3 and Escherichia coli strain SlRl to
suppress P. ultimum causing damping-off of cucumber. When applied as a seed
treatment, both strains were effective in a concentration of approximately 107 cfu per
seed. Cucumber plants were significantly less susceptible to P. ultimum when seeds were
pre-germinated for 29 h before being planted in infested soil. Examining the effect of root
colonizations on the ability to protect plant from disease revealed that E. coli strain
Sl7R1 was not an effective root colonizer compared with the E. cloacae strain 501R3.
Whereas strain S l 7R1 increased about 65 fold at 96 h when applied as a seed treatment,
strain 501 R3 increased 24000-fold in the same time. Results indicated that E. coli was
able to suppress P. ultimum.

In another study of control of P. ultimum, chitin waste-based composts were used

by Labrie et al., (2001). The composts were prepared in two phases, peat moss and

13

sawdust were mixed with chitinous waste or cow manure and incubated until end of
thermophilic phase, then a shrimp waste was added and incubated for the second
thermophilic phase. Filter-sterilized water extracts of the composts Were used against two
oomycetes pathogens, P. ultimum and P. fragariae var. rubi. Occurrence of cucumber
damping-off was significantly reduced comparing with water extracts of commercial
compost made from shrimp waste and peat moss only. Results suggested that the two-

phase compost promoted the increase of gram-positive bacteria that inhibited P. ultimum.

l4

Chapter 2

Chronological ultrastructural analysis of the hyphae of three Pythium species treated with
the biocontrol Pseudomonasfluorescens Q2-87

producing 2,4-diacetylphloroglucinol

Abstract

Pseudomonasfluorescens Q2-87 has potential antifungal activities against
soilborne plant pathogens including true fungi and Oomycetes. The mechanism of action
against these two different groups of fungi is expectantly different. In this study, the
effects of the compound 2,4-diacetylphloroglucinol on Oomycetes were studied under the
light and scanning electron microscopes (SEM) as a preliminary step to examining the
mechanism of inhibition. P. fluorescens Q2-87 was tested against different Pythium
species including Pythium ultimum, P. aphanidermatum, and P. intermedium in toxic
plate studies. The three phytopathogens were unable to recover after the treatment. The
antagonism experiment showed that the compound has toxic effect on the pathogens.
Examination of the Pythium species with light microscopy revealed that growth stopped
and hyphae had areas of compression and localized swelling. Lysis was observed near the
margin of dual cultures. Examination by scanning electron microscopy (SEM) revealed
gradual loss of the rough hyphal surface, and presence of hyphal exudation on the second

and third day, respectively. Cell wall degradation exposing the hyphal cell wall

15

microfibrils was observed on the fifth day, and hyphae lost turgor and collapsed. Cell

wall degradation likely was the result of hydrolytic enzymatic activity.

Introduction

Pseudomonasfluorescens has been reported to produce the antibiotic 2,4-
diacetylphloroglucinol (2,4—DAPG) (Shanahan et al., 1992; Bonsall et al., 1997;
Rodriguez and Pfender, 1997; Vincent et al., 1991), which has potential biocontrol
activities (Harrison et al., 1993; Duffy and Défago, 1997; Keel et al., 1992; Nowak-
Thompson, et al., 1994; Sharifi-Tehrani et al., 1998). The compound 2,4-
diacetylphloroglucinol has broad antiviral (Tada et al., 1990), antibacterial (Keel et a1 .,
1992; Reddy and Borovkov, 1970), antifungal (Keel et al., 1992; Nowak-Thompson, et
al., 1994; Vincent et al., 1991) and antihelminthic (Bowden et al., 1965) properties. 2,4-
DAPG has been classified as a phloroglucinol antibiotic that are known to cause
membrane disruption (Tomas-Lorente et al., 1989), consequently preventing the
pathogens from causing disease. However, detailed information concerning the
mechanism of action of 2,4-DAPG produced in situ by P. fluorescens on Pythium species
is not known.

Pythium species are members of the kingdom Chromista, division Oomycota.
Their cell walls are composed mainly of B-1,3- and 0-1 ,6-glucans and cellulose B-1 ,4-
glucan ( Bartnicki-Garcia, 1968; Blaschek et al., 1992) rather than chitin, the major cell
wall component in true fungi. It can be speculated that the mechanism of control in these

two groups may be different.

16

Our objective in this study was to clarify the mechanism by which P. fluorescens
Q2-87 controls Oomycetes by determining the sequence of events involved in the

antagonism of Pythium species by 2,4-DAPG.

Material and Methods
Fungal culture and grth media

Pythium species used in the experiment were Pythium ultimum, P.
aphanidermatum and P. intermedium. The isolate P. ultimum was purified from cowpea
in Georgia and kindly supplied by Dr. Jeffret Hoy (Agricultural Experiment Station,
LSU). P. aphanidermatum and P. intermedium were isolated from turfgrass in Michigan
and cultures were maintained on V8 agar: V8 juice 200 ml, CaC03 3g, agar 15g, water

820 ml.

Bacterial culture and grth media

P. fluorescens strain Q2-87 was obtained from ARS Culture Collection, Peoria,
Illinois (NRRL , B-23374). Cultures were maintained at 28 °C on tryptone glucose yeast
medium (TGY): tryptone 5.0g, yeast extract 5.0g, glucose 1.0g, KzHPO4 1.0g, agar 15.0g
in lliter distilled water, pH 7.0. The experiments were conducted on potato dextrose agar

(PDA, Difco, Detroit, Michigan, USA), pH 6.6.

Antagonism experiment preparation
Bacterial cultures were grown PDA, at room temperature (24°C) for 24 h prior to
use as a seed culture. Fungal cultures were grown for 3-4 days on PDA, and hyphal tips

from the actively growing mycelium were used. Sterilized slides (50 x 75 mm) were

17

placed in petri dishes and covered with molten FDA 2 mm thick. After solidification,
slides were inoculated with bacteria and fungi. Control slides were prepared without
bacteria. Observation of inhibition and morphological changes were recorded with the

light microscopy every 24 h for five days.

Scanning Electron Microscopy (SEM)

In dual culture, bacteria and fungi were placed approximately 2 cm apart on the
media. Each slide was placed in a sterilized petri dish and incubated for five days at room
temperature. Streaks of the bacteria and plugs of the fungi were inoculated at the same
time, and samples were taken every day for five days for examination. The control slides
were not inoculated with the bacterium. Slides were processed for examination using the
scanning electron microscope as follows: samples were fixed at 20° C for 1-2 h in 4%
glutaraldehyde and then buffered with 0.1 M sodium phosphate, pH 7.4, at room
temperature. Following a brief rinse in the buffer, samples were dehydrated in ethanol
series (25, 50, 75, and 95%) for 10-15 min at each gradation and with three 10 min
changes in 100% ethanol. Samples were critical point dried (Balzers critical point dryer)
using liquid carbon dioxide as the transitional fluid. Then, they were mounted on
aluminum stubs using adhesive tabes. Samples were coated with approximately 24.5 nm
of gold for 3.5 min (Emscope Sputter Coater, model SC 500), and purged with argon gas.
Samples were examined in a JEOL 6300 SEM, manufactured by Japan Electron Optics

Limited. The experiment was repeated three times and pictures taken for each replication.

18

Results
Antagonism in vitro

Inhibition of fungal growth and morphological changes were examined under a
light microscope. After 24 h, an inhibition zone was observed when the bacterium and the
fungus were inoculated at the same time. However, slides that were inoculated with the
bacterium only and incubated for 24h prior to inoculation with the fungus showed no
fungal growth. For the duration of the experiment, particularly in the early days, the
fungus produced arial hyphae, an attempt to grow away from the antibiotic amended
media. This phenomenon occurred in cultures with all the Pythium species, but not in the

control.

Morphological changes under light microscope.

Observation of cultures under the light microscope revealed cessation of hyphal
growth and morphological changes (Fig 1A and B). Examination of the fungal margins
in dual culture revealed hyphal alterations such as areas of depression or swelling. These
features were most severe in P. intermedium. In addition, some abnormal bulb shapes
were observed. The shape and the size of the control hyphae remained regular, long, and
branched for the duration of the experiment. When fimgal plugs from the dual plates were

transferred to fresh medium on the fifth day, Pythium hyphae were unable to recover.

Ultrastructural analysis of Pythium cell wall (SEM)
SEM observations revealed that the fungal cell wall is made of at least two
different structural elements, long microfibrils and an amorphus matrix. P.uItimum

hyphae from the control plates were rough and exhibited uniform folds over the entire

19

hyphal surface in all three Pythium species. The folding patterns varied with the species
(Fig. 2 A and B). The cell wall components of, B-1, 3- and B-1, 6 -glucan and cellulose,
were tightly bounded together. On day one of the dual culture, changes in hyphal shape
and cell wall surface could not be distinguished from the control (Fig. 2 C and D). On
day two, hyphae began to show visible changes in the surface of the cell wall. The
surface became smoother than the control and the folds of the cell wall became more flat
(Fig. 2 E and F). Some exudates were observed along the hyphae. On day three, hyphae
exhibited complete loss of the rough surface with the folds flattened. The matrix of the
cell wall degraded (Fig 3 A&B). On days four and five, dramatic changes were evident
including pealing off of a surface hyphae and exposure of microfibrils (Figs. 3 C, D, E

and F). The irregular shape of hyphae of P. intermedium , day four and five, was more

evident in SEM ( Fig 4 A & B).

Discussion

The results confirmed the ability of 2,4-DAPG produced by the bacterium P.
fluorescens Q2-87, to inhibit the growth of P. ultimum, P. aphanidermatum, and P.
intermedium. In fact, the antagonism experiment showed that the 2,4-DAPG was
fungicidal, since the plates were kept for 40 days without any sign of fungal recovery
even where aerial hyphae were produced. Our data consistence with F enton er al., (1992)
and Shanahan et al. , (1992) that P. fluorescens Q2-87 has the ability to inhibit the growth
of P. ultimum.

Studies with the light microscope revealed dramatic changes in the fimgal hyphae

at the margin and throughout the colony of the dual culture. P. intermedium showed the

20

bulbs swelling of the hyphae more clearly than the others, suggesting that slight
differences in reaction to 2,4-DAPG can be expected among species. Alterations of P.
ultimum hypha following treatment with antifungal compounds isolated from P.
aureofaciens have been reported (Paulitz et al., 2000). The nature of these morphological
changes was described as swollen and abnormal appearance of the hyphae.

Scanning electron microscopy revealed the details of the nature of the mechanism
of action of the antifimgal compound 2,4-DAPG, produced by P. fluorescens Q2-87,
against the plant pathogenic Pythium species. The hyphae from the control plates of the
three Pythium species showed a rough surface with a specific folding pattern for each
species. Mares et al., (2000) showed that P. ultimum cell walls posses a smooth surface at
X 720 magnification. However, at a magnification one order of magnitude higher (X
8,000) the surface was rough and had many folds, which are species specific. All Pythium
species treated with 2,4-DAPG showed alteration of hyphal morphology such as
smoothing of the hyphal surface, loosening of the surface folds, hyphal collapse and loss
of turgor pressure, and degradation of the cell wall. These results are consistent with
those obtained by Benhamou et al. (1996) with P. ultimum exposed to P. fluorescens.
However, cell wall changes were not observed probably because samples were examined
two days after the treatment. Collapse and loss of turgor pressure were similar in both
studiesThe early symptoms were most dramatic in P. intermedium (Fig. 4 A & B),
including irregular hyphal shape and swollen bulbs. By the fifth day, cell wall
degradation was evident as the fibrils and cell wall skelet were seen in all three Pythium

species.

21

Mares et al. (2000) studied the effectiveness of four antifungal compounds
belonging to pyrazole-pyrimidines on Pythium morphology. They also found that
treatment by 1-(3) nitrophenyl-6-trifluoromethylpyrazolo [3,4-d] pyrimidine-4(5H)-
thione caused the hyphae to be asymmetrical and to produce exudates. These results are
similar to our data where asymmetrical hyphal shape and exudates were observed.

Oomycete cell wall components are mainly [3- 1,3- and B—1,6- glucans and
cellulose (B-1,4-linked glucan) ( Wang and Bartnicki-Garcia, 1966; Bartnicki-Garcia,
1968; Bartnicki-Garcia and Lippman,l973). Degradation of the cell wall requires
production of hydrolytic enzymes (Bartnicki-Garcia, 1966; Bartnicki-Garcia and Wang,
1983). Since Pythium cell wall degradation was observed in our study with SEM,
enzymes could be contributing to the degradation, possibly glucanase or/and cellulase.

Bartnicki-Garcia (1968) stated that glucan was the major component of the
Oomycete cell wall in the different genera he examined for cell wall components,
including the genus Pythium. Furthermore, removal of cellulose from the cell wall of the
Oomycetes Phytophthora species by cuprammonium extraction did not change the
hyphal morphology visibly. Thus, glucan was probably responsible for the skeletal
network of the cell wall. Pythium cultures examined immediately after treatment with
2,4-DAPG exhibited high glucanase activity (data not shown), which suggested that
glucan had already been degraded by the fifth day of the experiment. Therefore, the cell
wall skeleton visible on the fifth day was probably not glucan. Hunsley and Burnett
(1970) suggested the cell wall microfibrils of Phytophthora parasitica were cellulose
since they could be removed by cellulase enzyme following treatment with glucanase.

Our results at day four showed that degradation of the cell wall occurred by peeling off

22

the hyphal surface layer, exposing loosened fibrils, which probably were cellulose. It has
been stated (Farkas, 1990; Wessels, 1990; Fleet and Phaff, 1981) that the interior wall
layers of hyphae have polysaccharides that are resistant to extraction by alkali and acid
and form fibrils that are responsible for cell wall strength and rigidity. The microfibrils
are B—glucans, including cellulose.

Our results suggest that there are enzymes involved in the cell wall degradation of
the Pythium hyphae. The source and nature of these enzymes are unknown but the
bacteria may possibly induce the fungus to produce these enzymes. Further examinations

of the mechanism of action of degradation of the cell wall are being conducted.

23

 

Fig. 1. Light micrograph of Pythium ultimum exposed to bacteria Pseudomonasfluores-
cence Q2-87. A. Control hypha showing normal growth. The hyphae are long, branched
and regular in diameter. B. After 5 days of treatment, hyphae show growth stopping,
irregular shape and expand in hyphal size.

24

 

Fig. 2. Scanning electron micrographs showing chronological events on Pythium ultimum
hypha after treatment with Pseudomonasfluorescens Q2-87. A. A control hypha at
8,000X magnification shows normal growth. B. A control hypha at 30,000X shows the
roughness of the hyphal surface with some folds. C & D. One day after treatment, no
visible changes are observed on the hyphal surface. E & F. Two days after treatment, the
hyphal surface was smoother. 30,000X mag. shows that the folds increase in size, and
became more flattened.

25

 

Fig. 3. Scanning electron micrographs showing chronological events on Pythium ultimum
hypha after treatment with Pseudomonasfluorescens Q2-87. A. Three days after treat-
ment, Pythium hyphae had lost most of their folds and rough surface. B. At 30,000X
surfaces become flatten and exudates are visible along the hyphal surface. C. Four days
after the treatment, peeling of the outer layer of the surface of the cell wall can be seen.
D. Close observation at 30,000X revealed loosened and disconnected cell wall. E & F.
By the fifth day of treatment, complete degradation of the cell wall outer layer occurred
exposing the fibrils of the cell wall.

26

 

Fig. 4. Scanning electron micrographs of Pythium intermedium hyphae after treatment
with Pseudomonasfluorescens Q2-87. A & B. Pythium hyphae shows irregular shape and
swelling on some areas on the hyphae forming “bulbs” along with some exudates from
the cell wall are evident.

27

Chapter 3

Induction of cell wall degrading enzymes in Pythium ultimum by 2,4-

diacetylphloroglucinol produced by Pseudomonasfluorescens Q2-87

Abstract

In previous studies we have shown that degradation of Pythium species cell walls
occurred after treatment with 2,4-diacetylphloroglucinol (2,4-DAPG) produced by
Pseudomonasfluorescens Q2-87. The source of the enzymes that caused the cell wall
degradation, B-1,3- and B-l,4-glucanase, was unknown. In this study, we determine the
source and identify the enzymes. A crude extract of broth cultures of P. fluorescens was
examined using thin-layer chromatography (TLC), and the presence of 2,4-DAPG was
confirmed by comparing the Rf value to that of an authentic sample .of 2,4-DAPG.
Enzyme assay of B-1,3- and B-l,4-glucanases showed enzymatic activities in mycelium
at various times following treatment with fraction C of the extract of 2,4-DAPG.
Enzymatic activities were several fold higher in 2,4-DAPG treated mycelium than that in
the control. The glucanase activity reached its highest peak at 12 h; however, cellulase
activity reached its highest peak at 24 h after treatment. Native PAGE revealed the
presence of the two enzymes in Pythium mycelium, including one isozyme of glucanase
and two isozymes of cellulase. 2,4-DAPG appears to induce Pythium to produce the cell

wall degrading enzymes, B-1 ,3 and B-1,4-glucanase.

28

Introduction

Many reports have long-established the potential of Pseudomonasfiuorescens as a
biocontrol agent on different phytopathogens via production of antibiotics (Thomasow
and Weller, 1988; Hill, et al., 1994; Cronin, et al., 1997a, Cornin, et al., 1997b, Duffy
and Défago, 1999). In general, modes of action of these biocontrol agents include
competition, production of lytic enzymes, antibiotics, and siderophores. 2,4-
diacetylphloroglucinol (2,4- DAPG) is considered to be the only antibiotic produced by
P. fluorescens strains F113, CHAO, and Q2-87 (Shanahan, et al., 1992; Keel, et al., 1992;
F enton, et al., 1992; Harrison et al., 1993; Bonsall et al., 1997) It is well documented that
P. fluorescens is effective in controlling the plant pathogen Pythium ultimum (Shanahan
et al., 1992; Dunne et al., 1998).

Production of hydrolytic enzymes biocontrol fungi such as T richoderma spp.
(Thrane et al., 2000, Elad and Kapat, 1999, Thrane et al., 1997), mycophilic fungi
Sporotrichum mycophyllum (Pisarevskaya, 1997) and biocontrol bacteria (F raidlender et
al., 1993, Velazhahan et al., 1999, Meena et al., 2001) is well documented. Studies by
F raidlender et al. (1993) have documented the ability of the bacterium Pseudomonas
cepacia to control several plant pathogens in the greenhouse including Pythium ultimum,
F usarium oxysporum, Rhizoctonia solani, and Sclerotium rolfisii by producing hydrolytic
enzymes.

The cell wall is the primary contact between the cell and its environment.

Oomycete cell walls are composed mainly of polysaccharides, glucan (B-1,3- and B-1,6-

glucan) and cellulose (B-l,4 linked glucan) (Bartiniki-Garcia, 1968). Thallus grth

29

occurs at the hyphal tip in fungi due to the apical superiority. Thomas and Mullins (1967)
suggested that hydrolytic enzymes may play an important role in plasticizing the fungal
cell wall and allowing expansion. Thus, B —1 , 3-glucanases and cellulases, capable of
degrading B-1,3 glucan and B-1,4 linked glucans, are naturally present in fungal cells
controlling hyphal grth and branching. The production of these enzymes is regulated
by the fungus because of their potential to degrade the fungal cell wall (Bartiniki-Garcia
and Lippman, 1973; Potgieter and Alexander, 1966; Févre, 1977; Schroth and Hancock,
1981)

Earlier studies in our laboratory showed that there was cell wall degradation of
several Pythium species including P. ultimum, P. aphanidermatum, and P. intermedium
after the treatment with P. fluorescens Q2-87.The source of the enzymes that caused cell
wall degradation was unknown. Therefore, the objectives of this study are (i)
determination the source of the enzymes that cause the Pythium cell wall degradation, (ii)
confirmation of the secondary metabolite produced by P. fluorescens Q2-87 that cause
the production of cell wall degrading enzyme and, (iii) identification and quantitation of
the enzymes involved in Pythium cell wall degradation during specified times afier

treatment.

Materials and Methods
Organisms and culture conditions

P. fluorescens strain Q2-87 was obtained as a dried culture from the USDA-ARS
Culture Collection, Peoria, Illinois (N RRL, B-23374). Cultures were grown in 100 ml

tryptic soy broth (TSB, 30g/ L. Difco, Detroit, M1) for 24 h. Cultures were maintained at

30

28°C in tryptone glucose yeast medium (TGY, tryptone 5.0g, yeast extract 5.0g, glucose
1.0g, K2HPO4 1.0g, agar 15.0g in 1Liter-distilled water, pH7.0.) P. ultimum was isolated
from cowpea in Georgia and kindly supplied by Dr. Jeff Hoy (Louisian State University).
Fungal cultures were grown in V8 medium (V8 juice 200 ml, CaCO3 3g, agar 15 g,
distilled water 820 ml) and experiments were conducted in potato dextrose broth (PDB,

24g/L. Difco) and potato dextrose agar (PDA, 39g/L. Difco).

Isolation of 2,4-DAPG antibiotic substances

Bacteria were grown for five days in PDB in a rotary shaker at room temperature.
Cultures were centrifuged at 10,000 rpm for 45 min and then filtered through 0.45 and
0.2 pm nitrocellulose filters. Three liters of media were lyophilized that yielded 50 g of
dry powder. The lyophilized cell free media obtained was stirred with methanol (MeOH)
ratio was 1:4, and then centrifuged at 3000 rpm for 30 min. The supernatant was
removed. The precipitate, labeled fraction A, was dried and kept at 4°C. The supernatant
was evaporated in vacuo. After evaporation, the residue (22.372 g) was dissolved in
methanol (30 ml) and precipitated with chloroform (CHC13) to remove sugars. The
precipitate was filter of off, dried under vacuum and labeled fraction B. The filtrateing
MeOI-I/ CHCL3, was evaporated to dryness and labeled fraction C (10.55 g). Fraction C,
an authentic sample of 2,4-DAPG, (purchased from Toronto Research Chemicals Inc.
Toronto, Canada), and phloroglucinol (Aldrich) were subjected to thin-layer
chromatography (TLC) with chlorophorm: methanol (12:1 v/v) as mobile phase on silca

gel plates. The plates were viewed under UV (254 nm) afier developing. The UV visible

31

spots were marked and further confirmed by spraying by 25% sulfuric acid followed by

charring. R; values of synthetic 2,4-DAPG and fraction C were calculated.

Oomycete inhibition assay

The inhibition assay of P. ultimum in vitro was carried out on PDA plates as
described by Shanahan et al., (1992). 100 ml of PDA media amended with 0.4 g of
fraction C were poured onto petri plates. Three-day old colony plugs were placed in the
middle of the plates. The plates were incubated at 28°C for two days and inhibition zone
was measured. For the Pythium inhibition assay using synthetic 2,4-DAPG, serial
dilutions of the compound were made and added to PDA after autoclaving the media and
pouring into plates. P. ultimum plugs from a fully grown plate were placed in the middle
of the plates. Plates were incubated at 28°C for two days and the inhibition zone was
measured. Pythium inhibition assay included P. fluorescens as a positive control. Bacteria
were streaked in the middle of PDA plates and culture plugs were placed about 2 cm
from the bacteria. Plates were incubated under the same conditions mentioned above.

Pythium inhibition assay of phloroglucinol was carried out in the same manner as 2,4-

DAPG.

Treatment and sampling

Pythium mycelia were grown in PDB for four days, then treated with the fraction
C of the extraction for 2,4-DAPG and then incubated at room temperature in a rotary
shaker. Treated and untreated samples were collected at 3, 6, 9, 12, 24, 48 and 72 hours,

lyophilized and stored at -20°C until analysis.

32

Enzyme extraction

For determination of B-1,3-glucanase activity, dried mycelium was homogenized
in 50mM potassium acetate buffer, pH 5.0 containing lmM EDTA in a chilled mortar
and pestle. For determination of cellulase activity mycelium homogenized in cold
deionized water. The homogenate was centrifuged at 10,000x g at 4°C for 15 min. The
supernatant was decanted and the pellet was re-suspended in 0.2 ml of the same buffer
and re-centrifiiged under the same conditions as above. The two supernatants were

combined and used immediately for enzyme assays.

13- l ,3-glucanase spectrophotometric assay

B-1,3-glucanase activity was measured by a modification of the published method
of Lima et al., (1997) using Azurine-crosslinked pachyman (AZCL-pachyman;
Megazyme) as the substrate. The reaction mixture contained 0.4 ml potassium acetate
buffer 10mM, pH 5.0, and 0.1 ml enzyme crude sample. Samples were equilibrated in a
30°C water bath for 3-4 min, 100.0 uL of Laminarin (100.0 mg/3ml IOmM potassium
acetate buffer pH 5.0) was added, and the mixture incubated for 30 min at 30°C. The
reaction was stopped with 700 uL of Tris (20% w/v), incubated at room temperature for
3-4 min and then briefly centrifuged. The amount of soluble dyed fragments released
from Laminarin was measured spectrophotometrically at 595nm. Quantifiction of enzyme
units was achieved using a calibration curve constructed by plotting the amount of
glucanase units and their optical densities and performing a linear regression. The method
was linear (R>0.99) in the analysed amount range of 0.005-.025 units of glucanase. The

activities were expressed as units h'lg'l dry weight (DW).

33

Cellulase (B-l, 4-glucanase) spectrophotometric assay

Cellulase was measured by a modification of the method of Worthington (1988)
using cellulose as a substrate (Sigrnacell type 20, Sigma). The reaction mixture contained
4 ml of 5% cellulose solution in 50 mM potassium acetate buffer, pH 5, at 37°C and 1 ml
sample. Mixtures were incubated at 37°C for 120 min with moderate shaking. After
incubation, samples were centrifuged for 5 min at 10,000 rpm. In a cuvette, 3 ml Infinity
Glucose Reagent (Sigma) was added and equilibrated to 25°C, then 0.1 ml of supernatant
was then supplemented. Enzyme activities were measured by recording the increase in
absorbance for 5 min at 340 nm. Units/ml enzyme was calculated as AA340 test- AA340
Blank) (3.1)(5)(df)/ (6.22)(2)(1)(0.1) where: 3.1=final volume, 5=total volume,
df=dilution factor, 6.22=millimolar extinction coefficient of B-NADH at 340nm,
2=conversion factor from 2 h to 1h as per the unit definition, l=volume of cellulase,
0.1=volume of used cellulase afier incubation, and AA340 Blank / Test: AA340 final-
initial. The experiment was repeated three times, each time with three replicates.

Cellulase activity was expressed as units/h/g fresh weight (FW).

Detection of B—1, 3- glucanase and B—l,4-glucanase isoforrns by PAGE

The molecular mass of glucanase and cellulase were determined using SDS-
PAGE according to Laemmli (1970) except that samples were not boiled before loading
as described by Alba (2000). The gel was stained with Coomassie blue.
Detection of B-l, 3-glucanase activitiy was made in native gel.
After electrophoresis, the gel was equilibrated in 50mM potassium acetate buffer, pH 5,
and then was incubated in 25 mM potassium acetate buffer amended with 0.5% larninarin

(Sigma), pH 5 for 45 min at 37° C. After incubation, the gel was washed with distilled

34

water several times and incubated with 1 M NaOH amended with 0.15% 2,3, 5-triphenyl
tetrazolium chloride in boiling water for 5 min until red bands appeared (Kook Hwang et
al., 1997). The amount of total soluble proteins loaded on the gel for adjusted to the
concentration used for each sample was 30 ug protein as described by Kim and Hwang
(1994). Protein contents of extracts were determined by the method of Bradford (1976)
using the BioRAD dye reagent (Bio-Rad, Hercules, California, USA) with Bovine Serum

Albumin as the standard.

Results
Determination of 2,4—DAPG presence in bacterial cell free medium

TLC plates developed with fraction C containing putative 2,4-DAPG, synthetic
phloroglucinol, and synthetic 2,4-DAPG revealed that fraction C contained a band that
corresponded to the authentic phloroglucinol and authentic 2,4—DAPG as detected under
UV at 254 nm and visualized after spraying with sulfuric acid (Fig.1). The Rf values for
both putative and authentic 2,4-DAPG compounds were 0.82. The band that

corresponded to the authentic phloroglucinol had Rf value of 0.24. Another band was

present that had Rf value of 0.52.

Pythium inhibition bioassay

The bioassay of fraction C, authentic 2,4-DAPG, phloroglucinol and the bacteria
showed differences in their ability to inhibit Pythium growth. Phloroglucinol at 30 ppm
was unable to inhibit the grth of P. ultimum. However, fraction C and authentic 2,4-
DAPG at 30 ppm completely inhibited Pythium growth. Plates streaked with the

biocontrol agent P. fluorescens Q2-87 inhibited fungal growth (Fig.2).

35

Effect of bacterial 2,4-DAPG on glucanase activity

The cell wall degrading enzyme [3-1,3-glucanase, released by treated mycelium,
showed higher activity after the treatment by fraction C, than the untreated mycelium.
After three hours of the treatment, the enzyme activity increased about three fold.
Enzymatic activity continued to increase and reached a peak at 12 h of treatment, a five
fold increase. Between 12 and 24 h, the enzymatic activity decreased considerably and

after 24 h, it further decreased until it reached near zero at 72 h (Fig. 3).

Effect of bacterial 2,4-DAPG on cellulase activity

The assay of mycelium treated with fraction C showed production of [3-1 ,4-
glucanase (cellulase) activity by P. ultimum after the treatment that was much higher than
untreated mycelium. The treated mycelium produced a two fold increase in the enzyme
production at 3 h. The increase in the activity was consistent and reached a peak of 5 fold
at 24 h. A decrease in the enzymatic activity was observed afier 24 h. It is notable that

after 72h, the enzyme activity was still two fold higher than the control (Fig. 4).

Detection and visualization of 13-1, 3- and B-1, 4- glucanase by PAGE

To elucidate the nature of glucanase and cellulase enzymes present in Pythium mycelium
after induction with fraction C, crude enzymes were extracted and subjected to PAGE
analysis. Determination of glucanase molecular mass in mycelium after treatment at 24 h
with fraction C by PAGE showed that there was one band similar to one of the isozymes
in the enzyme standard at a molecular weight of less than 29 KD. The same band was

present in the untreated mycelium when sample was loaded 4 fold more than the treated

36

sample. The cellulase showed two different bands at a molecular weight of less than 68
KD that were similar to two isozyme bands in the cellulase standard and the untreated
(Fig. 6). For detection of 13-1, 3- glucanase activity in the gel, a red band was developed
after boiling the gel in a water bath. The treated and untreated mycelium showed bands,
however, the intensity of the bands was higher in the treated samples than the control.
The intensity of bands represents the amount of protein expressed since the amounts of

total soluble proteins loaded were same for all samples (Fig. 5).

Discussion

The mechanism of action of fluorescent Pseudomonads differs depending on the
bacterial strain. Many antibiotic substances such as pyrrolnotrin, phenazines and 2,4-
DAPG are produced by fluorescent Pseudomonads (Shanahan et al., 1992). Although
some members of the Pseudomonads are able to produce lytic enzymes, especially B—1, 3-
glucanases, to control phytopathogens (F ridlender et al., 1993), 2,4—DAPG has been
shown to be the only antifungal compound produced by P. fluorescens strains F113,
CHAO, and Q2-87 (Shanahan, et al., 1992; Keel, et al., 1992; Fenton, et al., 1992;
Harrison et al., 1993; Bonsall et al., 1997) Morever, it has been confirmed that mutant-
negative strains of these bacteria lost the ability to inhibit phytopathogens (Shanahan et
al., 1992; Keel et al., 1992; Fenton, et al., 1992; and Carroll, et al., 1995). In our
experiments with fraction C, we demonstrated that the inhibitory substance produced by
P. fluorescens was 2,4-DAPG. This was confirmed by comparison with the synthetic 2,4-
DAPG. In addition, synthetic phloroglucinol was tested in the Pythium inhibition assay

and showed no inhibitory activity. This supports the argument that the inhibitory activity

37

was due only to the presence of 2,4-DAPG, as suggested by Shanahan et al., (1992), Keel
et al., (1992), and Fenton, et al., (1992). In addition, the bioassay with the synthetic 2,4-
DAPG, fraction C, and bacteria showed no difference in Pythium inhibition. Our results
substantiate the results obtained by the previous researchers.

The enzyme assays of 8-1, 3- and B-1 , 4- glucanase showed activity in the
mycelium. The peak of activity or production of B-l, 3-glucanase was at 12 h after
treatment, whereas the peak for B—1, 4- glucanase was at 24 h. The activities of both
enzymes exceeded the control by 5 fold. Downer, et al., (2001), while studying the
effects of cellulytic enzymes on Phytophthora cinnamomi, found 50-100 units/ml of
cellulase was able to fully dissolve the mycelium. In another report by Velazhahan, et al.,
(1999), it was found that eight Pseudomonas isolates were able to produce B—l , 3-
glucanase. The production of B-l, 3-glucanase was significantly different among isolates,
and the highest production of the enzymes reached more than 80 units/mg protein/hour.
Although an accurate comparison of the number of units between these results and ours
cannot be made due to the differences in units of expression, confirmation of the
destructive ability of these enzyme was clear. Our results showed that there were high 15-
1, 3- and 13-1, 4- glucanase activities in Pythium mycelium after treatment with fraction
C.

F ridlender et al. (1993) showed that B-l, 3-glucanase produced by P. cepacia was
responsible for the antibiotic activity that resulted in successful biological control of P.
ultimum. Their results showed the potential of this enzyme in degrading the cell wall,

especially of a pathogen belonging to the Oomycetes. Our results are in agreement with

38

the published reports and confirm the potential of this enzyme as an anti-oomycetes
agent.

Increasing B-1, 3- and 13-1, 4- glucanase activities in Pythium mycelium after
treatment by fraction C indicatesthat these enzymes have been induced by the presence of
this fraction in the media, or its diffusion onto the fungal cells. Since the extraction with
organic compounds has eliminated any enzymatic activity that may be present in fraction
C, and since phloroglucinol showed no biological activity, the results support the notion
that the source of these enzymes is in the mycelium. In addition, glucanase and cellulase
were assayed in fraction C and no enzymatic activity was detected. The induction is
probably due to the two-acetyl groups present in the compound 2,4-DAPG. The
mechanism of induction of these enzymes is not clear, but a hypothesis can be made that
the acetyl group may induce the transcription or translation of the glucanase and
cellulase. Studying the DNase and RN ase may be useful in explaining this hypothesis.

The native gel for P. ultimum samples treated with fraction C for 24 h, with
presence of glucanase and cellulase standard showed the presence of one band similar to
one of the glucanase isozymes standard at molecular weight less than 29 KD. Samples
showed two bands similar to two cellulase isozymes standard at molecular weight of less
than 68 KD. Although the molecular weight in this particular experiment is not totally
dependable due to the lack of enzyme purification, the gel confirmed the presence of the
two enzymes in the mycelium after treatment with 2,4-DAPG.The samples were
extracted in the same buffer as the one used for assaying the enzymatic activity with

spectrophotometry to insure consistency.

39

Presence of these enzymes in the Pythium cells is essential for facilitating growth
such as hyphal branching, however; their expression should be regulated to avoid
uncontrolled cell wall degradation (Bartiniki-Garcia and Lippman, 1973). The loss of the
ability to control the expression of these enzymes in the presence of 2,4-DAPG is
probably caused by disruption of the regulatory mechanism. Over production of cell wall

degrading enzymes probably explains 2,4-DAPG mode of action.

40

” ii
\ocrra

OH OH

2,4-DAPG
o¢ \ocn3

H 0

H
0H 0H 2-MAPG

H

Phloroglucinol

OH OH

 

Fig. 1. A TLC plat shows phloroglucinol, fraction C and synthetic 2,4-DAPG. Fraction C
shows a similar spot to 2,4-DAPG and another to phloroglucinol. B. Chemical structure
of phloroglucinol, 2, 3 ‘ L‘ g‘ ‘ and 2,4 diacetylphloroglucinol.

41

NV”

 

Fig. 2. Fungal inhibition assay of P. ultimum on PDA. A. control, B. P. fluorescence Q2—
87 . C. fraction C, and D. synthetic 2,4-DAPG. In C&D the fungus could not grow.

42

Glucanase activity

3.5 7
3.0 I
2.5!: I
2.0 I “E“
1.5
1.0 I

 

F-l

 

“I'

 

 

Glucanase activity (unitslhlg DW)
ri

 

 

 

 

 

 

 

 

 

 

 

 

0.0 -. - ~- ,,_- . l—I .—-._;

0 3 6 9 12 24 48 72
Time

 

 

 

 

 

 

Fig. 3. Effect of 2,4-DAPG produced by P. fluorescence on glucanase activities of P.
ultimum. Initial sample was taken before the treatment to serve as a control reference.
Samples were collected at different times and enzymatic activities expressed as units/h/ g

dry weight.

43

 

Cellulase activity

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

I

I

I

I

I A250. 2." ~44

IE- I II

I§20.0~ T

If I ‘ II
£15.0~ , i

.5, "' II

I; II

Io1o.oI .

I z I + *
s 5, II

I: ' II

.0 II

I 0.0' ’f I T T I I a ""

o 3 6 9 12 24 43 72

Time I

L_.____,__.__. ___. ,7 _. * ___.. _* ,_- __.. ____ .‘._

 

 

Fig. 4. Effect of 2,4-DAPG produced by P. fluorescence on cellulase activities of P.
ultimum. Initial sample was taken before the treatment to serve as a control reference.

Samples were collected at different times and enzymatic activities expressed as units/h/g
fresh weight.

 

Fig. 5. Native gel shows glucanase activity of Pythium ultimum treated with
fraction C containing 2,4-DAPG. Lane 1,2,3,4,5,6,7,8,9 and 10 (control, 30 min,
1, 2, 3, 6, 9, 12 and 24h, respectively).

45

Standard
Control
Glucanase
standard
Treatment
Cellusase
standard

200.0

97.4
68.0 a

43.0

29.0

 

1 2 3 4 5

Fig. 6. SDS-PAGE stained with comassi showing the presence of glucanase and cellulase
bands on P. ultimum samples treated for 24 h with crude extract. Lane A is the protein
standard, B. untreated P. ultimum sample C. glucanase standard, D. P. ultimum sample
extracted and E. cellulase standard.

46

Chapter 4

Ultrastructural, hydrolytic enzymes and molecular analysis of the effect of 2,4-

Diacetylphloroglucinol produced by Pseudomonasfluorescens on Pythium ultimum cells

Abstract

Pseudomonasfluorescens Q2-87 is a producer of the 2,4-diacetylphloroglucinol
was used as a biocontrol agent in this study. Examination of P. ultimum cell with the
transmission electron microscopy (TEM) following exposure to P. fluorescens Q2-87,
revealed correlations between length of exposure and cell response. After three days
treatment, Pythium cell walls showed disruption and degradation. Mitochondrion
membranes and nuclei show disorder. By the fifth day the cytoplasm and plasma
membranes aggregate and the organelles appeare disorganized. Moreover, many cells are
empty of their components. Assaying the enzymatic activities of enzymes such as
protease and RN ase reveal an increase of activity as early as 30 min after exposure to the
partially purified 2,4-DAPG, fraction C. Protease show a 2 fold increase in activity 30
min after treatment and reach a 5 fold increase over the control at 48 hrs. Determination
of total soluble proteins in the same sample reveal a change in protein content during the
exposure time with increase in the amount of soluble protein immediately after exposure
to fraction C. The maximum amount of the protein content occurs 2 h after exposure.
RNase also showed similar increase in activity after 30 min of exposure to fraction C.

After 48 hrs exposur, RNase reached 5 fold higher than the control. Measuring the total

47

soluble proteins in the same samples showed changes in the amount of proteins. The
maximum protein content recorded was two and a half fold after 2 h of exposure to the
antifungal compound. Changes in protein patterns compared to the untreated control are

evident with polyacrylamide gel electrophoresis.

Introduction

Numerous studies have demonstrated the antifungal potential of Pseudomonas
fluorescens strains that produce the antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) as
a biocontrol agent (Shanahan et al., 1992; Bonsall et al., 1997; Rodriguez and Pfender,
1997; Vincent et al., 1991; Harrison et al., 1993; Duffy and Défago, 1997; Keel et al.,
1992; Nowak-Thompson, et al., 1994; Sharifi-Tehrani et al., 1998). 2,4-DAPG has broad
antiviral, antibacterial and antifungal activities (Tada et al., 1990, Keel et al., 1992;
Reddy and Borovkov, 1970; Nowak-Thompson, et al., 1994; Vincent et al., 1991). 2,4-
DAPG producing strains have been used effectively in biological control of plant
pathogenic fungi.

Studies have showed that microbial antibiotics cause cell disruption in Erysiphe
graminis var. tritici (Hajlaoui and Bélanger, 1993) and Erysiphe graminis f. sp. Hordei
(Klecan er al., 1990) after exposure to the biocontrol Sporothrixflocculosa and
Tilletiopsis pallescens respectively and in Penicillium digitatum after the treatment with
the biocontrol Verticillium lecanii (Benhamou and Brodeur, 2000.)

Additionally, production of hydrolytic enzymes by Pseudomonas species that act
against soilborne pathogens are well documented (Fraidlender et al., 1993, Velazhahan et

al., 1999; Meena et al., 2001). Combining proteolytic and phloroglucinol producing

48

Pseudomonads as a biocontrol product has provided protection of sugar beet against
Pythium ultimum (Dunne et al., 1998).

Protease production is known to be one of the main mode of action of the
biocontrol agent Trichoderma spp. (Haab et al., 1990). Examination of T. viridi against
Sclerotium rolfsii revealed that production of proteolytic enzymes by T. viridi could
control the pathogen in an autoclaved soil (Rodriguez-Kabana, 1978). In addition, T.
harzianum produces a considerable amount of protease as a mode of action against
Botrytis cinerea (Elad and Kapat, 1999). Stenotrophomonas maltophilia W81 was able to
control Pythium ultimum by producing extracellular proteolytic enzymes (Dunne et al.,
1997). Dunne et al also showed that Pythium cultures exposed to cell free supematants of
S. maltophilia caused hyphal lysis and loss of consequent growth ability upon transferred
to fresh media.

Our previous studies showed that 2,4-DAPG produced by P. fluorescens Q2-87
caused induction of glucanase and cellulase activity that degraded P. ultimum cell wall,
chapter 3. The effect of this antibiotic metabolite on cell cytology as well as any self-
destructive processes inside the cell has not been studied. The objectives of the present
research was to; (i) delineate the ultrastructural changes in the Pythium cell after
treatment with 2,4-DAPG, (ii) determine if the metabolite affects the production of self-
destructive enzymes inside the cell and (iii) determine the metabolites affect on the

Pythium cell at the molecular level in relation to the changes in protein patterns.

49

Materials and Methods
Organisms and culture conditions

P. fluorescens strain Q2-87 was obtained as a dried culture from USDA-ARS
Culture Collection, Peoria, Illinois (NRRL, B-23374). Cultures were grown on 100 m1
Tryptic soy Broth (TSB, 30g/ L. Difco, Detroit, M1) for 24 h. Cultures were maintained
at 28°C in Tryptone Glucose Yeast medium (TGY): tryptone 5.0g, yeast extract 5.0g,
glucose 1.0g, K2HPO4 1.0g, agar 15.0g in 1Liter distilled water, pH 7.0. P. ultimum was
isolated from cowpea in Georgia and kindly supplied by Dr. Jeff Hoy (Louisiana State
University). Pythium cultures were grown in V8 medium: V8 juice 200 ml, CaCO3 3 g,
agar 15 g, distilled water 820 ml and experiments were conducted in Potato Dextrose

Broth (PDB, 24g/L. Difco) and potato dextrose agar (PDA, 39g/L. Difco).

Antagonism experiment and TEM sample preparation

Bacterial cultures were grown on PDA, at room temperature (24°C) for 24 h. prior
to use as a seed culture. Pythium cultures were grown for 3-4 days on PDA, and hyphal
tips from the actively growing mycelium were used. Sterilized glass slides (50 x 75 mm)
were placed in petri dishes containing PDA. Afier solidification, slides were inoculated
with bacteria and Pythium. Control slides were prepared without bacteria. Hyphae from
the outer edge of the treated colonies were cut and immediately fixed in 2.5%
glutaraldehyde + 1% sucrose in 50 mM cacodylate pH 7.1 for 2 hr. at room temperature.
After fixation, the samples were washed 5 times in 50 mM cacodylate buffer, pH 7.1, for
20 minutes. All samples were post fixed for 1 hr. in 1% osmium tetroxide at 4°C.

Samples were then washed 4 times in distilled water (20 min each wash), fixed with 1%

50

uranyl acetate for one hour at room temperature, and subsequently washed 2 times in
distilled water (15 minutes). The samples were dehydrated through a graded acetone
series, one change every 30 minutes each in 25, 50, 70, 85 and 95% acetone and five
changes for 20 minutes each in 100 % acetone, and subsequently infiltrated and
embedded in Spurtol resin. Samples were infiltrated with a mixture of 2:2 acetone resins
for two hours and 3:1 mixture of acetone resin overnight. Several changes in 100% resin
were made including 2 changes of 4 hours each, one change overnight, two changes 4
hours each, one change overnight, one change one hour and finally one hour embedded in
quetol. Blocks were made using silicone molds and polymerized for 48 hours at 60 °C.
Thin sections were collected on copper grids and stained with uranyl acetate for 30 min
and lead citrate for 3 min. Examinations were made using a JOELIOOX (JOEL Ltd.,

Tokyo, Japan) at accelerating voltage 120 KV.

Isolation of 2,4-DAPG antibiotic substance

The bacterium was grown in PDB for five days in a rotary shaker at room
temperature. Cultures were centrifirged at 10,000 rpm for 45 min and then filtered
through 0.45 and 0.2 pm nitrocellulose filters. Three liters of media were lyophilized,
yielding 50 g dry weight. The lyophilized crude extract was subject to further
purifications as described previously in chapter 3. Fungus mycelia were treated with the

partially purified antifimgal compound as previously described in chapter 3.

51

Treatment and sampling

Mycelia were grown for four days in PDB, then treated with the partially purified
2,4-DAPG, fraction C and incubated at room temperature in a rotary shaker. Treated and
untreated samples were collected at 3,6,9, 12, 24, 48 and 72 hours, lyophilized and stored

at —20°C until analysis.

Protein determination

Total soluble protein was determined by using the method of Bradford with

Bovine Serum Albumin (BSA) as a standard (Bradford 1976).

Protease extraction and assay

For determination of Protease activity, dry mycelium were homogenized in
50mM Tris-HCl buffer, pH 7.0 containing lmM EDTA, 3 mM MgC12, 0.02% (v/v) 13-
mercaptoethanol and 1% PVP (polyvinylpyrrolidone). The homogenate was centrifuged
at 10,000x rpm at 4°C for 30 min. The supernatant was decanted and the pellet was re-
suspended in the same amount of the same buffer, and re-centrifuged under the same
conditions as above. The two supematants were combined and used immediately for
enzyme assays. The assay was carried out using the methods described by Sankhla et al.,
(1976). The assay mixture consisted of 1 ml of 0.5% casein, 3 ml of 0.2 M phosphate
buffer pH 7.0 and 0.2 ml enzyme extract. The assay was then incubated at 37°C for 25
min. The reaction was terminated by adding 1 ml of 10% trichloroacetic acid (TCA).
After centrifugation, the absorbance was measured at 280 nm. One unit of enzyme

activity was equal to an increase in absorbance of 1 in 1 h under the above specified

52

conditions. Protease activity was expressed as units/h/g fresh weight (FW) and as

units/mg protein.

RNase extraction and assay

RN ase was extracted in 25 mM Tris-Hcl buffer at pH7.5. The homogenate was
centrifuged at 10,000x rpm at 4°C for 30 min. The supernatant was decanted and pellets
were re-suspended in the similar amount of the same buffer and re-centrifuged under the
same conditions as above. The two supematants were combined and used immediately
for enzyme assays. RNase activity was determined by a slight modification of the method
of Altiman et al. (1977). The reacton mixture contained 0.4 ml of RNA substrate (15 mg
highly polymerized RNA, Sigma, dissolved in 16 ml of 6.2 mM acetate buffer pH 5.5),
which was added to 0.1ml of enzyme extract and incubated at 28°C for 30 min. The
reaction was terminated by adding 0.5ml of 2.5% w/v TCA containing 0.3% uranyl
acetate. The samples were then placed in ice for 15 min and centrifuged at 10,000 rpm for
15 min. The supernatant was diluted appropriately and absorbance was read at 260 nm.

RNase activity was expressed in the same manner as protease

Soluble protein extraction and changes of protein patterns on SDS-PAGE

Treated and untreated samples were lyophilized and ground in a chilled mortar
and a pestle with 50mM Tris-HCl buffer pH 7.5. Samples were extracted at 4°C for 12 h
then centrifuged at 4°C for 30 min at 14,000 rpm. The supernatant was decanted and
pellets were re-suspended in the same buffer and re-centrifuged under the same
conditions as above. The two supematants were combined and used- for recording of

protein patterns. SDS-PAGE was made with 10% (w/v) polyacrylamide separating gels

53

and 5% stacking gel as described by Lamli (1977). Gels were stained with coomassie
blue and the experiment was repeated five times. Glucanase and cellulase standards were

loaded along with a molecular marker range from 200 -14.7 kb (GibcoBRL).

Results
Histological observation by TEM

P. ultimum grown on unamended PDA showed typical ultrastructure with regular
cylindrical hyphae. Cells had a normal cell wall and enriched cytoplasm with many
organelles, including nuclei, mitochondria, vacuoles and storage vesicles (Fig. 1A). P.
ultimum grown in dual cultures with P. fluorescens Q2-87 showed alteration in the cell
wall structure as well as internal disruption. At three days after dual culture, the number
of vacuoles increased considerably (Fig. 13). Examination of the mlclei on a control cell
showed presence of the double membrane and nucleolus. In contrast, the nucleus in the
dual culture was disrupted in its shape, the nucleolus was absent, and the chromatin was
disrupted inside the nucleus (Fig. 1C & D). Close examination of the mitochondria
showed that the double membrane was disrupted and possibly dissolved. A regular
double membrane was present in the control cells (Fig. 1E & F). The number of cristae
present in mitochondria of the dual cultures was fewer compared to number present in the
control cells. The cell wall in the untreated sample was evident and regular, however in
the treated samples the cell wall was disrupted and in some areas was totally degraded
exposing the cell components (Fig. 2 A & B). At the fifth day of the treatment the main
features of these cells were irregular in shape and organelles were disorganization. The
cytoplasm and cell plasma membrane were notably aggregated (Fig. 2 C). Some P.

ultimum cells were devoid of cell components (Fig. 2 D).

54

Effect of 2,4-DAPG on Protease activity

Protease activity in the mycelium treated with the partially purified 2,4-DAPG extract,
fraction C, showed a higher activity than that of untreated mycelium. The maximum units
of the hydrolytic protease present in the control sample were 4 units/ g F W. After 30 min
of the treatment with fraction C, the enzymatic activity of the mycelium was 2 fold higher
than that of the control. There were no significant differences between 30 min and 1h of
treatment or between 1 h and 2 h, however the trend in activity approached 2.5 fold
increase at 2 h compared with the untreated mycelium. The activity 'of the enzyme
continued to increase with increasing incubation time with fraction C up to 72 h. At 3 and
6 h, the enzymatic activity increased about 3 fold. The maximum activity was recorded at
48 h at 5 fold increase compared to the control (Fig 3). The enzymatic activity showed a
decrease at 72 h but was still higher than that in the control. Fig. 3 also showed the total
soluble protein level changed over time-course of the treatment. In the untreated tissues,
there was about 0. 6 mg/ g FW, however after the treatment with 2,4-DAPG for 30 min,
the amount of total soluble proteins was significantly higher and reached 2 fold. The
maximum amount of the soluble proteins reached was about 2.5 fold at 2 h. The amount
of soluble proteins showed a gradual decrease until they reached the same amount as the
untreated tissues at 24 h. They continued to decrease until reached half the amount of
proteins present in the control at 72 h. Calculation of the specific activity of protease,
units /mg protein, showed that it increased gradually over time and reached its maximum

peak at the 72 h after treatment (Fig. 3).

55

Effect of 2,4-DAPG on RNase activity

Untreated samples had a low RNase activity compared to mycelium treated with
the partially purified 2,4-DAPG extract. The treated samples showed no significant
difference in RNase activity among samples taken at 30 min, 1, and 2 hrs, but they
differed significantly from the control. After I h, RNase activity reached 2 fold higher
than that in the control. The enzymatic activity reached three fold after 9 h and maximum
activity occurred at 48 h of treatment that increased to 5 fold compared with the control.
At 72 h, there was a sharp decrease in the activity (Fig. 4). Changes in total soluble
proteins over the treatment time were also shown in Fig. 4. The maximum amount of
total soluble proteins expressed was 0.6 mg/ g FW. However, in the treated sample at 30
min the amount of protein increased more than 2 fold and at 1 h the activity reached
about 2 .5 fold. The amount of protein showed a sharp decrease at 2 h, but was still
higher than that in the control. Less soluble protein was recorded at 72 h. Calculation of
the RN ase specific activity showed first a decrease at 30 min and 1 h after the treatment
compared to the control. However, at 2 h the specific activity of the enzyme showed an
increase of more than 5 fold as compared to the control. At 72 h, RNase specific activity

showed a sharp decrease (Fig. 4).

Detection of protein changes on PAGE

In SDS gel; protein patterns showed changes over time following treatment with
partially purified 2,4-DAPG. There was a band similar in migration to the band of the
glucanase standard (arrow) at a molecular mass of less than 29 kb. A second band was

similar to the cellulase standard, at molecular mass less than 68kb (double arrow heads)

56

(Fig. 5). The treated samples showed loss of most of the high molecular mass proteins

especially at 6 h after the treatment.

Discussion

P. fluorescens Q2-87 produce 2,4-DAPG that is capable of inhibiting different
phytopathogens (Harrison et al., 1993; Duffy and Défago, 1997; Keel et al., 1992;
Nowak-Thompson, et al., 1994; Sharifi-Tehrani et al., 1998). Studies have confirmed that
mutant negative strain, that have lost the ability to produce 2,4-DAPG, also lost the
ability to inhibit plant pathogens (Shanahan et al., 1992; Keel et al., 1992; Fenton, et al.,
1992; and Carroll, et al., 1995). Observation of P. ultimum cells with TEM, after
treatment with a partial purified 2,4-DAPG, showed a sharp increase of vacuoles, cell
disorder, cell membrane aggregation and organelle disruption. Klecan et al., (1990) found
that Tilletiopsis pallescens caused increase of vacuoles in Bluronia graminis f. sp. hordei
and that increase was related to sign of senescence. They hypothesized that the mode of
action of the biocontrol agent was via production of toxic compound. Their results are
similar to our observations of P. ultimum and are also in agreement with the result
obtained by Benhamou and Garand (2001). In another study by Haijlaoui and Bélanger,
(1993) they reported that treatment with antibiotics caused cell disruption. Moreover,
Askary et al., (1997) suggested that production of antibiotics by the biocontrol agent of
Verticillium lecanii caused cytoplasm disruption and aggregation in the cucumber
powdery mildew pathogen Sphaerothecafuliginea. That disruption was the result of
antibiotic diffusion into cells. Weete (1980) explained that disruption of lipids and

phospholipids of cell plasma membrane affected the membrane permeability and caused

57

plasmolysis. The same conclusion of the affected plasma membrane was approached by
Lewis and Papavizas (1987). Our previous observations of Pythium cell wall degradation
in chapter 2 with SEM and induction of glucanase and cellulase, cell wall degrading
enzymes, in P. ultimum mycelium in chapter 3 were due to the act of 2,4-DAPG. We
hypothesize that 2,4-DAPG was able to diffuse into cells or act from outside the cell
without diffusion and caused the induction of these enzymes.

Alteration of P. ultimum cell ultrastructure is indication of changes at
physiological and molecular levels. Increase of enzymes production such as protease and
RNase occur on senescence related activities (Alteman et al., 1977). Since we found
marked changes in Pythium cells, we hypothesized that assaying the protease and RN ase
activity might elucidate when the senescence activity began that may explain whey the
fungus cells failed to recover after transferred to fresh media.

The results presented in this study indicated that the Pythium mycelium over
expressed the hydrolytic enzyme protease (Fig. 2). Because P. fluorescens Q2-87 does
not produce lytic enzymes or any other antibiotic compounds except 2,4-DAPG in
fraction C, the source of any enzyme is the fungus itself. Lytic enzymes such as protease
are able to inhibit P. ultimum Dunne et al., (1997). They reported that incubation of
Pythium with cell free supematants of Stenotrophomonas maltophilia W81 and other
materials containing proteases (such as commercial peotease) caused hyphal lysis and
loss of the ability to grow. They also found the protease negative mutant strain had no
effect on growth. In another study by Dunne et al., (1998) combining two biocontrol
agents, P. fluorescens producing 2,4-DAPG and S. maltophilia W81, against P. ultimum,

they found that the combination resulted in complete control of Pythium that was similar

58

to what they had obtained with fimgicide in the field experiment. Our results showed that
2,4-DAPG induced production of protease inside the Pythium cells which would probably
inhibit the Pythium more effectively. In addition, Sivan and Chet (1989) found that pre-
treating F usarium oxysporum hyphae with proteolytic enzymes increased their lysis
susceptibility by B-l , 3-glucanase execreted by T. harzianum. Their finding supports our
hypothesis that 2,4-DAPG diffused into Pythium cells and caused over production of
several self-destructive enzymes such as glucanase, cellulase, protease and RNase.

Ribonucleases (RNases) are ubiquitious components in the living cell and their
role is to terminate the life span of different RNA species by hydrolytic or phosphorolytic
action (D’ Alessio and Riordan, 1997). Increase of RNase activity was observed
associated with programmed cell death (Lehmann et al., 2001), senescence in tomato
(Lers et al., 1998) and Arabidopsis thaliana (Taylor et al., 1993). Galiana et al., (1997)
reported that S-like RNase in tobacco was able to inhibit Phytophthora parasitica from
colonize the leaves. They concluded that this extracellular ribonuclease was participating
in the defense mechanism of tobacco. Hugot et al., (2002) proposed that the inhibition
was mediated by interacellular degradation of the pathogen RNAs after translocation of
the protein through Golgi bodies. The results had obtained in this study suggest that the
observed increase in RNase activity probably was due to induction of programmed cell
death in the Pythium cells that were exposed to 2,4-DAPG.

In conclusion, 2,4-DAPG caused cell damaged that was evident by disruption of
nuclei, mitochondrion, cell walls, vacuoles, cytoplasm and plasma membranes. Induction
of senescence enzymes such as protease and RNase, and changes in protein patterns,

suggest that Pythium cells cannot recover after treatment with the antibiotic compound.

59

Future work may determine the minimum dose of 2,4-DAPG that can cause the

irreversible changes in Pythium cells.

60

 

Fig. 1. Transmission electron micrographs of Pythium ultimum samples collected from
the margin of colony treated with Pseudomonasfluorescens Q2-87. A. P. ultimum grown
in single culture. The hypha is cylinder and has organelle enriched cytoplasm. B. P.
ultimum in dual culture, 3 days after treatment, increase of vacuoles. C. Control hypha
has a normal nuclei surrounded with double membrane, presence of nucleolus is evident.
D. Treated P. ultimum hypha at day 3, the double membrane id dissolved and presence of
nucleolus is not evident. E. Normal mitochondrion on untreated sample. The double
membrane is present along with cristae. F. Mitochonderion from the dual culture at day 3,
the double membrane is dissolved.

61

 

Fig. 2. Transmission electron micrographs of Pythium ultimum samples collected from
the margin of colony treated with Pseudomonasfluorescens Q2-87 producing 2,4-DAPG.
A. Untreated hypha of Pythium ultimum showing presence of normal cell wall and plasma
membrane. B. Treated hypha from the dual culture at day3, cell wall is degraded and
plasma membrane begins to aggregate. C. At day 5, treated hypha shows cytoplasm
aggregation and disorganization of organelles. D. At day 5, treated hypha is free from its
components.

62

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64

 

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65

 

Summary and Conclusion

Pseudomonasfluorescens Q2-87, previously known as P. aureofaciens Q2-87,
was isolated from take-all suppressive soil near Quincy, Washington and inhibited
Gaeumannomyces graminis var. tritici. P. fluorescens Q2-87 produces a secondary
metabolite 2,4-diacetylphloroglucinol that has a broad antiviral, antifungal, antibacterial
and antihelminthic activity. There are 6 genes responsible of 2,4-DAPG production and
tranfering them to 2,4-DAPG non-producing strains resulted in production of the
compound. Although there are many reports about P. fluorescens Q2-87 and 2,4-DAPG,
little is known about its mode of action.

Research in the three chapter of this dissertation has been carried out to answer
questions about the mode of action of 2,4-DAPG in control of Oomycetous plant
pathogens.

Observation of the antagonistic experiment revealed that 2,4-DAPG was able to
inhibit Pythium species growth, additionally mycelium transfered to fresh media did not
recover. Examination of Pythium hyphae under the light microscopy revealed changing in
hyphal morphology. To answer the question of the nature of the changes seen in the
hyphae, scanning electron microscopy was used to examine the hyphae over 5
consecutive days. Observation of gradual changes in the cell wall revealed cell wall
degradation, and exudation was also observed from the hyphae.

Since the source of the enzymes that caused cell wall degradation was unknown,
bacterial growth media was examined for presence of any enzymes showed non were

present. That led to extraction of 2,4-DAPG compound with methanol and then methanol

66

and chloroform, and then testing of extracted material, called fraction, for inhibitory
properties. Fraction C was able to inhibit Pythium growth and had similar Rf to the
authentic 2,4-DAPG on TLC plates. Fraction C was further used in an antagonistic
experiment to assay the enzymatic activity in the Pythium mycelium. The cell wall
degrading enzymes glucanase and cellulase are assayed at different times, 3,6,9,12, 24,
48, and 72 h following treatment of mycelium with fraction C. A high enzymatic activity
was expressed in all treatments compared with untreated mycelium. For glucanase, the
enzymatic activity reached 5 fold 12 h after treatment and then decreased. For cellulase,
the enzymatic activity reached 5 fold at 24 h after treatment. Glucanase showed the same
activity in native-PAGE. Running samples of extracted mycelium along with glucanase
and cellulase standards in a PAGE gel revealed presence of one band in Pythium
mycelium similar to one of the glucanase isozymes whereas gels revealed presence of
two isozymes of cellulase in Pythium samples similar to the standard. Glucanase activity
was further confirmed in PAGE.

Transmission electron microscopy was carried out to examine Pythium cell
components to better understand the effect of the 2,4-DAPG compound unside the cell.
Changes in cell wall structure, and ultrastructure of mitochoneria, nuclei, cytoplasm and
plasma memberanes was observed. An increased number of vacoules was a sign of
programmed cell death, and examination of senescence enzymes could explain when that
process strated. Assaying protease activity revealed that increase in its enzymatic activity
started as early as 30 min after the treatment. The activity continued to increase until it

reached 5 fold at 3 h of the treatment.

67

RN ase activity increased over the control 30 min after the treatment, degrading
most of RNA in the cell. The highest activity recorded was 1 h after treatment.
Examination of the protin patterns in PAGE showed changes in protein expressions as
early as 1 h after treatment. In addition, high molecular weight proteins began to
dissappear following 9 h of the treatment.

A lot of changes occurred inside Pythium cells that explain the effect of 2,4-
DAPG on the fungus. Although induction of hydrolytic enzymes and programmed cell

death could be hypothesized as the mode of action, one important aspect that should be

 

persued is to determine where exactly 2,4-DAPG binds in the cell inducing these

changes.

68

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