interactions between plant growth-promoting rhizobacteria
TRANSCRIPT
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INTERACTIONS BETWEEN PLANT GROWTH-PROMOTING RHIZOBACTERIA (PGPR) AND SQUASH PLANTS – THE ROLE OF PGPR-MEDIATED SUPPRESSION
OF PHYTOPHTHORA BLIGHT
By
XIAODAN MO
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
2013
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© 2013 Xiaodan Mo
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This dissertation work is dedicated to my husband, Jiebin, for his endless love, support and encouragement during the challenges in research and life. This work is also
dedicated to my parents and my baby, who have always loved me unconditionally.
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ACKNOWLEDGMENTS
I am very fortunate to have performed my graduate work at University of Florida;
and there are many people to thank for their part in my dissertation work. I would like to
express my deepest appreciation to my advisor, Dr. Shouan Zhang, for giving me
extraordinary support on my research and to assist me in the dissertation writing. I am
grateful for his guidance and the opportunities he has afforded me. He is incredibly
organized and a great problem solver, both of these qualities were immensely helpful in
moving my project forward. I would also like to thank my dissertation committee
members, Dr. Bruce Schaffer, Dr. Pamela D. Roberts, Dr. Jeffrey B. Jones, and Dr.
Wenyuan Song, for serving on my committee and contribution to my dissertation. I am
very appreciated to Dr. Jodie V. Johnson in Chemistry Department, University of Florida
for his assistance in quantitative analysis of surfactins, and Dr. Joseph W. Kloepper in
the Department of Entomology and Plant Pathology, Auburn University for providing
Bacillus PGPR strains.
I would like to thank all members of Dr. Zhang’s lab who have contributed to the
progress I have made. Yuqing Fu and Dr. Xiaohui Fan have given their time to teach me
techniques and aid in experiments since the first day I joined the lab. I would like to
thank Dr. Zelalem Marsha and Dr. Jaimin S. Patel for sharing their own expertise and
enthusiasm for science.
Finally, I would like to thank my friends and family for their continued support and
encouragement. Thanks for listening to my problems, providing perspective, being
patient with me, and being there whenever I need you.
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TABLE OF CONTENTS page
ACKNOWLEDGMENTS .................................................................................................. 4
LIST OF TABLES ............................................................................................................ 9
LIST OF FIGURES ........................................................................................................ 11
LIST OF ABBREVIATIONS ........................................................................................... 13
ABSTRACT ................................................................................................................... 14
CHAPTER
1 LITERATURE REVIEW .......................................................................................... 16
Summer Squash ..................................................................................................... 16 Squash Crown and Root Rot Disease Caused by Phytophthora capsici ................ 18
Plant Growth-Promoting Rhizobacteria ................................................................... 22 Rhizosphere ..................................................................................................... 22
PGPR as Biocontrol Agents (BCAs) on Vegetable Crops ................................ 23 Root Colonization of PGPR .............................................................................. 26
Mode of Action of Biocontrol Agents ....................................................................... 28 Antagonism Mediated by Metabolites ............................................................... 28
Competition for Nutrient and Niches ................................................................. 30 Defense Mechanisms in Plants ........................................................................ 30
2 BIOLOGICAL MANAGEMENT OF PHYTOPHTHORA BLIGHT AND PLANT GROWTH PROMOTION BY SELECTED PGPR STRAINS UNDER GREENHOUSE CONDITIONS ............................................................................... 36
Introduction ............................................................................................................. 36 Materials and Methods............................................................................................ 40
PGPR Strains and Their Growth Conditions ..................................................... 40
Media Used in the This Study ........................................................................... 41 Growth of Squash Plants .................................................................................. 41
Application of PGPR Treatments in the Greenhouse ....................................... 42 Inoculum Preparation and Inoculation .............................................................. 42
Disease Rating ................................................................................................. 43 Evaluation of PGPR for Suppressing Phytophthora blight ................................ 43 Plant Growth Promotion by PGPR ................................................................... 44 Effect of PGPR Concentration on Disease Suppression .................................. 44 Effect of Timing of PGPR Application on Disease Suppression ....................... 44
Effect of PGPR on Chlorophyll Content in Squash Leaves .............................. 45 Statistical Analyses .......................................................................................... 45
Results .................................................................................................................... 46
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Susceptibility of Squash to Different P. capsici Isolates ................................... 46
Effect of PGPR on Phytophthora Blight in the Greenhouse.............................. 46 Effect of PGPR on Plant Growth in the Greenhouse ........................................ 47
Effect of Concentration of Selected PGPR on Disease Suppression ............... 48 Effect of Application Timing of PGPR on Disease Development and Age-
related Resistance to Phytophthora Crown and Root Rot on Squash Treated with IN937b ...................................................................................... 49
Effect of PGPR on Chlorophyll Content in Squash Leaves .............................. 49
Discussion .............................................................................................................. 50 Virulence Differentiation of P. capsici Isolates .................................................. 50 Control of Phytophthora Crown and Root Rot by Bacillus spp. ........................ 51 Enhanced Plant Growth by Bacillus spp. .......................................................... 53
Relation of Leaf Chlorophyll Content and Plant Health .................................... 53 Optimization of Application Rate and Timing .................................................... 54
3 THE ROLE OF BIOFILM FORMATION AND COLONIZATION IN THE RHIZOSPHERE BY BACILLUS SUBTILIS IN937B WITH SYNTHESIS OF SURFACTINS ......................................................................................................... 75
Introduction ............................................................................................................. 75 Materials and Methods............................................................................................ 79
PGPR Strains and Media ................................................................................. 79 Pellicle Formation ............................................................................................. 79
Colony Morphology Assay ................................................................................ 80 Biofilm Production Assay in Microtiter Plates ................................................... 80
DNA Extraction and Polymerase chain reaction (PCR) Analysis ...................... 80 Surfactin Extraction .......................................................................................... 82 HPLC/ESI-MSn Assay of Surfactin Production ................................................. 82
Screening IN937b Mutant for Rifampicin Resistance ....................................... 83 Root Treatment by IN937b Rifampicin-Resistance Mutant ............................... 83
Rhizosphere Sampling ..................................................................................... 84 Recovery of IN937b Mutant with Rifampicin-Resistance from Roots ............... 84
Results .................................................................................................................... 85 Architecture of Pellicles and Colonies of Bacillus spp. ..................................... 85 In Vitro Test for Biofilm formation ..................................................................... 86
Effect of Bacterial Density on Biofilm Formation ............................................... 86 PCR Detection of Lipopeptide Genes in Bacillus PGPR Strains in Relation
to Biofilm Formation ...................................................................................... 87 HPLC/MS Analysis of Surfactins Production in B. subtilis IN937b .................... 88
Survival of IN937b in the Rhizosphere ............................................................. 88 Survival of IN937b in Squash Roots ................................................................. 89
Discussion .............................................................................................................. 89
4 THE ROLE OF SUBTILOSIN A-LIKE ANTIMICROBIAL PEPTIDE IN SUPPRESSION OF PHYTOPHTHORA CAPSICI BY BACILLUS SUBTILIS IN937B .................................................................................................................. 110
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Introduction ........................................................................................................... 110 Materials and methods.......................................................................................... 113
Dual Culture Assay ......................................................................................... 113
Antibiotic Extraction and Analysis ................................................................... 113 Antifungal Activity of Methanol Extracts Against Phytophthora capsici .......... 114
Detached Leaf Assays ................................................................................... 115 HPLC/MS Assays of Surfactin Production ...................................................... 116 DNA Extraction and PCR Analysis ................................................................. 116
Results .................................................................................................................. 117 Dual Culture Assays ....................................................................................... 117 Methanol Extracts of IN937b Culture .............................................................. 117 Chromatographic Separation by HPLC .......................................................... 117 PCR Detection of sbo Genes in Bacillus PGPR IN937b ................................. 118
Assays of Hyphal Growth Inhibition ................................................................ 118 Effect of Methanol Extracts on Mycelium Morphology .................................... 119
The EC50 of the Extracts ................................................................................. 119
Effect of Methanol Extracts on Zoospore Germination ................................... 119 Effect of Methanol Extracts on Sporangial Formation .................................... 119 Effect of Methanol Extracts on Detached Leaf Infection ................................. 120
Discussion ............................................................................................................ 120 Hyphal Growth Inhibition ................................................................................ 121
Zoospore Germination and Sporangial Formation .......................................... 122 Detached Leaf Assays ................................................................................... 122 Identification of Subtilosin A ........................................................................... 123
5 ELICITATION OF PLANT SYSTEMIC DEFENSE BY BACILLUS PGPR STRAINS .............................................................................................................. 135
Introduction ........................................................................................................... 135 Materials and Methods.......................................................................................... 139
Inoculum Preparation and Inoculation ............................................................ 139 Application of Bacillus PGPR Treatment in the Greenhouse .......................... 139
Split-Root Assay ............................................................................................. 140
Enzyme Activity Assays.................................................................................. 140 Enzyme Activity Assays.................................................................................. 141
Results .................................................................................................................. 141 Induction of Systemic Resistance by PGPR with P. capsici Challenge .......... 141
Increased Activities of PO and PAL Induced Locally by PGPR Treatment with P. capsici Challenge ............................................................................ 142
Systemically Induced Enzyme Activity as Determined by Split-Root System without P. capsici Challenge ....................................................................... 143
Increased PAL and PO Activities Systemically in Response to P. capsici in Different Seedling Stages............................................................................ 143
Discussion ............................................................................................................ 144 Defense-Related Enzymes ............................................................................. 144 Locally Induced Enzyme Activity .................................................................... 146 Priming: Getting Ready for Pathogen Invasion ............................................... 147
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6 SUMMARIES AND FUTURE PERSPECTIVES .................................................... 158
Summaries ............................................................................................................ 158 Future Perspectives .............................................................................................. 160
LIST OF REFERENCES ............................................................................................. 163
BIOGRAPHICAL SKETCH .......................................................................................... 187
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LIST OF TABLES
Table page 1-1 Bacillus-based biofungicides on vegetables ....................................................... 35
2-1 Relevant information for PGPR strains tested in this study ................................ 58
2-2 Disease development in squash inoculated with different isolates of P. capsici ................................................................................................................ 59
2-3 Effect of the concentration of P. capsici isolate 151 on disease development .... 60
2-4 Efficacy of PGPR (1-8 PGPR strains) on suppression of Phytophthora crown and root rot under greenhouse conditions .......................................................... 61
2-5 Efficacy of PGPR (9-16 PGPR strains) on suppression of Phytophthora crown and root rot under greenhouse conditions................................................ 62
2-6 Effect of PGPR on plant growth under greenhouse conditions ........................... 63
2-7 Effect of IN937b concentration on crown and root rot disease ........................... 64
2-8 Effect of SE76 concentration on crown and root rot disease .............................. 65
2-9 Effect of IN937a concentration on crown and root rot disease ........................... 66
2-10 Effect of Bacillus spp. application timing (stage 1: seed) on crown and root rot disease .......................................................................................................... 67
2-11 Effect of Bacillus spp. application timing (stage 2: 10 days after emergence) on crown and root rot disease ............................................................................ 68
2-12 Effect of Bacillus spp. application timing (Stage 3: 17 days after emergence) on crown and root rot disease ............................................................................ 69
2-13 Effect of PGPR on chlorophyll content in squash leaves under greenhouse conditions ........................................................................................................... 70
3-1 List of primer sequences used for biosynthetic genes of surfactins, bacillomycin, iturin A, Fengycin, and Zwittermicin A. .......................................... 96
3-2 Biofilm formation by Bacillus strains with initial bacterial populations ................. 96
3-3 Detection of the genes related to biofilm formation ............................................. 96
3-4 Identification of surfactin ions by HPLC/ESI-MS ................................................. 97
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3-5 HPLC/(+)ESI-MS peak areas and relative areas for the detected surfactins in the crude extracts of IN937b............................................................................... 97
4-1 Antagonism of Bacillus PGPR strains against P. capsici on PDA plates .......... 125
4-2 Effect of Methanol Extracts of IN937b on hyphal growth of P. capsici .............. 126
4-3 Effect of Methanol Extracts of IN937b in Detached Leaf assays ...................... 127
5-1 Effect of Bacillus PGPR on number and size of lesions in the detached leave assay under the lab conditions ......................................................................... 149
5-2 Effect of different concentrations of IN937b on the number and size of lesions in the detached leave assay under the lab conditions ...................................... 149
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LIST OF FIGURES
Figure page 2-1 Regression analysis of the concentration of P. capsici isolate 151 on disease
development ....................................................................................................... 71
2-2 Regression analysis of Effect of PGPR strains concentration on crown and root rot disease ................................................................................................... 72
2-3 Effect of IN937b on squash root and shoot dry weight. ...................................... 73
2-4 Chlorophyll content in leaves of squash plants treated with PGPR. ................... 74
3-1 Architecture of pellicles and colony morphology of PGPR strains. ..................... 98
3-2 Pellicle development of Bacillus strains. ............................................................. 99
3-3 Biofilm development properties of Bacillus strains. ............................................. 99
3-4 Biofilm formation rate of Bacillus strains. .......................................................... 100
3-5 HPLC/MS analysis of the fraction from crude extraction from B. subtilis IN937b culture. ................................................................................................. 101
3-6 Colonization of the rhizosphere of squash plants by a mutant of IN937b with rifampicin resistance. ........................................................................................ 105
3-7 Colonization of the squash roots by a mutant of IN937b with rifampicin resistance ......................................................................................................... 106
3-8 PCR product profiles of the sfp gene ................................................................ 107
3-9 Comparison of the predicted amino acid sequence for the sfp gene present in several Bacillus strains. .................................................................................... 109
3-10 Phylogram of sfp homologs from several Bacillus strains ................................. 109
4-1 Antagonism of Bacillus spp. against P. capsici on PDA plates ......................... 128
4-2 Antagonism of methanol extracts of IN937b against P. capsici on CM agar plates ................................................................................................................ 129
4-3 Effect of methanol extracts of IN937b on morphology of P. capsici .................. 130
4-4 Effect of methanol extract concentrations of IN937b on zoospore germination 131
4-5 Effect of methanol extracts of IN937b on sporangial formationmg/mL) ............ 132
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4-6 Effect of methanol extracts on detached leaf infection ..................................... 134
5-1 Induction of PAL by PGPR in squash roots ...................................................... 150
5-2 Induction of PO by PGPR in squash roots ........................................................ 151
5-3 Induction of PAL by PGPR in roots against P. capsici ...................................... 152
5-4 Induction of PO by PGPR in roots against P. capsici ....................................... 153
5-5 Induction of PAL by IN937b in split root system ............................................... 154
5-6 Induction of PO by IN937b in split root system ................................................. 155
5-7 Induction of PAL by PGPR against P. capsici in different growth stages .......... 156
5-8 Induction of PO by PGPR against P. capsici in different growth stages ........... 157
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LIST OF ABBREVIATIONS
ASM Acibenzolar-S-methyl
BCA Biological control agent
CFU Colony forming units
ET Ethylene
ETI Effector-triggered immunity
HR Hypersensitive response
ISR Induced systemic resistance
JA Jasmonic acid
LPS Lipopolysaccharides
MAMP Microbe associated molecular pattern
PAL Phenylalanine ammonia lyase
PCR Polymerase chain reaction
PGPR Plant growth-promoting rhizobacteria
PO Peroxidase
PR Pathogenesis related
PTI PAMP-triggered immunity
R protein Resistance protein
SA Salicylic acid
SAR Systemic acquired resistance
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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
INTERACTIONS BETWEEN PLANT GROWTH-PROMOTING RHIZOBACTERIA
(PGPR) AND SQUASH PLANTS – THE ROLE OF PGPR-MEDIATED SUPPRESSION OF PHYTOPHTHORA BLIGHT
By
Xiaodan Mo
December 2013
Chair: Shouan Zhang Major: Plant Pathology
Phytophthora blight is a devastating disease in vegetable production worldwide.
This disease is caused by the soil-borne Oomycete Phytophthora capsici. The objective
of this study was to investigate the suppression of Phytophthora blight on squash and
plant defense induced by plant growth-promoting rhizobacteria (PGPR). The biocontrol
efficacy of 16 Bacillus PGPR strains to reduce disease severity was evaluated under
greenhouse conditions. A rifampicin-resistant mutant of Bacillus PGPR strain IN937b
was obtained and used to monitor its colonization on squash roots. The underlying
mechanisms of disease suppression by PGPR were determined by analyses of
secondary metabolites involving P.capsici inhibition and induced systemic resistance on
squash.
In this study, Bacillus PGPR strain IN937b were consistent in reducing
Phytophthora blight on squash when applied at the concentration of 108 CFU/mL and on
older plants, i.e. the first PGPR application was made at 17 days after emergence. The
growth promotion effect of IN937b only occurred under conditions without fertilization.
IN937b was capable of producing surfactins and forming biofilms. IN937b colonized the
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rhizosphere of squash and also occupied the internal roots. At the early stage of
colonization, the population of IN937b remained relatively high in the rhizosphere. The
bacterial population in the rhizosphere decreased 14 days after bacterization, while
bacterial cells in root tissues survived longer and accumulated gradually. IN937b
showed in vitro inhibition activities against P. capsici. Subtilosin A, an antimicrobial
peptide produced by B. subtilis IN937b was found to be involved in reduction in
zoospore germination, sporangial formation and hyphal growth of P. capsici, and the
suppression of lesion expansion on the detached leaves after being challenged with P.
capsici. Greater levels of Peroxidase and phenylalanine ammonia-lyase induced by
IN937b may have contributed collectively to induced resistance - a plant innate
resistance in squash plants against P. capsici, which eventually resulted in lower
disease severity compared to the non-treated control. This study provides future
researchers with insights into understanding biocontrol mode of action that can be used
to develop effective biocontrol agents and to manipulate rhizosphere ecosystems for
improved crop production.
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CHAPTER 1 LITERATURE REVIEW
Summer Squash
Summer squash (Cucurbita pepo L.), originating from the Americas, is one of
widely grown and consumed vegetable crops in the world. As a member of the highly
diverse species in the gourd family Cucurbitaceae, is closely related to both muskmelon
(Cucumis melo L.) and cucumber (Cucumis sativus L.) (Paris, 2008). The initial
domestication of squash occurred in Mexico and Central America about 10,000 years
ago, where it spread quickly to Asia, Europe, and Africa, and now it is distributed
throughout the world (Smith, 1997).
Summer squash can be used in a wide variety of dishes both as fresh and
processing products. The word “squash” comes from a Massachusetts Native American
word “askutasquash”, meaning eaten raw or uncooked (Kemble et al., 2005). The fruits,
seeds, flowers, shoots and roots of squash are eatable. Consumption of summer
squash increases every year (Locke et al., 2009). Summer squash is used as folk
medicine because of its nutritional benefits. Since squash contains calcium,
magnesium, potassium, vitamins B6 and E, the fruit may contribute to health of the
heart, lungs and bones. Squash is also a good source of fiber and protein with a low
sugar and sodium concentrations (NASS, 2011).
According to the Food and Agriculture Organization of the United Nations (FAO),
more than 1.8 million hectares (ha) of pumpkin, squash, and gourds are harvested
annually with a total yield of 24 million tons in 2010 (FAO, 2011). As an easy to grow,
short season crop, summer squash is adapted to temperate and subtropical climates
and is grown in many regions (Paris, 1996). Today, the largest producers of summer
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squash include China, India, Russia, Iran, USA, Egypt and Mexico (NASS, 2012).
According to the United States Department of Agriculture (USDA), the United States
grew 0.38 million tons of squash for fresh market valued at 248.7 million US dollars in
2012 (NASS, 2013). During 2011-2012, squash growers planted 18,210 ha for fresh
market in the United States. The average harvested yield of summer squash is 19.3
tons/ha. The cultivation of squash can be found in a wide latitudinal range from the
North (New York) to the South (Florida).
Florida ranks first nationally for the area harvested and value of production on
squash processing and fresh market. It is one of the few crops that is shipped every
month of the year throughout the United States (Mossler and Nesheim, 2001).The
growing acreage was 4,047 ha and the value of the production reached 66 million US
dollars in 2012 (NASS, 2012). In 2006, 60% of the squash acreage was in Miami-Dade
County (Li et al., 2006). In the Homestead area, where the majority of the state's squash
is produced, squash is grown from August to the following April (Mossler and Nesheim,
2001).
Squash grows best under conditions of high humidity, and temperature between
18 and 24 ℃, and properly fertilized soil. Squash plants are largely direct seeded in
Florida (Hochmuth and Hanlon, 2010). Sandy loam soil is suitable for squash due to the
shallow root system of this crop (Li et al., 2006). The water requirement for squash of
approximately one inch of water per week, is slightly lower than that for other vegetable
crops requiring (Ertek et al., 2004). Due to hot and dry weather in the winter, more than
90% of squash-producing farms are irrigated to increase production (Mossler and
Nesheim, 2001). Under favorable environmental and fertilized conditions, the squash
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plants grow rapidly and produce fruits continuously. Squash requires more phosphorus
and potassium than nitrogen (Ozoreshampton et al., 1994). Timely and appropriate
application of fertilizers can make a significant difference in the quality and quantity of
fruits, and may promote early fruit maturation, allowing the fruit to be harvest early.
In Florida, common diseases of squash in the greenhouse and field include
pathogens Fusarium spp., Rhizoctonia spp., Phytophthora spp., and Pythium spp
(Zhang et al., 2007). Diseases cause substantial losses of the yield, up to 20% annually
and also decrease the fruit quality (Agrios, 2005). Some diseases occur infrequently
such as Phytophthora blight (Phytophthora capsici), downy mildew
(Pseudoperonospora cubensis), powdery mildew (Podosphaera xanthii), angular leaf
spot (Pseudomonas syringae pv. lachrymans), gummy stem blight (Mycosphaerella
citrullina) and Alternaria leaf spot (Alternaria cucumerina) (Zhang et al., 2007).
However, when the weather is favorable for disease development, these diseases may
cause up to 100% yield loss (Babadoost, 2000).
Squash Crown and Root Rot Disease Caused by Phytophthora capsici
Phytophthora blight is a devastating disease of vegetable crops in Florida (Ploetz
et al., 2002). This disease is caused by the soil-borne Oomycete Phytophthora capsici.
Phytophthora capsici was first described on chili pepper at New Mexico in 1922
(Leonian, 1922). Phytophthora capsici which is widespread throughout the United
States has been reported to infect a wide range of plant taxa involving a total of more
than 50 species, including major vegetable crops such as cucurbits, peppers, eggplants,
tomatoes and snap beans (Granke et al., 2012). In recent years, vegetable producers in
eastern U.S. have experienced severe losses of vegetable crops due to P. capsici
infection (Hausbeck and Lamour, 2004).
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Summer squash is highly susceptible to P. capsici. Diseased plants are easily
pulled from the soil due to the rotted roots system (Padley et al., 2008). Water-soaked
lesions on the stem result in girdling and plant death because water and nutrient
transport are reduced (Gevens et al., 2008). Affected fruit can be completely covered by
sporangia causing fruit to rots, and can occur after harvest during shipment (Ando et al.,
2009).
Phytophthora capsici releases zoospores from sporangia, or sporangia
germinate directly to initiate infection on squash. The zoospores swim through water in
the soil and are chemotactically attracted by the exudates released by the host roots
(Granke and Hausbeck, 2010). After the zoospores have adhered to the root surface,
they encyst and produce a precisely oriented germ tube that grows into adjacent host
plant tissue (Raftoyannis and Dick, 2006). The development of zoospores, cysts, and
germ tubes is triggered by chemical signals in the environment, some which are
produced by the plant roots (Bishop-Hurley et al., 2002).
Phytophthora capsici has caused longstanding problems for agriculture. In the
field, the disease can spread rapidly in the presence of heavy rainfall and excessive
irrigation water. When surface moisture is present, zoospores landing on host plants
can invade plant tissues through natural openings such as stomata and hydathodes
(gas or water pores, respectively) (Granke and Hausbeck, 2010). The sexual spore of
P. capsici is the oospore. The oospore’s thick walls help it survive unfavorable periods
in the soil. It can also be the overwintering propagule in the soil and the primary source
of inoculum (Granke et al., 2009). Dissemination may also occur by contaminated
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equipment and through movement of plant debris or other hosts (Lamour and
Hausbeck, 2000).
Practices for managing of soil-borne pathogens include crop rotation, fungicide
applications, and the use of resistant or tolerant varieties. Field production of vegetable
crops in Florida is based on high-input, intensively managed production systems that
utilize a broad spectrum fumigants such as methyl bromide to manage soil-borne pests
(French-Monar et al., 2007). The fumigant methyl bromide has been used extensively to
control soil-borne pathogens, and is effective against the mycelia and the long-term
persistent oospores of P. capsici in the soil (Zhang et al., 2010). However, agricultural
emissions of methyl bromide is a significant cause of ozone depletion (Portmann et al.,
2012). Alternatives to methyl bromide either are not as effective as methyl bromide or
not economically practical, since squash is not a high value crop (Zhang et al., 2010).
With the phase-out of methyl bromide, the ban on this fumigant could potentially cost
about 1.5 billion US dollars in lost crop production annually in the U. S. (Sande et al.,
2011).
Crop rotation is an important component of integrated disease management;
however, the long-term survival of P. capsici oospores, even in the absence of a host,
limits the effectiveness of this strategy. Crop rotation is needed for more than 3 years to
avoid increasing inoculum levels (Hausbeck and Lamour, 2004). In Florida, the
oospores survive in the soil up to 5 years. It is very difficult to eliminate the oospores
from an infested field (French-Monar et al., 2007).
There are some fungicides registered for use on cucurbits, but none are highly
effective against P. capsici under hot and humid conditions of South Florida (Mossler
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and Nesheim, 2001). Among more than 50 fungicides tested, only six were effective for
controlling P. capsici (Babadoost et al., 2008). No single fungicide has consistently and
effectively suppressed losses caused by P. capsici. More importantly, P. capsici
developed resistance to some fungicides used for Phytophthora blight control. For
example, Phytophthora blight often has been managed with application of fungicides
containing the active ingredient mefenoxam. Since the mefenoxam-resistant isolates
have developed, the usefulness of fungicides containing mefenoxam in the field trials
has diminished (Lamour and Hausbeck, 2000).
Plant breeding for resistance is considered an economically viable control
method for Phytophthora blight. Commercial cucurbit varieties vary with respect to their
Phytophthora blight resistance, but highly resistant varieties with ideal horticultural traits
are still not available (Babadoost et al., 2008). Phytophthora capsici is a heterothallic
species with two mating types, designated as A1 and A2, which interact to produce
sexual oospores. The presence of both mating types in the same area provides more
potentially virulent phenotypes of the pathogen through sexual recombination. Variation
in genetics and virulence among isolates of P. capsici lead to costly and time-
consuming breeding endeavors (Granke et al., 2012).
At present, there is no single method which can provide adequate control of P.
capsici. Since the pathogen is difficult to eradicate, induction of plant resistance against
pathogen is considered as a potential method in reduction and control of the disease
(Shoresh et al., 2010). Plants interact continuously with biotic factors (mycorrhiza,
endophytes, insects, nematodes, plant growth-promoting rhizobacteria, and other root-
associated microbes) in the field, which might lead to the inhibit the growth of plant
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pathogens (Ghorbani et al., 2008), known as biological control or biocontrol. Biocontrol
can be developed as a component for sustainably integrated disease management with
reduced adverse impact to the environment.
Plant Growth-Promoting Rhizobacteria
Rhizosphere
The term, “rhizosphere” comes from rhizo or rhiza, a Greek word for root, and
“sphere” which means an environment or area influenced by the root (Morgan et al.,
2005). In the rhizosphere, plant roots can interact with soil microorganisms, and soil
microorganisms can, in turn, affect plant growth and health during the entire growth
season (Hartmann et al., 2008).
The rhizosphere is the location where beneficial microorganisms and soil-borne
pathogens compete to establish a relationship with the plant (Raaijmakers et al.,
2009a). Microorganisms compete with each other to acquire nutrients to grow and
multiply (Lugtenberg and Kamilova, 2009). During these processes, metabolic
processes of microorganisms result in rapid nutrient cycles and movement of
microorganisms in the soil (Raaijmakers et al., 2009a). Some beneficial rhizosphere
microorganisms can reduce population densities and activities of soil-borne pathogens
by competition, antagonism, and induced plant defense (Weller et al., 2002). Beneficial
microorganisms such as plant growth-promoting rhizobacteria (PGPR), positively affect
different crops including rice, pepper, tomato, tobacco, and bean (Kloepper and
Schroth, 1978). PGPR are a group of free-living rhizospheric bacteria that enhance
plant health. PGPR can promote plant health not only directly by facilitating mineral
nutrient uptake or phytohormone production, but also indirectly by protecting plants
against pathogens (Persello-Cartieaux et al., 2003; Zahir et al., 2004). There have been
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many reports that PGPR prevent diseases caused by Oomycetes and fungal pathogens
including root diseases (damping-off of tomato), foliar diseases (powdery mildews on
cucurbit and strawberry), and postharvest diseases (green, grey and blue molds)
(Kloepper et al., 2004).
PGPR as Biocontrol Agents (BCAs) on Vegetable Crops
PGPR isolated from different plant species belong to many genera including
Pseudomonas, Rhizobia, and Bacillus (Lugtenberg and Kamilova, 2009). Multiple
Bacillus and Paenibacillus spp. can be readily cultured from both bulk (free of root) and
rhizosphere (influenced by root) soils. The population of PGPR is affected by plant
species and root exudates in the rhizosphere. Plant roots release organic compounds
such as amino acids and sugars into the rhizosphere (Lugtenberg and Kamilova, 2009),
which have a positive effect on the population of many PGPR. The plant rhizosphere is
the preferred ecological niche for PGPR. Due to nutrient availability in the rhizosphere,
there are about 2.5 to 1,260 times more microorganisms in the rhizosphere than the
bulk soil (Rouatt et al., 1960). The population density of Bacillus spp. in the rhizosphere
generally range from 103 to 107 cells per gram of rhizosphere soil (Pandey and Palni,
1997).
PGPR are effective in controlling plant diseases caused either by soil-borne,
foliar or post-harvest pathogens (Raupach and Kloepper, 1998b; Kloepper et al., 2004).
PGPR were isolated from different crops (tomato, pepper, cucumber, etc.) based on
their multiple plant growth promoting and antifungal capabilities. These PGPR isolates
are well adapted when they are introduced to particular rhizospheric soil environment
(vanVeen et al., 1997). The target pathogens include soil-borne fungal pathogens
including Phytophthora, Fusarium, and Rhizoctonia, as well as foliar diseases caused
24
by bacterial pathogens Xanthomonas and Ralstonia. Several commercial PGPR
products are currently available for use on vegetables and new products are constantly
being developed and commercialized in the market (Kumar et al., 2011b). The details of
commonly used commercial PGPR products for managing important diseases of
vegetables are listed in Table 1-1.
PGPR as biocontrol agents have the following advantages: (1) PGPR which were
isolated from the rhizosphere or plants are environmentally friendly and nontoxic
(Beneduzi et al., 2012), (2) PGPR utilize root exudates leading to their rapid growth and
mass production (Lugtenberg and Kamilova, 2009), (3) PGPR adapt to the environment
by colonizing and multiplying in the rhizosphere and interior of the plant (Compant et al.,
2010), and (4) PGPR undertake a defined range of functions including antibiosis, growth
promotion, and induced systemic resistance (Compant et al., 2005).
The success of biocontrol agents mainly depends on the existing seed, soil, or
foliar application technologies that help integrate PGPR into the cropping system. Seed
inoculation with the P. fluorescens isolate CW2 positively affects tomato growth and
protects seedlings from damping-off disease caused by Pythium ultimum (Salman and
Abuamsha, 2012). A foliar spray of P. fluorescens Pf1, Py15, and B. subtilis Bs16
significantly reduced the incidence of early blight caused by Alternaria solani (Latha et
al., 2009). The most commonly used application method for PGPR is as a soil drench.
Soil application of Bacillus subtilis QST713 consistently reduced infection by
Plasmodiophora brassicae on canola roots. Antibiosis and induced resistance are
involved in clubroot suppression by QST713 (Lahlali et al., 2013). The PGPR strains
25
aggressively colonized plant roots, thus helping to inhibit pathogens in the rhizosphere
by producing antibiotics and inducing host resistance.
The dosage of the BCAs significantly influence the biocontrol efficacy through
their effect on establishment of pathogens and the development of diseases. To achieve
a beneficial effect on a target plant, PGPR have to be implemented into the field in a
timely manner and in high numbers. In the field, a cell concentration at 109 CFU per mL
of Pseudomonas brassicacearum MA250 was required for effective biocontrol of
Fusarium spp. on winter cereals (Levenfors et al., 2008). The implemented PGPR strain
had to reach a threshold population density in order to effectively suppress pathogens in
the rhizosphere. For some PGPR strains, the maximum root colonization was less than
106 CFU per gram of root in the nutrient-rich environment (Ahmad et al., 2011).
Pseudomonas putida strain WCS358 reached a threshold population density of
approximately 105 CFU per gram of root, which was required for a significant
suppression of Fusarium wilt on radish (Raaijmakers et al., 1995b).
Host age influences the effectiveness of PGPR against pathogens. Plants vary in
their susceptibility to certain pathogens due to their age or stage of development, known
as age-related resistance (Garcia-Ruiz and Murphy, 2001). In general, young plants are
susceptible to pathogens while adult plants have a certain degree of resistance. Age-
related resistance to P. capsici is found in cucumber. Immature fruits are more
susceptible to P. capsici than mature fruits (Meyer and Hausbeck, 2013). Plant age also
affects PGPR populations through a modification in the root exudation (Roesti et al.,
2006). At a specific age of the host plant and the appropriate timing of PGPR
application, a plant may respond to PGPR and pathogens by activating its resistance
26
locally and systemically (Katagiri and Tsuda, 2010b). Some proteins and metabolites
form during this particular period. A resistance protein (R protein) - mediated recognition
of P. syringae is involved in age-related resistance in Arabidopsis (Kus et al., 2002).
Salicylic acid (SA) signaling was involved in age-related resistance of Nicotiana
benthamiana against P. infestans (Shibata et al., 2010). Young and susceptible plants
failed to induce the salicylic acid (SA) - mediated signaling pathway. Pretreating young
plants with acibenzolar-S-methyl (ASM, Actigard®, Syngenta Crop Protection,
Greensboro, NC, USA) or SA analogs induced resistance to P. infestans.
Root Colonization of PGPR
The first step in all interactions between plants and PGPR is bacterial
colonization of plant root systems. The steps of colonization include recognition,
adherence, invasion, occupation and multiplication, and signals to establish the
interactions (Compant et al., 2010). Effective root colonization by PGPR depends on
their ability to survive and proliferate in the presence of indigenous microorganisms over
a period of time (Maheshwari, 2011). For example, P. fluorescens 2112 consistently
lasts for 18 weeks in the rhizosphere of pea (Kim et al., 2012).
Chemotaxis is one way that plants attract PGPR to the rhizosphere through the
release of carbohydrates and amino acids (Somers et al., 2004). The major chemo-
attractants in tomato root exudates for P. fluorescens WCS365 were identified as
leucine and dicarboxylic acids (Lugtenberg et al., 1999). Another organic acid, malate,
appeared to be the major chemo-attractant for B. subtilis FB17 in root exudates of
Arabidopsis thaliana (Rudrappa et al., 2008). Biocontrol agents may be distributed in
the specific crop or the different niches of the hosts (Beneduzi et al., 2008). For
example, B. amyloliquefaciens FZB42 primarily colonized in root hair and tips on
27
Arabidopsis. However, when FZB42 was applied to maize roots, the bacterial cells
concentrated at the base of primary roots indicating that FZB42 colonized plant roots in
different niches (Fan et al., 2011).
Some bacteria present in the rhizosphere are capable of entering the plants as
endophytes that do not cause harm and can establish a mutualistic association with
plants. Endophytic bacteria have been isolated from a wide range of plant species. In
general, endophytic bacteria occur at lower population densities than rhizospheric
bacteria (Compant et al., 2010). The population density of endophytes is greatly
variable, depending mainly on the bacterial species and genotypes of the host plant,
also influences by the developmental stage of the host, inoculum density, and
environmental conditions (Sturz et al., 2005).
Populations of PGPR usually develop and form biofilms in their natural
environments (Seneviratne et al., 2010). Biofilms are a group of highly structured cells
capable of sticking together on the surface of liquid or solid substances in a self-
produced extracellular matrix (Costerton, 1995). Biofilms change the growth rate of cells
and transform from free-living cell to sessile biofilm cells through nutrient limitation
(Morikawa, 2006). Biofilm formation is important for PGPR to act as a biocontrol agent
against plant pathogens and to respond quickly to environmental conditions
(Seneviratne et al., 2010). Paenibacillus polymyxa protected Arabidopsis thaliana
against Phytophthora palmivora and Pythium aphanidermatum by establishing biofilm
on plant roots to protect the colonization sites such as root tips. Competition for
nutritional elements in the root exudate, biofilm-forming PGPR act as a sink for the
nutrients can inhibit pathogen growth in the rhizosphere (Timmusk et al., 2005). In
28
addition, biofilm-forming PGPR can produce a variety of antimicrobial metabolites which
include broad spectrum lipopeptides such as surfactins that are potent biosurfactants for
maintaining structure of biofilms and are important for suppressing pathogens (Bais et
al., 2004b).
Although beneficial aspects of PGPR have been well documented, there are
some problems about variability in colonization efficiency, field performance, and
rhizosphere competence (Babalola, 2010). In the field, PGPR behave unpredictably in
different environments (Spadaro and Gullino, 2005). Variability in rhizosphere
colonization has been proposed to be a major obstacle to agronomic application of
promising PGPR strains (Cooper and Rao, 2006). Research into the mechanisms of
action has provided a comprehensive understanding of PGPR as biocontrol agents.
Research on the mechanism that is responsible for how the PGPR interact with the
hosts and pathogens would help to improve the efficacy of growth promotion and
biocontrol. Detailed studies of the whole system need to be undertaken in order to
reduce the variability of PGPR performance (Compant et al., 2005).
Mode of Action of Biocontrol Agents
The process of biocontrol is the action of suppressing pathogens by using PGPR
with a variety of mechanisms such as antagonism mediated by metabolites, competition
for nutrients and niches in the rhizosphere, and/or activation of plant defenses. Most of
successful commercial BCAs are based on the thorough knowledge of their modes of
action, not only under laboratory conditions, but also in greenhouse and field situations.
Antagonism Mediated by Metabolites
Antagonism is usually mediated by the production of secondary antimicrobial
metabolites (antibiosis), lytic enzymes, and/or effectors (Raaijmakers et al., 2009a). In
29
the case of PGPR, antibiotics are secondary metabolites with low-molecular weight that
are toxic to other microorganisms (Mannanov and Sattarova, 2001). The metabolites
released by PGPR are involved in directly inhibiting plant pathogens locally before they
reach the plant (Kessler, 2011). Only a small fraction of pathogen populations is
exposed to the antimicrobial compounds in the rhizosphere during a short period of its
life cycle (Raaijmakers et al., 2009b). Therefore, resistance in pathogens to
antimicrobial compounds produced by antagonistic PGPR is presumed not to develop,
or at least develops relatively slowly compared to resistance to chemicals in pathogen
populations (Ishii, 2006).
Pseudomonas and Bacillus species are known to produce antibiotics that are
inhibitory to the growth and/or activities of fungal pathogens of plants. Well-known and
characterized compounds produced by Pseudomonas are phenazine-1-carboxylic acid
(PCA) and 2, 4-diacetylphloroglucinol (DAPG), while cyclic lipopeptides surfactants,
zwittermycin A, and bacteriocins can be produced by Bacillus (Hayat et al., 2010). To
demonstrate the role of antibiotics in biocontrol, the mutants defective in structural
genes for synthesis of the antibiotic must be negative in biocontrol. In a previous study,
knock-out mutants of lipopeptides surfactin genes were severely defective in biofilm
formation in vitro and in colonization of tomato roots. At the same time, the mutant
decreased the efficacy of biocontrol against Ralstonia solanacearum on tomato under
greenhouse conditions (Chen et al., 2013). DAPG is a primary factor contributing to
biological control of plant disease by P. fluorescens strains CHA0 and several
Pseudomonas strains. The 2, 4-DAPG biosynthetic and regulatory mutants
demonstrated that DAPG has a broad spectrum of antifungal activities that inhibit
30
pathogens directly, and also induce systemic resistance in the host plants (Weller et al.,
2012).
Competition for Nutrient and Niches
An introduced PGPR strain can be defined as an effective root colonizer if it is
able to propagate and survive in the rhizosphere for several weeks. PGPR face the
challenge of carbon starvation in most soils (Knee et al., 2001). Competition for different
carbon sources generally occurs in the soil between the introduced PGPR strains and
the native inhabitants in the rhizosphere (Nihorimbere et al., 2011). For this purpose,
PGPR directly inhibited pathogens under highly competitive conditions (Cunniffe and
Gilligan, 2011). For example, the lack of carbon sources is assumed to inhibit fungal
spore germination in the soil providing a broad spectrum of protection on plants (Tang
et al., 2010).
PGPR can compete with pathogens for niches when they colonize the same host
tissues and develop in different root cortical cells, indicating some sort of competition for
space (Ongena and Jacques, 2008). A number of bacterial functions such as motility,
attachment, growth, stress resistance and the production of secondary metabolites have
been associated with competence for space occupation in the rhizosphere (Lugtenberg
and Kamilova, 2009).
Defense Mechanisms in Plants
Plants possess morphological barriers, secondary metabolites, and antimicrobial
proteins that prevent or overcome a pathogen’s ability invading a plant (van Loon et al.,
2006). Plants possess different strategies to recognize and counteract pathogen
attacks. As a first line of defense, the plant cell surface contains pattern recognition
receptors (PRRs) that recognize potential pathogens through flagella, outer membrane
31
lipopolysaccharides (LPS), and other cell wall or secreted components (Zipfel, 2008). In
addition to flagella and LPS, there are various bacterial compounds that can be
recognized by the plant, including N-acyl-homoserine lactones, biosurfactants,
siderophores, and the antibiotics DAPG and pyocyanin (Doornbos et al., 2012).
General elicitors that are designated as pathogen-associated molecular patterns
(PAMPs) are isolated from pathogens (Boller and He, 2009). Non-pathogenic
microorganism elicitors are designated as one of microbe-associated molecular patterns
(MAMPs), which are recognized in a similar way (Bittel and Robatzek, 2007). The
perception of general elicitors triggers a broad array of reactions, known as basal
resistance or PAMP-Triggered Immunity (PTI). However, some successful pathogenic
microorganisms may overcome basal resistance by inhibiting signaling pathways, thus
suppressing this first type of immunity (Zamioudis and Pieterse, 2012). In response,
plants have evolved a second line of defense through specific disease resistance (R)
genes, the so-called effector triggered immunity (ETI) (Katagiri and Tsuda, 2010a). R
gene-mediated resistance is expressed through similar defense responses as those that
are active in basal resistance, but on a much greater scale (Henry et al., 2012).
Therefore, PTI and ETI are considered as primary and secondary innate immunity,
respectively.
The phenomenon of induced resistance to pathogens in plants has been studied
intensively in recent years (Hammerschmidt, 1999). Induced resistance can be divided
into systemic acquired resistance (SAR) and induced systemic resistance (ISR). In
SAR, resistance can be activated by chemicals, avirulent pathogens, or attenuated
pathogens. In contrast to SAR, ISR is induced in roots by PGPR. During ISR, gene
32
expression is induced locally in roots or systemically in leaves upon colonization by
PGPR, and the treated plants are in primed conditions (i.e. defense genes respond
more rapidly and strongly after pathogen attack).
ISR Mediated by PGPR
To fully understand the specific mechanism of ISR, research on ISR in certain
crop species mediated by specific PGPR strains is required. Globally, local and
systemic defense responses triggered by microorganisms are controlled by a signaling
network in which the plant hormones salicylic acid (SA), jasmonic acid (JA), and
ethylene (ET) play important roles (Henry et al., 2012).
Induction of SA-mediated responses has been demonstrated to occur in the
rhizosphere (Doornbos et al., 2012). Several phytohormones have been demonstrated
to negatively cross-communicate with the SA signaling pathway and affect the outcome
of the immune response (Verhagen et al., 2004; Pieterse et al., 2009). Local JA and ET
signaling play a role in the control of beneficial interactions (Lopez-Raez et al., 2010).
ET also appears to have a crucial role in regulating the progress of root colonization
(Choudhary and Johri, 2009). For example, a significant reduction in the expression of
genes encoding ET-related transcription factors has been reported for Arabidopsis roots
colonized by P. fluorescens WCS417 and FPT9601-T5, which supports the hypothesis
that PGPR modulate host immune response by interfering with the ET signaling
pathway (Verhagen et al., 2004).
Most pathogenicity-related (PR) proteins are induced through the action of the
signaling compounds and possess antimicrobial activities (van Loon et al., 2006). B.
cereus strain BS107 against Xanthomonas axonopodis pv. vesicatoria systemically
induced plant defense genes CaPR1 and PR4 in pepper leaves (Yang et al., 2009). In
33
tobacco (Nicotiana tabacum cv. Samsun NN), at least 16 PR-1-type genes appear to be
present after being induced upon TMV infection (Riviere et al., 2008). These findings
indicate that PR proteins classified in the same family on the basis of sequence
homology can have different properties and hence may differ substantially in biological
activity. Examples of PR proteins include phenylalanine ammonia-lyase (PAL) (Mariutto
et al., 2011), peroxidase (Sarwar et al., 2011), and polyphenoloxidase (Mayer, 2006).
They are generally present constitutively and only increased the expression level during
most infections.
Plants recognize certain ISR determinants to elicit ISR. Volatile organic
compounds and more particularly 2,3-butendiol were the determinants for elicitation
identified from Bacillus spp. (Ryu et al., 2003). Massetolide A produced by P.
fluorescens retains ISR-eliciting activity in tomato plants for the control of Phytophthora
infestans, the causal agent of late blight (Tran et al., 2007). The compound 2,4-
diacetylphloroglucinol (DAPG) may also act as an elicitor of systemic resistance in
some Pseudomonas strains (Bakker et al., 2003). Another class of compounds
emerged recently as ISR elicitors is biosurfactant such as rhamnolipids and
lipopeptides. Pure fengycins and surfactins from B. subtilis provided a significant
protective effect that is similar to the one induced by living cells of the producing strain.
Experiments conducted on bean and tomato showed that overexpression of both
surfactin and fengycin biosynthetic genes in B. subtilis strain 168 (the strain is impaired
in lipopeptide production) was associated with a significant increase in the potential of
the derivatives to induce resistance (Ongena et al., 2007).
34
B. subtilis mutant strain M1 was constructed with a deletion in a surfactin
synthase gene and thus deficient in surfactin production (Bais et al., 2004b). Bacillus
subtilis M1 was ineffective as a biocontrol agent against P. syringae in Arabidopsis and
also failed to form robust biofilms on either roots or inert surfaces (Bais et al., 2004b).
The understanding of the physiological and biological basis of these induced immunity
mechanisms has greatly advanced the development of novel strategies for disease
control (Henry et al., 2012). The importance of these considerations is expected to have
practical applications. PGPR have the ability to control pathogens through complex
processes of colonization and antibiotic production. Studies of the population dynamics
and physiology of PGPR can be important and useful for applying PGPR for disease
management in vegetable production.
The aim of this dissertation was to investigate PGPR strains for controlling
Phytophthora blight on squash. The first objective was to determine the effectiveness of
Bacillus PGPR strains in the biocontrol of crown and root rot disease on squash caused
by P. capsici in soilless potting mixes under the greenhouse conditions. The second
objective was to focus on understanding of the ecology of Bacillus PGPR and pathogen
to enhance the effective use of Bacillus PGPR in the suppression of plant diseases. The
third objective was to gain insights into the role of antifungal antibiotics in disease
suppression via antagonism. The fourth objective was to gain an understanding of
mechanism in induced resistance by monitoring the defense-related enzyme activities
changes on squash to impair pathogen progression locally and systemically.
35
Table 1-1. Bacillus-based biofungicides on vegetables
Brands® Bacillus species
Target pathogen
Crop Mode of action
Reference
Kodiak B. subtilis GB03
Colletotrichum orbiculare
Cucumber Antagonism ISR Plant growth promotion
(Raupach and Kloepper, 1998a)
Rhapsody B. subtilis QST 708
Alternaria cucumerina
Muskmelon
Antagonism (Keinath et al., 2007)
Serenade B. subtilis QST713
Pseudomonas syringae
Tomato Antagonism ISR Plant growth promotion
(Gilardi et al., 2010)
Sclerotinia sclerotiorum
Soybean (Zeng et al., 2012b)
Sonata
B. pumilus QST 2808
Phytophthora infestans, Fusarium spp., Pythium ultimum
Potato ISR (Gachango et al., 2012)
Subtilex B. subtilis MBI 600
Phoma valerianellae
Lettuce ISR (Schmitt et al., 2009)
Yield Shield B. pumilus GB34
Rhizoctonia Fusarium
Legumes (Bradley, 2008)
Companion B. subtilis GB03, B. lichenformis and B. megaterium
Fusarium oxysporum
Tomato Antagonism ISR Plant growth promotion
(Myresiotis et al., 2012)
36
CHAPTER 2 BIOLOGICAL MANAGEMENT OF PHYTOPHTHORA BLIGHT AND PLANT GROWTH PROMOTION BY SELECTED PGPR STRAINS UNDER GREENHOUSE CONDITIONS
Introduction
Squash (Cucurbita pepo L.), in the family Cucurbitaceae, is a widely grown and
consumed vegetable crop in the United States and many other countries as well. In
2012, growers in the United States harvested 340,060 tons of squash for fresh market
valued at $248.7 million (NASS, 2011). Ranked No. 1 nationally in terms of the acreage
harvested and the value of processing and fresh market production, squash became a
valuable vegetable crop for Florida growers. Squash is vulnerable to bacterial and
fungal pathogens. The most devastating squash disease is Phytophthora blight
(Babadoost, 2000), which can cause yield losses up to 100% in warm and humid
conditions (Hausbeck and Lamour, 2004). Phytophthora blight poses major challenges
to growers by reducing yield and adversely affecting the fruit quality.
Phytophthora blight is a soil-borne disease which commonly affects numerous
crops in the United States including all cucurbits, peppers, tomatoes, snap beans and
several weed species (Ploetz and Haynes, 2000). The pathogen that causes this
disease is the Oomycete Phytophthora capsici Leonian (Leonian, 1922), which
produces motile zoospores. When it rains, or irrigation water splashes in the field,
zoospores can be spread with water to reach healthy squash tissues (Granke et al.,
2012). Zoospores and sporangia can infect all parts of squash plants including leaves,
roots, stems and fruits (Hausbeck and Lamour, 2004). The affected plants quickly die in
a week due to break down of the root and/or stem systems.
Symptoms of Phytophthora blight are first visible as water-soaking lesions, which
then turn dark over time (Kousik et al., 2012). Phytophthora capsici is easy to identify on
37
the fruit or stem as white mass of mycelia, sporangiophores and sporangia on the
surface of the tissue (Roberts et al., 2005). This diploid Oomycete is heterothallic with
two different mating types to complete sexual cycle (Erwin and Ribeiro, 1996). When
both mating types A1 and A2 are found in the same field, the sexual propagule
oospores are produced. Oospores can tolerate adverse environments and survive in the
soil for up to five years (French-Monar et al., 2007). In the spring, oospores are the
primary source for initial infection in the field (Pavon et al., 2008). Therefore, controlling
this disease has become increasingly challenging because oospores can survive a long
time in the soil, even without host tissues. Oospores have the ability to infect all parts of
plants, and more aggressive types of this pathogen may potentially cause frequent
outbreaks (Sanogo and Ji, 2012). So far, there is no single strategy that provides
adequate protection against this disease.
In an effort to prevent or control Phytophthora blight, resistant cultivars remain
the most economical and long-term approach. Although genetic resistance was
identified from some cucurbit species, there is no cucurbit cultivar commercially
available with measurable resistance to P. capsici (Hausbeck and Lamour, 2004).
Mapping genes for resistance to Phytophthora blight is challenging, and breeding for
resistant cucurbits is complex due to continuous changes in the genetics of P. capsici
(Babadoost et al., 2008).
In Florida, intensively managed production systems for vegetable production rely
on methyl bromide, a fumigant commonly used to manage soil-borne pests (French-
Monar et al., 2007). With the impending phase-out of methyl bromide (Noling and
Becker, 1994), growers must apply fungicides frequently to maintain their productivity.
38
However, overusing chemicals increases the risk of the pathogens developing
resistance to fungicides, resulting in the reduction of chemical efficacy. Chemicals
adversely impact non-target species and the environment, causing potential public
health concerns (Pimentel et al., 1992). As a result, there is an increasing demand for
the development of biologically based, environmentally safe alternatives to chemical
pest-control products.
The use of microorganisms to control plant disease is a promising strategy and
has potential for improving plant health (Cook, 1993). As an eco-friendly and cost-
effective strategy, biological control agents (BCAs) are usually isolated from natural
fields or the inside of plant tissues (Zhang et al., 2009). These BCAs are capable of
colonizing the root surface efficiently when they are reintroduced into the soil. Once
established, BCAs are capable of providing long-lasting and long-term defenses against
soil-borne pathogens attack.
Many members of the genus Bacillus have the ability to effectively control plant
diseases. Bacillus subtilis is an ideal example of a bacterial biological control agent,
which is able to control damping-off caused by Pythium ultimum (Asaka and Shoda,
1996), crown rot caused by Aspergillus niger (Podile and Prakash, 1996), and vascular
wilt caused by Fusarium oxysporum (El-Hassan and Gowen, 2006). Bacillus subtilis has
also been developed commercially for bio-control products including BioYield®
(Kloepper et al., 2007), Subtilex® (Schisler et al., 2004) and Serenade® (Lahlali et al.,
2013).
Bacillus strains have several characteristics making them good candidates for
developing competitive BCAs. First, Bacillus spp. have the potential to produce more
39
than 20 antibiotics with strong antimicrobial activity (Stein, 2005). Antibiotics help
Bacillus strains successfully survive and multiply in the soil and to compete with other
microorganisms for nutrients and space (Nihorimbere et al., 2011). Bacillus PGPR have
been shown to increase seed emergence, plant weight, and yield (Kloepper et al.,
2004). Bacillus spp. secrete plant hormones such as indole-3-acetic acid (IAA),
cytokinin and gibberellin which enhance plant growth (Spaepen et al., 2007). Bacillus
strains also increase plant uptake of available minerals, nitrogen, and phosphorus from
the soil (Toro et al., 1998). Bacillus strains form heat- and desiccation- resistant
endospores that promote survival in stressful environments (Lim and Kim, 2010).
Commercialization and the use of most bio-control products remains limited due
to inconsistency in their efficacy, particularly under field conditions (Ramaekers et al.,
2010). To exert their beneficial effects, the application method and timing is critical for
avoiding an outbreak of the pathogen prior to the introduction of Bacillus spp. into the
soil (Spadaro and Gullino, 2005). To solve this problem, the appropriate methods and
timing for applying BCAs need to be determined. Application timing is important
because BCAs need to be applied prior to a pathogen establishing itself on a host plant
in order to achieve the best control. Host may regulate its defense response to a
pathogen during its different growth stage, known as age-related resistance (Kus et al.,
2002). Appling BCAs to a plant host prior to the plant’s maturation is imperative to
impede the contact of the pathogen with the host plant. A positive correlation has been
shown between a plant age and resistance to Phytophthora megasperma var sojae in
soybean(Hanley et al., 1995). Thus, the application timing of BCAs may directly
influenced by age-related resistance.
40
It is generally assumed that the biocontrol efficiency and yield are higher when
BCAs are used in a high dosage. Application rate can be altered to maximize BCAs
efficacy. However, it is probable that introducing too many of microbes could negatively
affect the plant or the rhizosphere (Strigul and Kravchenko, 2006). For example,
population growth of PGPR is suppressed when PGPR population density is high (over
109 CFU/g soil) due to competitiveness for resources and production of secondary
metabolites. Additional work is needed to determine the optimal application rate of
BCAs in for efficient control.
In previous research, PGPR strains from Auburn University (Dr. Joseph
Kloepper’s lab) isolated originally from the plant rhizosphere were efficient in plant
growth promotion and disease control against multiple pathogens on different vegetable
crops under greenhouse and field conditions (Zhang et al., 2010). The overall goal of
the present study was to evaluate bio-control agents including PGPR for suppressing
the Phytophthora blight on squash. The specific objectives were to (1) investigate the
potential of Bacillus spp. for bio-control of Phytophthora blight on squash; (2) optimize
the application rate and timing for better control of this disease.
Materials and Methods
PGPR Strains and Their Growth Conditions
Sixteen bacilli PGPR strains provided by Dr. Joseph W. Kloepper at Auburn
University, AL were isolated from the soil rhizosphere, root surfaces, and roots of
different crops. Information on these PGPR strains are listed in the Table 2-1. PGPR
strains used in this study were stored in Nutrient Broth (NB, pH=7.0) containing 20%
glycerol at -80 ℃.
41
PGPR strains were streaked on nutrient agar (NA) and incubated at 28 ℃ for 24
h. Single colonies were inoculated in NB and cultured for 24 h in a shaker incubator
(SteadyShake 757, Amerex Instruments, Inc., Lafayette, USA) at 150 revolutions per
minute (rpm) at 28 ℃. The bacterial culture was centrifuged at 3,000 rpm for 10 minutes
(min) and the cells were suspended in 20 milliliter (mL) of sterilized deionized water (DI
water). The concentrations of the bacterial suspensions were adjusted to 108 cells/mL
based on the optical density (OD600) with a spectrophotometer (UNICO SpectroQuest
TM 4802 UV/VIS Double Beam, United Products & Instruments Inc., NJ).
Media Used in the This Study
Pimaricin–ampicillin–rifampicin–pentachloronitrobenzene with hymexazol
cornmeal agar (PARPH) medium was modified (Mitchell and Kannwischer-Mitchell,
1992): corn meal agar (Difco, 17 g/L) amended with: ampicillin (100 mg/L), hymexazol
(50 mg/L), pimaricin (10 mg/L), PCNB (100 mg/L), rifampicin (10 mg/L).
Clarified V8 broth was prepared with 250 mL V8 juice by mixing with 0.2 g
CaCO3 and centrifuging at 3,000 rpm for 20 min at room temperature. The supernatant
was filtered through Whatman® No. 1 filter paper. The final volume of supernatant was
200 mL and mixed with 800 mL DI water.
V8 agar (unclarified) was prepared with 10% V8 juice, 1% CaCO3 and 15 g/L
Agar. The mixture was heated and stirred prior to autoclaving in order to dissolve the
solutes completely.
Growth of Squash Plants
Zucchini (Cucurbita pepo cv. Senator) seeds were obtained from S&B Inc.
(Homestead, FL). The seeds were washed in DI water (pH 2.0, adjusted with 6M HCl) to
remove fungicides in the coating, and grown in the plastic pots (7.62 cm high and 10.16
42
cm at the opening with 8 drain holes) containing potting mix (Fafard® Lightweight Mix 2,
Conrad Fafard, Inc., Aquawam, MA) in the greenhouse (24 ± 10 ℃) during the
experiments. After the squash plants had two completely expanded leaves, each
seedling was fertilized with 50 mL (3.5g/L) of a liquid soluble fertilizer (Peter’s 20:20:20
NPK, Peters Fertilizer Products, Fogelsville, PA) every two weeks.
Application of PGPR Treatments in the Greenhouse
To evaluate the effect of PGPR on Phytophthora blight and plant growth, PGPR
were applied two times in the greenhouse. For the first PGPR application, 20 mL of
each PGPR suspension at 1 × 108 CFU/mL was applied as a soil drench around the
base of the stem. One week later, the plants were treated with the same PGPR a
second time by soil drench to enhance the PGPR population and potential of disease
control.
Inoculum Preparation and Inoculation
Isolates (#121, #146, and #151) of P. capsici were provided by Dr. Pamela D.
Roberts, Southwest Florida Research and Education Center, University of Florida,
Immokalee, FL. The inoculum of P. capsici was prepared according to (Ploetz et al.,
2002) with slight modifications. Phytophthora capsici was grown on the selective
medium, PARPH, containing a mixture of fungicide and antibiotics to prevent
contamination by bacteria and other soil-borne pathogens. A disc (5-mm diameter) was
cut from the edge of the medium with P. capsici, transferred to V8 agar and grown for
one week at 25 ℃. Ten discs (5 mm) with P. capsici were cut from the V8 agar plate,
transferred to a Petri dish (90-mm diameter) containing 20 mL of V8 broth and
incubated for one week at 25 ℃ in the dark. The mycelium in the V8 broth was washed
twice with DI water, and 15 mL of DI water was added to merge the mycelium. Then the
43
plates were placed under light at room temperature for 2 days to induce sporangial
formation. The plates were chilled at 4 ℃ for 1 hour, before they were moved into an
incubator at 28 ℃ for 30 minutes to enhance the release of zoospores. The
concentration of zoospores was determined under an Olympus BH2-RFCA microscope
(Olympus, Tokyo, Japan) using a hemocytometer after the sporangial solution was
stirred in a vortex mixer for 1 min to break flagella of the zoospores.
For inoculation with P. capsici, the concentration of the inoculum was adjusted to
2×104 zoospores/mL using DI water. Three days after the second PGPR treatment, 5
mL of the zoospore inoculum was carefully added to the soil around the base of plant
stems. Inoculated plants were maintained on benches in the greenhouse with high soil
moisture by hand watering daily.
Disease Rating
Symptoms of Phytophthora blight usually started at the base of the stem about 4-
5 days after inoculation. The disease was rated according to a 0-5 scale (Zhang et al.,
2010), where 0=healthy plant, 1= water soaked lesions at the bottom of the stem, 2=
stem was girdled and had lesions or started to exhibit white mycelium on the surface of
diseased tissue, 3= all leaves turned yellow and the whole plant started to collapse, 4=
plants wilted and fell down, and 5= dead plant.
Evaluation of PGPR for Suppressing Phytophthora blight
Sixteen PGPR strains were tested in two groups. Each group included eight
PGPR strains, one standard fungicide (Presidio®) and a non-treated control. There were
ten plants in each treatment with each plant as a replicate. The experiment was
designed as a randomized complete block. Each experiment was conducted three
44
times. The replications and duration of experiments were similar in all trials unless
specified.
Plant Growth Promotion by PGPR
To evaluate the effect of PGPR on plant growth promotion, soil was drenched
with PGPR suspensions (1×108 CFU/mL) twice after two plant leaves were completely
expanded. In order to test the influence of fertilizers on plant growth promotion by
PGPR treatment, each pot received 20 mL (3.5 g/L) of the liquid fertilizer 20:20:20 NPK
mixture (Peters General Purpose 20-20-20; Peters Fertilizer Products, Fogelsville, PA)
3 days after the first PGPR application. In another experiment, plants were grown in the
potting mix without fertilization. Ten days after the second PGPR treatment, plant
height, stem diameter, fresh and dry weight of shoot and root, root length were
measured and recorded. The experiment was designed as a randomized complete
block, and ten plants were used in each treatment with each plant as a replicate.
Effect of PGPR Concentration on Disease Suppression
The effect of PGPR concentration on disease control efficacy was determined by
soil drench with PGPR suspensions at 105, 106, 107 and 108 CFU/mL. The different
PGPR concentrations were prepared by serial dilution from 108 CFU/mL with DI water.
The standard plant resistance inducer Actigard® was included in this experiment. Each
pot was treated with 10 mL of Actigard (30 mg/L) at the same time that the PGPR
treatment was applied.
Effect of Timing of PGPR Application on Disease Suppression
The effect of timing of PGPR application on Phytophthora blight in squash was
evaluated at different plant growth stages. The first PGPR application was applied 10
(Stage 1), 17 (Stage 2) and 24 (Stage 3) days after emergence in each individual trial.
45
The second PGPR application was applied 7 days after the first application.
Phytophthora capsici inoculation was the same as described previously.
Effect of PGPR on Chlorophyll Content in Squash Leaves
The chlorophyll content in squash leaves was estimated with a Minolta SPAD
502 meter (Minolta Camera Co., Osaka, Japan) to determine the effect of PGPR
treatment on chlorophyll contents. The data were collected one day before inoculation,
and five days after inoculation when plants started to show symptoms. The readings
were taken from the 3rd, 4th, 5th leaf from apex in all treatments. Data were averaged
from three different leaves for each plants.
Statistical Analyses
Data of disease severity was collected using the rating system from 0 (no
disease) to 5 (dead plant). The area under the disease progress curve (AUDPC) was
calculated for each replicate plant in each treatment using the formula
∑ [𝑦𝑖+𝑦𝑖+1
2] (𝑡𝑖+1 − 𝑡𝑖)
𝑛−1𝑖 , where yi = disease severity at the ith day, ti = time at the ith day,
and n=total number of evaluation times (Ristaino, 1991).
All analyses were performed by using the statistical analysis system (SAS
version9.1, SAS Institute, Cary, NC). Data of AUDPC and growth variables in response
to different PGPR strains in the greenhouse tests were analyzed by a one-way analysis
of variance (ANOVA). Significant differences among means were determined with
Fisher’s protected LSD test (p = 0.05).
46
Results
Susceptibility of Squash to Different P. capsici Isolates
Isolate 121 was the most aggressive among three P. capsici isolates (121, 146,
and 151) tested, resulting in highest disease severity and AUDPC value at 103, 104 and
105 zoospores/mL 8 days after inoculation (Table 2-2). Isolate 146 was had a slight
effect on inducing disease symptoms at 105 zoospores/ mL, and isolate 151 had a
moderate effect based on the fact that Phytophthora symptoms were the lowest at 104
and 105 zoospores/mL. Based on the result, isolate 151 was chosen to use in the
following experiments. The isolate 151 at 1x 105 zoospores/mL incited significantly
greater disease than 104 zoospores/mL, indicated as AUDPC values (Table 2-3). There
was no significant difference in AUDPC values by P. capsici from 2×104 to 8×104
zoospores/mL. Next, we analyzed the correlation between zoospores levels and
disease development, there would have a positive relationship between zoospores
concentrations and AUDPC values (Figure 2-1). Based on the results above, 2×104
zoospores/mL of isolate 151 was selected as the P. capsici inoculum concentration for
testing the effect of PGPR on suppression of Phytophthora crown and root rot, unless
indicated otherwise.
Effect of PGPR on Phytophthora Blight in the Greenhouse
Sixteen selected Bacillus strains of PGPR were evaluated in the greenhouse for
suppressing Phytophthora blight - in this case, Phytophthora crown and root rot. In the
progression of disease development, some PGPR strains significantly (P≤0.05) reduced
AUDPC value of Phytophthora blight compared to the non-treated control. PGPR strains
IN937b and SE76 consistently provided significant (P ≤ 0.05) protection against P.
capsici in all three trials (Table 2-4). When plants were treated with IN937b and SE76,
47
symptoms of crown rot caused by P. capsici developed more slowly than it did on non-
treated control plants. PGPR strains SE56, 1PC-11, T4 and GB03 significantly reduced
the AUDPC value in two of the three trials, PGPR strains SE52, INR7, IN937a, SE49,
1PN-19, and Companion significantly reduced the disease AUDPC in one of the three
trials, whereas the strains from commercial products Actinovate AG, BU EXP 1216S,
BU EXP 1216C, and SE34 were the least effective treatments for suppressing
Phytophthora blight on squash in this greenhouse study (Table 2-4 and Table 2-5).
However, no PGPR completely protected the plants from infection by P. capsici. At the
end of the evaluation, plants were dead in all treatments except for the standard
fungicide Presidio®.
Effect of PGPR on Plant Growth in the Greenhouse
When fertilization was adequate for plant growth, there was no difference in all
treatments concerning growth parameters (shoot and root dry weights, shoot height,
root length, etc.) (Table 2-6). Strain IN937a was added as a positive control because it
was shown to improve tomato plants growth (Adesemoye et al., 2009a). Mean plant
height (33.0 cm), stem caliper growth (6.6 mm), fresh shoot weight (34.8 g) and dry
shoot weight (4.2 g) observed in IN937a treated plants were highest in all treatments
(Table 2-6). IN937b numerically increased plant height by 4.5% when compared with
the non-treated control, albeit not significantly. Dry root weight in IN937b and SE76
increased by 50% compared with the non-treated control. Interestingly, IN937b and
SE76 significantly reduced root length by 12% and 7%, respectively. This indicated that
IN937b and SE76 did not result in the longest roots, but produced more lateral roots.
The ability of plant growth promotion by IN937b appears to be, at least partially, due to
the change of root architecture such as lateral roots to increase nutrient and water
48
uptake by the roots. The ASM treatment did not increase dry root weight or root length
indicating that ASM did not improve lateral root production.
When no fertilizer was added, squash treated with IN937b had significantly
greater dry shoot and root weight compared to the non-treated control (Figure 2-3). In
the experiment to evaluate the effect of fertilization with PGPR treatment on plant
growth promotion, no significant difference in plant growth was observed among strains
and the non-treated control after fertilizer applied, which are consistent with the result in
the Table 2-6.
Effect of Concentration of Selected PGPR on Disease Suppression
Application of IN937b at 106, 107 and 108 CFU/mL significantly reduced
Phytophthora blight severity compared to the non-treated control. IN937b applied at 108
CFU/mL significantly suppressed disease development by 45% compared to the non-
treated control based on the AUDPC values (Table 2-7). When the concentration was
reduced to 105 CFU/mL, disease development was similar to that in non-treated control
plants. A significant positive correlation (r2=0.92) was found between the IN937b
inoculum concentrations and AUDPC value (Figure 2-2).
SE76 at 108 CFU/mL had significantly lower AUDPC values when compared with
the untreated control (Table 2-8). Similarly as IN937b, treatment with SE76 at the
highest concentration (108 CFU/mL) had the highest efficacy to reduce disease severity
by 42% 6 days after inoculation, which was the last day of the investigation.
IN937a had significant disease control in one of the three trials (Table 2-4).
IN937a at high concentrations (108 and 107 CFU/mL) showed disease symptoms earlier
and had greater AUDPC values than the non-treated control (Table 2-9). In this trial, the
low concentration (105 and 106 CFU/mL) significantly reduced disease severity by 25%
49
and 27%, respectively, compared to the non-treated control. In this study, there is no
significant correlation (r2=0.022) between the IN937a inoculum concentrations and
AUDPC value (Figure 2-2).
Effect of Application Timing of PGPR on Disease Development and Age-related Resistance to Phytophthora Crown and Root Rot on Squash Treated with IN937b
Symptom development of crown and root rot in squash treated with IN937b
varied with the timing of application. When IN937b was applied at growth stage 1 (10
days after plant emergence) and stage 2 (17 days after plant emergence), symptoms
developed as early as 3 days after inoculation. Water-soaked lesions formed initially on
the young seedlings causing the whole plant to wilt quickly (Table 2-10 and 2-11);
whereas when applied at the growth stage 3 (24 days after emergence), symptom
development was delayed, and symptoms were first visible 7 days after inoculation
(Table 2-12). Lesions had visible signs of P. capsici, including mycelia and
sporangiophores with sporangia being easily found in the late stage of development.
IN937b and SE76 significantly reduced disease severity compared to the
untreated control when they were applied at all growth stages. In the trial with best
control efficacy, IN937b and SE76 significantly suppressed the disease by 54% and
57%, respectively, when they were applied at the stage 2 i.e. the first PGPR application
was made at 17 days after seeding (Table 2-11).
Effect of PGPR on Chlorophyll Content in Squash Leaves
The chlorophyll content (as estimated with a SPAD meter) is an indirect indicator
of plant health. The chlorophyll content was significantly greater in leaves of IN937b and
ASM (Actigard) treated plants when compared to the untreated (Figure 2-4). There was
50
no difference in chlorophyll content among the treatments of SE76, IN937a and the non-
treated control.
Five days after inoculation, significant differences in chlorophyll content were
observed in P. capsici infected plants in the treatments SE76 and ASM compared with
the untreated control (Table 2-13). Damage of roots and stems led to a drastic decrease
in SPAD values in the leaves of IN937a treated plants and the non-treated control. On
the other hand, plant treated with IN937b showed only a moderate decrease compared
with SE76 and ASM treatments, although IN937b treated plant did not show any
symptoms at the time of data collection.
Discussion
In the present study, select Bacillus PGPR strains (IN937b, SE76, SE56, SE49,
1PC-11, and GB03) showed effective biological control activity against P. capsici which
causes crown and root rot on squash in this greenhouse. We also investigated the
effect of Bacillus PGPR strains IN937b, SE76 and IN937a on squash growth promotion.
However, the growth promotion effect only occurred under conditions without
fertilization. Moreover, in our system, PGPR strains IN937b and SE76 were consistent
in improving the performance in managing of Phytophthora crown and root rot on
squash, when applied at the concentration of 108 CFU/mL and a longer time period i.e.
the first PGPR application was made at 17 days after seeding.
Virulence Differentiation of P. capsici Isolates
In this study, the susceptibility of the squash cultivar, Senator, to Phytophthora
crown and root rot differed significantly among three isolates 121, 146, and 151. Based
on the results of disease severity and AUDPC value, there appeared to be differences
in virulence among these P. capsici isolates. Isolate 121 was highly virulent, causing
51
crown and root rot. Isolate 146 was the least virulent based on slow symptom
development and lower disease incidence compared to plants treated with the other two
isolates. Different levels of virulence among P. capsici isolates led to similar conclusions
in previous studies with pepper (Oelke et al., 2003). Knowing that different levels of
virulence exist among P. capsici isolates infecting squash, we used isolate 151 with
moderate virulence caused symptoms to develop and tissue to collapse more than one
week after inoculation in the greenhouse.
In our tests, zoospores of P. capsici were used for inoculation. The development
of Phytophthora crown and root rot was affected by inoculum density (CafeFilho and
Duniway, 1996). When P. capsici was tested at 1, 2, 4, 6, 8, and 10 ×104 zoospores/mL
to determine the optimum concentration for the future experiments, high levels of crown
and root rot developed, and plant disease increased very quickly the first week after
inoculation when the concentrations were 6, 8, and 10 ×104 zoospores/mL. However,
only low levels of crown and root rot developed, and disease did not develop in some
plants inoculated with 1×104 zoospores/mL. Optimum disease levels were observed in
plants inoculated with P. capsici at the concentrations of 2 to 8 ×104 zoospores/mL.
Consequently, 2 ×104 zoospores/mL was chosen for further greenhouse experiments to
guaranty optimum plant infection and symptom development.
Control of Phytophthora Crown and Root Rot by Bacillus spp.
The development of biological control agents (BCAs) effective for control of
Phytophthora blight depends on the selection of an efficient strain, the introduction of
adequate bacterial cells into the soil, the interaction between the pathogen and the bio-
control strain, and the timing and method of application. In general, introducing large
populations of BCAs might provide a competitive advantage over residential
52
microorganisms. Sometimes, the bacterial strains that showed the greatest potential for
plant growth promotion were not necessarily the best for inhibiting pathogens. Bacillus
plays an important role in the control of soil-borne plant pathogens such as in
suppressing of crown and root rot caused by P. capsici (Zhang et al., 2010).
Competition for nutrients and space between PGPR and the pathogen is considered to
be one of the mechanisms whereby PGPR biologically control plant diseases.
In our greenhouse trials, some PGPR strains provided strong suppression of
Phytophthora crown and root rot, whereas others showed only moderate activity or no
activity at all. Two PGPR strains IN937b and SE76 consistently showed significant
suppression of Phytophthora blight on squash, which agrees with earlier studies
(Zhang et al., 2010). These Bacillus strains were most effective among the PGPR
strains tested based on data from our greenhouse trials. PGPR strains IN937b and
SE76 showed strong disease suppression, which may make them promising candidates
for the development of BCAs that manage Phytophthora blight on squash. In particular,
B. subtilis IN937b has shown potential to suppress disease and to promote plant
growth, which makes it a potentially promising BCAs for management of Phytophthora
blight on squash.
The effect of disease suppression by IN937b and SE76 was greater at high
concentrations (108 CFU/mL) than low concentrations (107 and 106 CFU/mL), although
these two PGPR strains applied at low concentrations were also effective in some of the
greenhouse trials. The efficacy of the lowest concentration (105 CFU/mL) was
decreased due to the lack of adequate viable cells for optimal control of crown rot.
53
Similar results were reported when 105 CFU/g root was present in the case of
Pseudomonas spp. (Raaijmakers et al., 1995a).
Enhanced Plant Growth by Bacillus spp.
The definition of PGPR comes from the fact that PGPR are able to increase plant
growth. Improvements of plant growth by PGPR on many different crops are well
documented (Kloepper et al., 2004). The ability of Bacillus strains IN937b, SE76 and
IN937a to increase squash plant biomass only occurred in the present study only when
plants were not fertilized, indicating that plant growth was strongly influenced by
fertilization. When the nutrient concentrations in the soil were sufficient, it was difficult to
promote plant growth by application of Bacillus PGPR. On the other hand, Bacillus spp.
could reduce fertilizer application rates due to its ability to increase nutrients available to
crops without any significant reduction of yield (Adesemoye et al., 2009b).
IN937b had a positive effect on plant growth, especially for lateral root growth.
Root biomass is commonly used to indicate growth promotion by PGPR and root
functions such as water and nutrient uptake (Lopez-Bucio et al., 2003). The increase in
lateral root formation by PGPR strains helps to explain the observed growth promotions.
Nutrient uptake by plant generally depends on the amount of root surface area
(Simunek and Hopmans, 2009).
Relation of Leaf Chlorophyll Content and Plant Health
The present study showed that chlorophyll content increased in leaves of IN937b
treated plants under greenhouse conditions, which agrees with the previous report
indicating that treating lettuce leaves with PGPR increased leaf chlorophyll content
(Arkhipova et al., 2005). In this present study, the increase in chlorophyll content in
IN937b treated squash plants indicated that foliar nitrogen content in plant leaves was
54
increased by IN937b, which might indirectly influence plant growth improvement. The
increase in chlorophyll content might be due to the changes in the plant’s metabolism.
Five days after inoculation with P. capsici, squash plants began to show
symptoms of water-soaked lesions on the stem, especially the leaves turned yellow.
Treatments with PGPR strain SE76 significantly reduced disease severity compared
with the untreated control, while leaf chlorophyll content was also significantly higher
than in the untreated control. IN937a had no effect on disease suppression and similar
leaf chlorophyll content as the untreated control.
Optimization of Application Rate and Timing
Loss of biocontrol efficiency observed after 2 weeks is associated with a
decrease in the populations of PGPR (Strigul and Kravchenko, 2006). Maintain a high
number of PGPR as well as the timing of applications is possible to obtain an optimal
long-term control of diseases. This requires to assess the behavior of a specific BCA.
Understanding the behavior of a BCA in the environment may lead to improved
performance of the BCA, including nutrient use (Ji et al., 1997) and site preference (Fan
et al., 2011). If certain behaviors can be correlated with better disease control, then it
may be possible to promote these behaviors and, hence, improve disease control.
BCAs can be made into more reliable and predictable products by promoting the
behaviors associated with improved disease control and by optimizing application
methods.
Our results indicated that 108 CFU/mL is the optimum concentration for PGPR
strains IN937b and SE76 to consistently promote plant growth and suppress disease
development. Bacterial concentrations of PGPR above 108 CFU/mL were not tested
55
since the highest vegetative cells of PGPR strains cultured within 24 h in NB are
approximately 1 × 108 CFU/mL.
It is important to determine a threshold of inoculum density for IN937b and SE76
at which they significantly suppress the Phytophthora blight on squash. Compared to
the low concentration of 105 CFU/mL, treatments with PGPR at high concentrations
demonstrated greater control efficacy. This is because symptom development was
delayed due to the high population of Bacillus spp. in the potting mix which may play a
long-term role in disease suppression. Some studies have suggested that specific
PGPR strains secreted secondary metabolites to prevent pathogen invasion and spread
(Compant et al., 2013). Thus, the low severity of disease in PGPR treatments was
probably due to low frequency of infection by the pathogen, reducing the potential for
pathogens to attack and colonize the root tissues of plants (Nihorimbere et al., 2010). In
general, two applications of PGPR were generally more effective than one treatment,
since the seedling stage highlighted the importance of maintaining a high population of
PGPR in the soil. This high population may reduce infection by pathogens (Poleatewich
et al., 2012). This requirement, however, poses a challenge to maintain high PGPR
populations in the soil because vegetative cells of PGPR strains lose efficacy quickly
under dry soil conditions due to poor colonization (West et al., 1985). Repeated
applications of PGPR may enhance the survival of PGPR in the soil (Schmidt et al.,
2004). In the present study, treatments of two applications likely helped to maintain high
populations of PGPR throughout the experiments.
The effect of age-related resistance in squash plants treated with PGPR remains
unknown (Sunwoo et al., 1996). Results from our study demonstrated that the timing of
56
applying PGPR can be crucial in the efficacy of PGPR against infection by P. capsici.
Application to the stage 2 (stage 1 was the youngest stage) seedlings was remarkably
more efficient in disease suppression than when it was applied at the two older stages,
suggesting that the PGPR strains IN937b and SE76 exhibited strong reactions starting
from early growth stage. It also suggested that PGPR strains need time to establish a
relationship with the host plant. In particular, the younger seedlings developed more
crown rot disease, similar to that described by Morin for gorse (Ulex europaeus L.)
plants (Morin et al., 1998). The gorse plants tested were susceptible to pathogens, but
younger plants were more easily killed. The age of plants influenced the capability of
pathogens to infect and spread due to thickening of the cuticle and inducing resistance
response. However, the effectiveness of Bacillus strains in suppressing pathogens
decreases as plants mature.
During the past decade, several studies have focused on bio-control activities of
B. subtilis in controlling plant diseases. Antimicrobial metabolites are often associated
with BCAs in the genus Bacillus, which may act directly on the pathogen. Indigenous
bacteria that are antagonistic to plant pathogens in the soil could make a substantial
contribution to preventing plant diseases, and therefore represent an alternative to using
chemical pesticides. It is clear that BCAs are still too variable in their performance to be
successfully used as a common practice for disease management. This inconsistency
has been attributed to a number of factors including fluctuations in the environment and
the variable expression of genes involved in disease suppression and root colonization
by the BCA. The present suggests that further investigations are needed regarding the
role of environmental factors in modulating performance of Bacillus PGPR strains in
57
order to improve their efficacy and performance in suppressing phytopathogens under
field conditions. A better understanding of the bio-control mechanisms may be helpful
for guiding future efforts in BCA development.
58
Table 2-1. Relevant information for PGPR strains tested in this study
Tested PGPR strains a
Identity
Protected plant
Target pathogen Reference
SE34
B. safensis Rice Xanthomonas oryzae pv. oryzae
(Chithrashree et al., 2011)
SE49
B. safensis Cucumber Colletotrichum orbiculare
(Wei et al., 1996)
SE52
B. safensis Loblolly pine Cronartium quercuum f. sp. fusiforme
(Enebak and Carey, 2000)
SE56 Lysinibacillus
boronitolerans Pepper
Colletotrichum gloeosporioides
(Jetiyanon and Kloepper, 2002)
SE76
B. safensis Tomato Colletotrichum gloeosporioides
(Jetiyanon and Kloepper, 2002)
IN937a
B. subtilis Tomato Ralstonia solanacearum
(Jetiyanon, 2007)
IN937b
B. subtilis Pepper Sclerotium rolfsii (Jetiyanon, 2007)
INR7
B. pumilus Pepper Xanthomonas axonopodis pv. vesicatoria
(Yi et al., 2013)
T4
B. safensis Tobacco Peronospora tabacina
(Zhang et al., 2002)
GB03
B. subtilis Arabidopsis thaliana
Pseudomonas syringae pv. tomato DC3000
(Kumar et al., 2012)
1PC-11
B. macauensis Squash Phytophthora capsici
(Zhang et al., 2010)
1PN-19
B. subtilis Squash Phytophthora capsici
(Zhang et al., 2010)
Companion (GB03)
B. subtilis
Arabidopsis thaliana
Pseudomonas syringae pv. tomato DC3000
(Kumar et al., 2012)
BU EXP 1216S (MBI600)
B. subtilis Rice
Rhizoctonia solani
(Kumar et al., 2010)
BU EXP 1216C (MBI600)
B. subtilis Squash
Podosphaera xanthii
(Zhang et al., 2011)
Actinovate AG
Streptomyces lydicus WYEC 108
Soybean Sclerotinia sclerotiorum
(Zeng et al., 2012a)
a PGPR strains were provided by Dr. Joseph W. Kloepper.
59
Table 2-2. Disease development in squash inoculated with different isolates of P. capsici
Zoospore concentration a
Disease severity b AUDPC c
121 146 151 121 146 151
102 0.0 b d 0.0 b 0.0 b 0.0 e 0.0 e 0.0 e
103 3.3 a 0.0 b 0.0 b 1.6 d 0.0 e 0.0 e
104 5.0 a 0.0 b 3.8 a 5.3 b 0.0 e 3.8 c
105 5.0 a 3.8 a 5.0 a 7.5 a 1.9 d 7.3 a
a Zoospore solutions of three isolates (121, 146, 151) were applied in the pots as a soil drench when plants had four completely expanded leaves. Each plant was inoculated with 5 mL of P. capsici inoculum. b. Disease severity was rated based on the scale from 0 (healthy) to 5 (dead). Data were collected 7 days after pathogen inoculation. c AUDPC was calculated based on the disease severity rated 5, 6, and 7 days after pathogen inoculation d In each column in disease severity, each value represents the mean of four replicates. Values followed by the same letter are not significantly different according to Fisher’s protected Least Significant Difference (LSD) test (P = 0.05).
60
Table 2-3. Effect of the concentration of P. capsici isolate 151 on disease development
Zoospore concentration (×104) a
Disease severity b Time after inoculation (Day)
AUDPC c 5 7 9
1 0.6 a d 3.0 a 3.0 b 9.6 b
2 0.6 a 3.0 a 3.8 ab 10.4 ab
4 0.8 a 3.0 a 3.5 ab 10.3 b
6 1.1 a 2.8 a 3.3 b 9.9 b
8 1.8 a 3.0 a 4.5 a 12.3 ab
10 1.8 a 4.0 a 4.5 a 14.3 a
a The concentration of P. capsici inoculum was diluted to 1, 2, 4, 6, 8, and 10 × 104 zoospores/mL. Squash plants were inoculated with 5 mL of P. capsici inoculum. b Disease severity was rated based on the scale from 0 (healthy) to 5 (dead). c AUDPC was calculated based on the disease severity rated 5, 7, and 9 days after pathogen inoculation. d Each value represents the mean of four replicates. Means followed by the same letter within a column are not significantly different according to Fisher’s protected LSD test (p = 0.05).
61
Table 2-4. Efficacy of PGPR (1-8 PGPR strains) on suppression of Phytophthora crown and root rot under greenhouse conditions
Treatment a AUDPC b
Trial 1 Trial 2 Trial 3
SE34 14.4 a c 17.4 ab 24.8 a
SE52 10.0 b 15.6 b 24.4 a
SE56 12.0 ab 14.7 b 20.1 b
SE76 5.8 c 15.6 b 17.1 c
INR7 13.6 a 15.3 b 22.9 a
1PN-19 13.6 a 15.3 b 23.7 a
IN937a 13.6 a 15.9 b 23.9 a
IN937b 5.6 c 15.3 b 19.6 b
Presidio® 1.0 d 0.0 c 0.0 d Non-treated control 12.8 ab 19.2 a 23.9 a
a PGPR strains were applied twice (1.0 ×108 CFU/mL; 20mL) as a soil drench. Presidio® (1.2 mL/L; 20mL) was applied as a standard fungicide which provide control of Phytophthora blight. b P. capsici inoculum on crown and roots was diluted to 2 × 104 zoospores/mL. Plants were inoculated by 5 mL of P. capsici inoculum. Disease severity was collected based on the rating scale from 0 (healthy) to 5 (dead). AUDPC calculated based on the disease severity. Trial 1: 11, 13, and 15 days after pathogen inoculation; Trial 2: 9, 12, and 15 days after pathogen inoculation; Trial 3: 7, 9, and 14 days after pathogen inoculation. c Each value represents the mean of five replicates. Means followed by the same letter within a column are not significantly different according to Fisher’s protected LSD test (p = 0.05).
62
Table 2-5. Efficacy of PGPR (9-16 PGPR strains) on suppression of Phytophthora crown and root rot under greenhouse conditions
Treatment a AUDPC b
Trial 1 Trial 2 Trial 3
SE49 15.6 ab c 10.3 bc 5.8 b
Com 9.6 cd 12.4 abcd 8.6 ab
AG 14.4 ab 16.1 a 8.4 ab
GB03 8.6 d 7.8 c 8.8 ab
1216S 14.4 ab 13.2 abc 8.4 ab
1216C 14.8 ab 13.8 ab 9.6 ab
T4 12.6 bcd 9.8 bc 11.4 a
1PC-11 13.6 bcd 11.7 bcd 8.2 ab
Presidio® 0.0 e 0.0 d 0.0 c Non-treated control 18.2 a 16.8 a 10.2 ab
a PGPR strains were applied twice (1.0 ×108 CFU/mL; 20mL) as a soil drench. Presidio® (1.2 mL/L; 20mL) was applied as a standard fungicide which provide control of Phytophthora blight. b P. capsici inoculum on crown and roots was diluted to 2 × 104 zoospores/mL. Plants were inoculated by 5 mL of P. capsici inoculum. Disease severity was collected based on the rating scale from 0 (healthy) to 5 (dead). AUDPC calculated based on the disease severity. Trial 1: 11, 15, and 17 days after pathogen inoculation; Trial 2: 12, 14, and 17 days after pathogen inoculation; Trial 3: 7, 9, and 11 days after pathogen inoculation. c Each value represents the mean of five replicates. Means followed by the same letter within a column are not significantly different according to Fisher’s protected LSD test (p = 0.05).
63
Table 2-6. Effect of PGPR on plant growth under greenhouse conditions
Treatment a
Parameters of plant growth b
Plant height cm
Stem diameter mm
Fresh shoot g
Dry shoot g
Fresh root g
Dry root g
Root elongation cm
IN937b 32.3 ab c 6.4 a 33.6 ab 3.9 a 4.6 a 0.3 a 14.3 c
SE76 31.6 ab 6.2 a 34.0 ab 3.9 a 4.3 a 0.3 a 15.0 bc
IN937a 33.0 a 6.6 a 34.8 a 4.2 a 4.3 a 0.2 a 15.3 b
Actigard® 32.9 a 6.5 a 32.6 ab 4.1 a 4.3 a 0.2 a 14.4 c Non-treated control
30.9 ab 6.3 a 34.1 ab 4.0 a 4.4 a 0.2 a 16.2 a
Non-treated control (-F)
30.3 b 6.2 a 30.7 b 2.9 b 3.3 b 0.1 b 13.3 d
a PGPR strains were applied twice (1.0 ×108 CFU/mL; 20 mL) as a soil drench. Actigard® (30 mg/L; 20 mL) was applied as a positive SAR inducer. Water soluble fertilizer 20:20:20 once a week (3.5 g/L; 20 mL per plant) was applied to all treatments except for the untreated control (-F). The untreated control (-F) represents the plants grown without fertilization. b Plants were investigated at 40 days after planting, the root and shoot were separated, and the biomass was measured. The length of root tissues was recorded to determine the root elongation. c Each value represents the mean of ten replicates. Means followed by the same letter within a column are not significantly different according to Fisher’s protected LSD test (p = 0.05).
64
Table 2-7. Effect of IN937b concentration on crown and root rot disease
Treatment a
Disease severity b Time after inoculation (Day) AUDPC c
3 4 5 6
105 CFU/mL 0.3 a d 1.4 b 2.1 ab 3.5 ab 5.4 ab
106 CFU/mL 0.1 b 1.2 b 1.9 bc 3.2 b 4.8 bc
107 CFU/mL 0.0 b 1.0 b 2.0 bc 3.7 ab 4.8 bc
108 CFU/mL 0.0 b 1.0 b 1.5 c 2.4 c 3.7 c
Non-treated control 0.0 b 2.1 a 2.6 a 4.0 a 6.7 a
Actigard® 0.0 b 0.0 c 0.0 d 0.0 d 0.0 d
Presidio® 0.0 b 0.0 c 0.0 d 0.0 d 0.0 d a PGPR were applied twice as a soil drench. The concentration of IN937b was adjusted to 105, 106, 107, and 108 CFU/mL, and 20 mL was applied to each plant. Presidio® (1.2 mL/L; 20mL/plant) and Actigard® (30 mg/L; 20mL/plant) were applied as a positive fungicide and SAR control, respectively, of Phytophthora blight. b P. capsici inoculum on crown and roots was diluted to 2 × 104 zoospores/mL. Plants were inoculated by 5 mL of P. capsici inoculum. Disease severity was collected at 3, 4, 5, and 6 days after pathogen inoculation based on the rating scale from 0 (healthy) to 5 (dead). c AUDPC calculated based on the disease severity 3, 4, 5, and 6 days after pathogen inoculation. d Each value represents the mean of ten replicates. Means followed by the same letter within a column are not significantly different according to Fisher’s protected LSD test (p = 0.05).
65
Table 2-8. Effect of SE76 concentration on crown and root rot disease
Treatment a
Disease severity b Time after inoculation (Day) AUDPC c
3 4 5 6
105 CFU/mL 0.0 b d 1.0 a 1.4 ab 2.9 b 3.9 ab
106 CFU/mL 0.5 a 1.1 a 1.7 ab 3.8 a 4.9 ab
107 CFU/mL 0.0 b 1.5 a 1.0 b 2.7 bc 3.8 ab
108 CFU/mL 0.0 b 1.1 a 1.1 b 2.2 c 3.2 b
Non-treated control 0.0 b 1.8 a 2.1 a 3.8 a 5.8 a
Actigard® 0.0 b 0.0 b 0.0 c 0.0 d 0.0 c
Presidio® 0.0 b 0.0 b 0.0 c 0.0 d 0.0 c a PGPR were applied twice as a soil drench. The concentration of SE76 was adjusted to 105, 106, 107, and 108 CFU/mL, and 20 mL was applied to each plant. Presidio® (1.2 mL/L; 20mL/plant) and Actigard® (30 mg/L; 20mL/plant) were applied as a positive fungicide and SAR control, respectively, of Phytophthora blight. b P. capsici inoculum on crown and roots was diluted to 2 × 104 zoospores/mL. Plants were inoculated by 5 mL of P. capsici inoculum. Disease severity was collected at 3, 4, 5, and 6 days after pathogen inoculation based on the rating scale from 0 (healthy) to 5 (dead). c AUDPC calculated based on the disease severity 3, 4, 5, and 6 days after pathogen inoculation. d Each value represents the mean of ten replicates. Means followed by the same letter within a column are not significantly different according to Fisher’s protected LSD test (p = 0.05).
66
Table 2-9. Effect of IN937a concentration on crown and root rot disease
Treatment a
Disease severity b Time after inoculation (Day) AUDPC c
3 4 5 6
105 CFU/mL 0.0 b d 1.3 c 1.6 b 3.6 bc 4.7 b
106 CFU/mL 0.0 b 1.6 bc 1.6 b 2.9 d 4.6 b
107 CFU/mL 0.4 a 2.2 a 2.8 a 4.0 a 7.2 a
108 CFU/mL 0.5 a 2.3 a 2.6 a 3.4 c 6.9 a
Non-treated control 0.0 b 1.9 ab 2.4 a 3.9 ab 6.3 a
Actigard® 0.0 b 0.0 d 0.0 c 0.0 e 0.0 c
Presidio® 0.0 b 0.0 d 0.0 c 0.0 e 0.0 c
a PGPR were applied twice as a soil drench. The concentration of IN937a was adjusted to 105, 106, 107, and 108 CFU/mL, and 20 mL was applied to each plant. Presidio® (1.2 mL/L; 20mL/plant) and Actigard® (30 mg/L; 20mL/plant) were applied as a positive fungicide and SAR control, respectively, of Phytophthora blight. b P. capsici inoculum on crown and roots was diluted to 2 × 104 zoospores/mL. Plants were inoculated by 5 mL of P. capsici inoculum. Disease severity was collected at 3, 4, 5, and 6 days after pathogen inoculation based on the rating scale from 0 (healthy) to 5 (dead). c AUDPC calculated based on the disease severity 3, 4, 5, and 6 days after pathogen inoculation. d Each value represents the mean of ten replicates. Means followed by the same letter within a column are not significantly different according to Fisher’s protected LSD test (p = 0.05).
67
Table 2-10. Effect of Bacillus spp. application timing (stage 1: seed) on crown and root rot disease
Treatment a
Disease severity b Time after inoculation (Day) AUDPC c
3 5 6 7
IN937b 0.4 b d 1.4 b 2.8 bc 3.6 bc 7.2 bc
SE76 0.0 b 1.1 b 2.5 c 3.0 c 5.3 c
IN937a 0.5 b 2.5 a 3.6 ab 4.6 ab 9.5 ab
Non-treated control 1.3 a 3.0 a 3.9 a 5.0 a 11.6 a
Actigard® 0.0 b 0.0 c 0.0 d 0.0 d 0.0 d
Presidio® 0.0 b 0.0 c 0.0 d 0.0 d 0.0 d a PGPR strains were applied twice (seed soaking and 10 days after emergence) as a soil drench (1.0 ×108 CFU/mL; 20mL). Presidio® (1.2 mL/L; 20mL) and Actigard® (30 mg/L; 20mL/plant) was applied as the positive control of Phytophthora blight. b P. capsici inoculum on crown and roots was diluted to 2 × 104 zoospores/mL. Plants were inoculated by 5 mL of P. capsici inoculum. Disease severity was collected at 3, 5, 6, and 7 days after pathogen inoculation based on the rating scale from 0 (healthy) to 5 (dead). c AUDPC calculated based on the disease severity 3, 5, 6, and 7 days after pathogen inoculation. d Each value represents the mean of ten replicates. Means followed by the same letter within a column are not significantly different according to Fisher’s protected LSD test (p = 0.05).
68
Table 2-11. Effect of Bacillus spp. application timing (stage 2: 10 days after emergence) on crown and root rot disease
Treatment a
Disease severity b Time after inoculation (Day) AUDPC c
3 5 7 9
IN937b 0.8 ab d 1.2 b 1.2 b 3.1 b 8.6 b
SE76 0.7 bc 1.2 b 1.3 b 2.5 b 8.0 b
IN937a 1.6 a 2.5 a 2.7 a 3.0 b 14.9 a
Non-treated control 1.2 ab 3.0 a 3.5 a 4.7 a 18.8 a
Actigard® 0.0 c 0.0 c 0.0 c 0.0 c 0.0 c
Presidio® 0.0 c 0.0 c 0.0 c 0.0 c 0.0 c a PGPR strains were applied twice (10 days and 17 days after emergence) as a soil drench (1.0 ×108 CFU/mL; 20mL). Presidio® (1.2 mL/L; 20mL) and Actigard® (30 mg/L; 20mL/plant) was applied as the positive control of Phytophthora blight. b P. capsici inoculum on crown and roots was diluted to 2 × 104 zoospores/mL. Plants were inoculated by 5 mL of P. capsici inoculum. Disease severity was collected at 3, 5, 7, and 9 days after pathogen inoculation based on the rating scale from 0 (healthy) to 5 (dead). c AUDPC calculated based on the disease severity 3, 5, 7, and 9 days after pathogen inoculation. d Each value represents the mean of ten replicates. Means followed by the same letter within a column are not significantly different according to Fisher’s protected LSD test (p = 0.05).
69
Table 2-12. Effect of Bacillus spp. application timing (Stage 3: 17 days after emergence) on crown and root rot disease
Treatment a
Disease severity b Time after inoculation (Day) AUDPC c
7 9 11 13
IN937b 1.4 b d 1.4 c 2.6 b 3.0 c 11.3 c
SE76 1.8 ab 2.1 b 2.7 b 3.4 bc 14.1 b
IN937a 2.0 a 2.1 b 2.9 b 3.9 b 15.9 ab
Non-treated control 1.9 a 2.8 a 3.6 a 5.0 a 18.6 a
Actigard® 0.5 c 0.7 d 1.0 c 1.1 d 4.9 d
Presidio® 0.0 d 0.0 e 0.0 d 0.0 e 0.0 e a PGPR strains were applied twice (17 days and 24 days after emergence) as a soil drench (1.0 ×108 CFU/mL; 20mL). Presidio® (1.2 mL/L; 20mL) and Actigard® (30 mg/L; 20mL/plant) was applied as the positive control of Phytophthora blight. b P. capsici inoculum on crown and roots was diluted to 2 × 104 zoospores/mL. Plants were inoculated by 5 mL of P. capsici inoculum. Disease severity was collected at 7, 9, 11, and 13 days after pathogen inoculation based on the rating scale from 0 (healthy) to 5 (dead). c AUDPC calculated based on the disease severity 7, 9, 11, and 13 days after pathogen inoculation. d Each value represents the mean of ten replicates. Means followed by the same letter within a column are not significantly different according to Fisher’s protected LSD test (p = 0.05).
70
Table 2-13. Effect of PGPR on chlorophyll content in squash leaves under greenhouse conditions
Treatment a Disease severity b SPAD reading c
IN937b 0.0 b d 21.7 b
SE76 0.3 b 29.3 a
IN937a 1.0 a 16.6 b
Actigard® 0.0 b 32.9 a
Non-treated Control 1.0 a 16.8 b
a PGPR were applied twice as a soil drench. The concentration of IN937b was adjusted to 108 CFU/mL, and 20 mL was applied to each plant. Actigard® (30 mg/L; 20mL/plant) was applied as a positive SAR control of Phytophthora blight. b Phytophthora capsici inoculum was diluted to 2 × 104 zoospores/mL. Plants were inoculated with 5 mL of P. capsici inoculum. Disease severity was rated at 5 days after pathogen inoculation based on the rating scale from 0 (healthy) to 5 (dead). c The SPAD measurement were carried out 5days after inoculation. SPAD values were measured on three leaves at the same position on each plant. d Each value represents the mean of 5 replicates. Means followed by the same letter within a column are not significantly different according to Fisher’s protected LSD test (p = 0.05).
71
Figure 2-1. Regression analysis of the concentration of P. capsici isolate 151 on
disease development. AUDPC was calculated based on the disease severity rated 5, 7, and 9 days after pathogen inoculation. Each value represents the mean of four replicates. Means followed by the same letter within a column are not significantly different according to Fisher’s protected LSD test (p = 0.05).
y = 0.4482x + 8.8175R² = 0.741
0
2
4
6
8
10
12
14
16
0 2 4 6 8 10 12
AU
DP
C
Zoospore concentration (×104 /mL)
72
A.
B.
C.
Figure 2-2. Regression analysis of Effect of PGPR strains concentration on crown and root rot disease. A: IN937b, B: SE76, C: IN937a. PGPR were applied twice as a soil drench. The concentration of PGPR was adjusted to 105, 106, 107, and 108 CFU/mL, and 20 mL was applied to each plant. Presidio® (1.2 mL/L; 20mL/plant) and Actigard® (30 mg/L; 20mL/plant) were applied as a positive fungicide and SAR control, respectively, of Phytophthora blight. P. capsici inoculum on crown and roots was diluted to 2 × 104 zoospores/mL. Plants were inoculated by 5 mL of P. capsici inoculum. Disease severity was collected at 3, 4, 5, and 6 days after pathogen inoculation based on the rating scale from 0 (healthy) to 5 (dead). AUDPC calculated based on the disease severity 3, 4, 5, and 6 days after pathogen inoculation. Values with same letter within a column are not significantly different according to Fisher’s protected LSD test (p = 0.05). Vertical bars represent mean ± S.D. (n = 5).
y = -0.3371x + 6.833R² = 0.9209
0.0
5.0
10.0
0.0 2.0 4.0 6.0 8.0 10.0A
UD
PC
IN937b concentration (log10 CFU/mL)
y = -0.2892x + 5.8237R² = 0.7674
0.0
5.0
10.0
0.0 2.0 4.0 6.0 8.0 10.0
AU
DP
C
SE76 concentration (log10 CFU/mL)
y = 0.0582x + 5.6371R² = 0.0220.0
5.0
10.0
0.0 2.0 4.0 6.0 8.0 10.0
AU
DP
C
IN937a concentration (log10 CFU/mL)
73
A
B
Figure 2-3. Effect of IN937b on squash root and shoot dry weight. A, Average dry weight of squash shoots measured 30 days after emergence. B, Average dry weight of squash roots measured 30 days after emergence. In the fertilization experiment, plants were fertilized with water soluble fertilizer (20:20:20, 3.5 g/mL, 20 mL/plant) once each week. Values with same letter within a column are not significantly different according to Fisher’s protected LSD test (p = 0.05). Vertical bars represent mean ± S.D. (n = 10).
a aa
b
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
With fertilization Without fertilization
Dry shoot
Dry
sho
ot
wei
ght
(g)
IN937b Non-treated control
a
aa
b
0.00
0.05
0.10
0.15
0.20
0.25
With fertilization Without fertilization
Dry root
Dry
ro
ot
wei
ght
(g)
IN937b Non-treated control
74
CholorophyII
IN937b SE76 IN937a ASM CK
SP
AD
0
10
20
30
40
50
a
c
bc
ab
c
Figure 2-4. Chlorophyll content in leaves of squash plants treated with PGPR. PGPR
strains were applied twice as a soil drench to squash roots. The concentration of IN937b, SE76, and IN937a was adjusted to 108 CFU/mL, and 20 mL was applied to each plant. Actigard® (30 mg/L; 20mL/plant) was applied as a positive SAR control of Phytophthora blight. The SPAD measurement were carried out 5days after inoculation. SPAD values were measured on three leaves at same position on each plant. Values with same letter within a column are not significantly different according to Fisher’s protected LSD test (p = 0.05). Vertical bars represent mean ± S.D. (n = 5).
75
CHAPTER 3 THE ROLE OF BIOFILM FORMATION AND COLONIZATION IN THE RHIZOSPHERE
BY BACILLUS SUBTILIS IN937B WITH SYNTHESIS OF SURFACTINS
Introduction
Plants attract beneficial rhizobacteria such as PGPR by exudates released from
plant roots (Raaijmakers et al., 2009b). PGPR are directly involved in the nutrition,
health and quality of plants. They have a role in cycling carbon, nitrogen, and
phosphorus, as well as suppressing soil-borne plant pathogens (Garbeva et al., 2004).
The PGPR-mediated nutrient cycling is related to increase nitrogen-fixation and the
availability of soluble phosphates in the soil. The mechanism by biocontrol-PGPR
protects plant roots from pathogens include microbial antagonism, biofilm formation and
the stimulation of induced systemic resistance in the host plants. The functional
activities and community diversities of PGPR are positively affected by plant roots.
Roots are able to release a wide range of organic and inorganic compounds into the
rhizosphere including sugars, amino acids, phenolics, enzymes, siderophores, etc.
(Pinton et al., 2001). The root exudates create specific habitat conditions suitable for
microbial development, and play an important role in affecting microbial populations,
availability of nutrients,
PGPR colonize the plant roots steadily by chemotaxis attraction and biofilm
formation (Yaryura et al., 2008). The process includes bacterial attachment to the root,
movement and proliferation of PGPR along the elongating root, and penetration of the
epidermis (Liu et al., 2011). PGPR are likely to locate roots through particular signals
emanating from the roots. Carbohydrates and amino acids also stimulate PGPR to
move towards roots (Bais et al., 2006). PGPR can also produce biofilms to initiate
beneficial interactions with host plants. Biofilm foster increased resistance to certain
76
environmental stresses as well as antimicrobial tolerance (Danhorn and Fuqua, 2007).
Biofilm is a multicellular structure which adheres to the root surface as a protective
mechanism against adverse environmental conditions. The formation of biofilm is
associated with root colonization by bacteria such as PGPR (Ahmad et al., 2011).
Root colonization by PGPR is considered integral for PGPR to be successful as
biocontrol agents (Whipps, 2001). The biocontrol effect depends on population densities
of PGPR applied for suppressing pathogens (Kumar et al., 2011a). For example, P.
fluorescens Pf1TZ could protect grape vine (Vitis vinifera L. cv. Chardonmy) plantlets
against Botrytis cinerea after 3 weeks of bacterial inoculation (Kilani-Feki et al., 2010).
The population density of Pf1TZ colonized roots required for the bacteria to protect
plants from the pathogen was 104 CFU/mg of fresh roots. However, shortly after PGPR
inoculation, the population declines progressively over time (vanVeen et al., 1997).
Natural complexity of the rhizosphere (soil pH, temperature, nutrients, competitors, and
predators) poses challenges to PGPR survival in different soil environments (Strigul and
Kravchenko, 2006). As effective biocontrol agents, PGPR must establish themselves in
the rhizosphere, which is affected by many abiotic and biotic factors.
Bacillus PGPR strains are widely considered to be capable of colonizing roots
(Compant et al., 2010). For example, B. pumilus SE34 grew on the root surface and
intercellularly penetrated roots of pea plants, allowing B. pumilus SE34 to get nutrients
from the plant and compete for root colonization (Benhamou et al., 1996). Many Bacillus
strains are capable of adopting and switching between free-living (planktonic) and
biofilm (surface-attached) forms. The planktonic form can rapidly proliferate as free-
floating individual cells, whereas the biofilm form are a unit of well-organized bacterial
77
cells which helps to tolerate adverse environments (O'Toole et al., 2000). As highly
structured microbial communities, biofilm helps bacterial strains adhere to a soild
surface where nutrients in an aqueous environment tent to concentrate. It is a survival
strategy that designed to anchor PGPR in a nutritionally advantageous environment and
permit their escape when growth factors have been exhausted.
Bacillus strains have the ability to form biofilm both in the laboratory and in nature
(O'Toole et al., 2000). Under laboratory conditions, B. subtilis 3610 produced
architecturally complex colonies on solid medium and formed floating biofilm called
pellicles at the interface of the liquid medium and air (Lopez et al., 2009). Bacillus
subtilis 3610 forms robust biofilms on the root surfaces of tomato plants (Chen et al.,
2013). Biofilm formation by Bacillus plays an important role in protecting plants from
pathogen attacks. B. subtilis ATCC 6051 suppress Pseudomonas syringae on
Arabidopsis by competition on colonization (Bais et al., 2004). Paenibacillus polymyxa
B1 and B2 protect Arabidopsis thaliana from infections by Pythium aphanidermatum by
biofilm formation on root tips (Timmusk et al., 2005).
Biofilm formation by Bacillus spp. is associated with production of surfactins,
which are well-known secondary metabolites with antifungal activities. Surfactins
produced by Bacillus spp. are required for biofilm formation and for the successful bio-
control of plant pathogens. A knock-out mutant of surfactin synthase resulted in the loss
of swarming motility of B. subtilis which involves the differentiation of vegetative cells
and population migration across solid root surfaces (Kinsinger et al., 2003; Koumoutsi et
al., 2004). Mutants deficient in surfactin synthesis were unable to suppress pathogens
(Bais et al., 2004b). B. amyloliquefaciens FZB 42 produces surfactins. Knock-out
78
mutants of surfactin production were unable to repress Fusarium oxysporum grown on
agar plates (Koumoutsi et al., 2004). Moreover, surfactins obtained from B.
amyloliquefaciens strain S499 were capable of triggering an oxidative burst which is
mediated perception by tobacco root cells that induce a defensive state in the plant
(Henry et al., 2011). These results indicate that surfactins play an important role in root
colonization by Bacillus spp. and in disease suppression via direct inhibition and
stimulation of the host plant’s immune system (Ongena and Jacques, 2008).
The possible role that surfactins play in biofilm formation includes functioning as
bio-surfactants, which have hydrophobic properties for water-insoluble substrates (Ron
and Rosenberg, 2001), and influences on attachment and detachment of
microorganisms to roots (Branda et al., 2001). Bacillus subtilis has long served as the
powerful model organism for laboratory studies the mechanisms of biofilm formation
(Vlamakis et al., 2013). However, studies need to be done to expand our understanding
of Bacillus PGPR biofilm formation on plant roots, or whether biofilm influences root
colonization by Bacillus PGPR.
In the present study, we focused on in vitro assays of biofilm formation and
colonization of selected Bacillus PGPR strains in the rhizosphere of squash. The
selected strains of Bacillus PGPR were able to suppress Phytophthora blight disease
caused by P. capsici through soil inoculation (see Chapter 2). The aim of this work were
the following: (1) to evaluate the ability of Bacillus PGPR to form biofilm in vitro, (2) to
investigate the timing and concentration of B. subtilis IN937b required for biofilm
formation, (3) to determine biosynthesis of surfactin characteristics of PGPR strains
79
corresponding to biofilm formation, (4) to assess whether IN937b would colonize and
form biofilm on squash roots.
Materials and Methods
PGPR Strains and Media
All Bacillus PGPR strains used in this study were stored in nutrient broth (NB)
containing 15% glycerol at -80 ℃ prior to use. Bacterial cultures were prepared first by
streaking each PGPR strain taken from ultra-cold storage onto nutrient agar (NA; Difco)
plates, then incubating them at 28 ℃ for 24 h.
For pellicle formation experiments, a minimal medium MSgg (5 mM potassium
phosphate, pH 7, 100 mM MOPS, pH 7, 2 mM MgCl2, 700 µM CaCl2, 50 µM MnCl2, 50
µM FeCl3, 1 µM ZnCl2, 2 µM thiamine, 0.5% glycerol, 0.5% glutamate, 50 µg ml-1
tryptophan, 50 µg ml-1 phenylalanine and 50 µg ml-1 threonine) was used (Branda et al.,
2001).
Surfactin production by PGPR was elicited using No. 3S medium containing 10 g
of soybean peptone, 10 g of glucose, 1 g of KH2PO4, and 0.5 g of MgSO4·7H2O per liter
of the medium (pH 7.0) (Tsuge et al., 2005).
Bacteria were grown in Luria broth (LB) medium for DNA extraction: 10 g of
bacto tryptone (Difco), 5 g of yeast extract (Difco), 10 g of NaCl per liter of the medium
(pH 7.0) (Gellert et al., 1976).
Pellicle Formation
PGPR strains were grown in 5 mL Nutrient Broth (NB; Difco) medium on a rotary
shaker incubator at 150 rpm overnight at 28 ℃. Five microliters of cell cultures were
transferred into 1 mL liquid MSgg medium in 10 mL plastic tubes (or 30 mL liquid MSgg
medium in 100 mL beakers). The plastic tubes and beakers were placed in the
80
incubator at 28 ℃ for 3 days when the pellicle, a biofilm formed at the air-liquid
interface, was observed and photographed with a Canon EOS Rebel T1i digital camera
(Canon, Tokyo, Japan).
Colony Morphology Assay
For colony architecture analysis on the solid media, PGPR strains were streaked
on NA plates to get the single colonies. The plates were incubated at 28 ℃ for at least
24 h to observe morphology of the single colonies. The architecture was clearly
observable under a dissecting microscope (Olympus DF Planapo Ⅸ, Olympus, Tokoya,
Japan). Colonies were imaged using a Canon EOS Rebel T1i digital camera.
Biofilm Production Assay in Microtiter Plates
Bacillus PGPR strains were grown in 10 mL NB medium at 28 ℃ on a rotary
shaker incubator at 150 rpm overnight. Biofilm production assays were performed with
MSgg medium (Branda et al., 2001). The inoculum for the microtiter plate assays was
obtained by diluting the Bacillus cells to 106 CFU/mL. Final inocula were diluted to an
identical density and calculated based on OD600. One hundred microliters of the
inoculum were grown in each well of the 6-well polyvinylchloride (PVC) microtiter plates,
each containing 10 mL liquid MSgg medium. The bacterial cells were grown at room
temperature for 7 days when they reached the stationary growth phase. Biofilm
production and morphologies were imaged using a Canon EOS Rebel T1i digital
camera.
DNA Extraction and Polymerase chain reaction (PCR) Analysis
Bacillus strains were inoculated into 10 mL LB medium and incubated on a rotary
shaker incubator at 150 rpm overnight at 28 ℃. DNA extraction was carried out using
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the Qiagen kit (Qiagen QIAamp DNA mini kit, Qiagen, Valencia, CA) according to the
manufacturer’s instructions. Polymerase chain reaction (PCR) reactions were carried
out based on the methodology and specific primers established by Hsie et al. (2004).
Primers used are listed in Table 3-1, that amplify genes encoding the antibiotics
synthetases of iturin A, bacillomycin D, surfactin, zwittermicin A, and fengycin (Hsieh et
al., 2004; Ramarathnam et al., 2007).
The PCR reaction was performed in a final volume of 25 µL containing 2.5 µL of
10X PCR buffer, 0.5 µL of dNTP mix, 1.0 µL each of PCR primers, 0.5 µL of Taq DNA
polymerases, and 1 µL each of template genomic DNA. Amplification was done with a
conventional thermal cycler (BioRad, Laboratories, Hercules, CA) with an initial
denaturing at 94 ℃ for 1 min, annealing at 46 ℃ for 30s and extension at 72 ℃ for 1
min, for a total of 30 cycles, and followed by a final extension of 72 ℃ for 10 min.
Following amplification, the PCR reaction mix was separated through 1% agarose gel
electrophoresis.
PCR products were purified using a Qiagen PCR purification kit (Qiagen,
Valencia, CA). DNA sequencing was conducted at the Interdisciplinary Center for
Biotechnology Research, University of Florida, Gainesville, FL. Sequence data were
processed and analyzed with Basic Logal Alignment Search Tool (BLAST) software of
National Center for Biotechnology Information (NCBI)
(http://www.ncbi.nlm.nih.gov/BLAST). The amino acid sequence of sfp in IN937b was
determined using the translation tool in the software DNAMAN version 7 (Lynnon
Biosoft, Quebec, Canada). The amino acid sequences for B. amyloliquefaciens FZB42,
B. cereus E33L, B. megaterium QM B1551, B. polymyxa SC2, B. pumilus ATCC 7061,
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B. subtlis 168, B. subtilis IN937b, Brevibacillus brevis NBRC 100599, and B.
licheniformis ATCC14580 were obtained from GenBank (http://www.ncbi.nlm.nih.gov/).
Multiple sequence alignment and Phylogenetic analysis were done with DNAMAN
version 7. Phylogenetic analysis was performed using a non-rooted neightbor-joining
tree with Poisson-correction (Bootstrap values: 1000 replicates) distances.
Surfactin Extraction
Surfactin was extracted from PGPR cultures according to Yakimov et al. (1995).
Bacterial cells were grown in No. 3S medium on a rotary shaker incubator at 150 rpm
for 48h at 28 ℃. Cells were separated from No.3S medium by centrifugation at 3,000
rpm for 30 min at room temperature. The supernatant was subjected to acid
precipitation by adding 6 M HCl to achieve a final pH of 2.0 and allowing the precipitate
to form at 4 ℃ overnight (Yakimov et al., 1995). The pellet was collected by
centrifugation at 3,000 rpm for 30 min at room temperature, and washed two times with
DI water (pH 2.0, adjusted by 6M HCl). The pellet was dissolved in methanol (pH 7.0,
adjusted by 1 M NaOH) by stirring for 2 h, and was placed in the refrigerator at 4 ℃
overnight. The crude material was collected for further analyses.
HPLC/ESI-MSn Assay of Surfactin Production
The crude material extracted as described above and the surfactin standard
(Sigma-Aldrich, St. Louis, USA) were sent to Dr. Jodie V. Johnson’s laboratory in the
Chemistry Department at the University of Florida, Gainesville, FL for detecting the
presence of surfactins and for further quantitative analyses. The extracts and surfactin
standard were analyzed via high performance liquid chromatography combined with
electrospray ionization tendem mass spectrometry (HPLC/ESI-MSn).
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Both (+) and (-) ESI-MSn collision-induced dissociations (CID) were conducted to
quantify surfactins produced in the testing samples. Both positive and negative ion
modes are provided in the Table 3-4. The gradient was changed to allow better
separation in (+)ESI-MS compared to (-)ESI-MS. (+)ESI-MS was chosen for further
analyses. The relative amount of different surfactin isoforms was detected in the crude
extracts of IN937b expressed in terms of HPLC/(+)ESI-MS peak areas and relative
areas. The sum of the intensities of the [M+H]+ and [M+Na]+ were used to produce the
ion-peaks for the surfactins. The peak area was obtained by integration of the
appropriate peak in the relative abundance of surfactins in the ion chromatograms
(Table 3-5).
Screening IN937b Mutant for Rifampicin Resistance
Spontaneous mutants with rifampicin-resistance were selected from nutrient agar
amended with rifampicin (NAR) plates according to Bolstridge et al. (2009) with
modifications. Wild type of B. subtilis IN937b was plated on NA amended with 10 µg/mL
rifampicin (NAR) and incubated at 28 ℃ for 48 h. The mutants with the same
morphology as the wild type were transferred to NAR with the higher concentration of
rifampicin (25 µg/mL). And the above step was repeated until the mutants had the ability
to resist 75 µg/mL rifampicin. Stability of the rifampicin resistance was confirmed by
sub-culturing ten times on NA and then on NAR (75 µg/mL). All mutants were stored in
20% glycerol at -80 ℃ (Bolstridge et al., 2009).
Root Treatment by IN937b Rifampicin-Resistance Mutant
A spontaneous mutant of IN937b resistant to 75 µg/mL rifampicin was used to
monitor changes in the IN937b population introduced into the rhizosphere. A single
colony of IN937b with rifampicin resistance was inoculated in 10 mL NB containing 50
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µg/mL rifampicin in 50 mL centrifuge tubes. After the tube was incubated at 28 ℃ on a
rotary shaker incubator at 150 rpm overnight, 1 mL of the bacterial suspension was
inoculated into 200 mL NB in 500 mL flasks, and incubated for 24 h under the same
conditions as mentioned above. The cell pellet was collected by centrifugation at 3000
rpm for 15 min and washed twice with sterile DI water. The cell concentration used in
this experiment was adjusted to 108 CFU/mL, determined by serial dilutions and plating.
Squash seeds were planted in plastic pots (7.62 cm high and 10.16 cm at the
opening with 8 drain holes) filled with potting mix. One seed was placed in each pot. In
the greenhouse, two-week-old squash plants were treated with rifampicin resistant
IN937b by soil drench at 107 CFU/g soil per plant.
Rhizosphere Sampling
Samples of the squash rhizosphere were taken from each of the pots 1, 3, 5, 7,
and 14 days after IN937b treatment. The roots of the squash plants were shaken
vigorously to get rid of bulk soil that did not tightly adhere to the roots. Four replicates in
each treatment (IN937b and non-treated control) were obtained at each sampling time.
The rhizosphere soil is defined as the soil still adhering to the roots after vigorous
shaking. The roots and rhizosphere soil were collected together, and 50 g of soil and
the roots were placed in a 500 mL plastic container with 100 mL sterile DI water. Total
bacterial populations were assessed by serial dilutions and plating of the resulting soil
suspension on NAR medium after the container was shaken on a rotary shaker
incubator at 150 rpm for 30 min at room temperature.
Recovery of IN937b Mutant with Rifampicin-Resistance from Roots
For measuring internal root colonization, roots were separated from the
rhizosphere samples mentioned above and were collected by rinsing the roots three
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times with DI water. Root samples were surface disinfected with sodium hypochlorite
(1%) for 5 min followed by ethanol (70%) for 30s. Then the roots were chopped and
ground with 2 mL sterile DI water in a sterile mortar using a pestle. In order to make
sure that no surface bacteria remained after surface-disinfection, one intact root for
each sample before chopping was placed on the NAR medium for surface bacteria
growing. The population of inoculated bacteria in internal root tissues was determined
by serial dilutions and plating of the homogenate on the NAR plates and incubated at 28
℃ for 48 h.
Results
Architecture of Pellicles and Colonies of Bacillus spp.
Tubes made of PVC which stimulate biofilm formation (Pedersen, 1990), were
used to test if selected Bacillus PGPR had the ability to form biofilm on an abiotic
surface. The Bacillus strains were allowed to grow on the surface of the liquid MSgg
medium. Five days after inoculation, the biofilm was observed as a flat layer at the
interface between the air and liquid medium (Figure 3-1 A.). PGPR strains IN937b,
1216S, GB03, IN937a, INR7 and 1PC-19 had the ability to form a thick, floating biofilm
composed of a large group of bacterial cells. However, strains SE76, 1PC-11, SE34,
SE49, SE52 and T4 exhibited poor growth of the layer, and they were unable to form a
biofilm initially.
When spotted on NA plates, the Bacillus strains produced colonies with
morphological characteristics of their corresponding pellicles (Figure 3-1 B.). Colonies
varied widely in morphology among Bacillus strains. Five days after inoculation, PGPR
strains IN937b, 1216S, GB03, IN937a, INR7 and 1PC-19 had wrinkled (because of its
rough appearance) colonies which were consistent with the result of pellicle formation in
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the liquid medium test (Figure 3-1 A.). SE76, 1PC-11, SE34, SE49, SE52 and T4
exhibited smooth (because of its small and flat appearance) colonies on NA plates
which were not able to form biofilm in the liquid medium. The wrinkled bacterial colonies
grew wider on the solid medium surface than the smooth colonies did.
Closer examination of the colonies under a dissecting microscopy (×10
magnification) revealed a highly structured bacterial community, with clearly observable
architecture. As shown in Figure 3-2, the wrinkled pattern of IPC-19 generated denser
layers compared to the other test PGPR strains. IN937b and 1216S consisted of many
wave-like columns that projected upward from the agar surface. IN937a, INR7 and
GB03 had aerial structures composed of cells bundled together in a flat form.
In Vitro Test for Biofilm formation
The highly structured biofilm was formed by the Bacillus strains on the surface of
a liquid MSgg medium in a beaker. Bacillus PGPR strains SE34, SE52, SE76, and T4
failed to form biofilm (Figure 3-3). GB03 and 1216S formed an extremely thin, fragile,
and smooth pellicle that lacked a distinctive architecture. Contrastingly, PGPR strains
IN937b and 1PC-19 formed a thick pellicle with an intricate vein-like appearance on its
surface, and a thick and highly folded structure of biofilm was clearly visible through the
beaker.
Effect of Bacterial Density on Biofilm Formation
Based on the observation of the in vitro test for biofilm formation, wherein
bacterial cells move in the liquid without a solid surface, we found that the process of
self-organization in biofilm has been linked to bacterial density and bacterial growth
speed in the liquid-air interface. In order to determine the influence of PGPR densities
on biofilm formation, we assayed the selected PGPR strains in liquid MSgg medium in
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polystyrene plates that has 6 wells each. Biofilm formation and progression were
visually inspected daily for 7 days. The initial concentration in liquid MSgg medium was
calculated as 104, 103, and 102 CFU/mL. At days 1 and 2 after bacterial inoculation,
IN937b had a high concentration (104 CFU/mL) and the positive strain 1PC-19 (104
CFU/mL) formed thin, loosely associated aggregations of bacterial cells at the surface
of the liquid medium in the well. At days 2 and 3, a thin pellicle comprised of both live
cells and extracellular materials formed on the air-surface interface in the wells
containing IN937b at 104 and 103 CFU/mL and 1PC-19 (Figure 3-4). From days 3 to 5,
the pellicle at the interface thickened and developed a thick mat of cording and
reticulations. In addition to bacterial cells, extracellular materials from biofilms produced
by the strain 1PC-19 adhered to and moved up the walls of the well during days 2 to 5
(Figure 3-4 and Table 3-2). IN937b at the lowest concentration (102 CFU/mL) and the
negative strain SE52 failed to form biofilm and the reticulation was incomplete, bacterial
cells were unable to disperse in the well and could not self-organize.
PCR Detection of Lipopeptide Genes in Bacillus PGPR Strains in Relation to Biofilm Formation
A 675-bp PCR product was amplified from IN937b using the primers specific to
the sfp gene which is responsible for surfactin production (Figure 3-8). Amplification of
the PCR products indicates that IN937b and IN937a harbor the sfp gene and may
produce surfactins. No PCR product was amplified from SE76 and SE52 using any of
the primers tested. No product was amplified from IN937b with primers specific to the
genes encoding the antibiotics iturin A, fengycin, zwittermicin A, and bacillomycin D
(Table 3-3).
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DNA sequences analyzed by BLAST software revealed that this PCR fragment in
IN937b had 100% similarity to B. subtilis 168 (Accession No. AL009126) whose
genome has been fully sequenced. The results indicated that IN937b is positive for the
presence of the 4’-phosphopantetheinyl transferase gene sfp for surfactin synthesis.
Alignment of the predicted amino acid sequences revealed that all the 5 amino acid
residues are conserved in all Bacillus for surfactin synthetase (Figure 3-9). The
phylogenetic tree based on amino acid sequences of sfp revealed a close relationship
among B. subtilis IN937b, B. subtilis 168, and B. amyloliquefaciens FZB42 (Figure 3-
10).
HPLC/MS Analysis of Surfactins Production in B. subtilis IN937b
The methanol extracts of No. 3S culture of strain IN937b were separated by
HPLC/ESI-MS, and a clear peak pattern of surfactins was eluted based on the
molecular weight range of the standard sample. The molecular weight (MW) of surfactin
isoforms was determined by ESI-MS (Figure 3-5 A-G), and the HPLC fractions that had
more than one peak were found to represent a cluster of isoforms at MW 993, 1007,
1021, 1035, 1049, and 1063 Da. The fatty acids in surfactins were determined (Table 3-
4). The structure of standard surfactins consists of a seven amino acid cyclopeptide with
a saturated fatty acid chain (R) attached. The chain length of R varies from C9 to C14
leads to detection of the major molecular weights. Surfactin isoforms with MW 1035 and
1021 Da contained the highest levels of relative abundance of 45.76% and 39.84%,
respectively (Table 3-5).
Survival of IN937b in the Rhizosphere
Recovery of IN937b from the rhizosphere began at 1 day after treatment to two
weeks after soil drench with IN937b (Figure 3-6). There was a high bacterial density at
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106 CFU/g soil per plant in the potting mix at 1 day after application. The population
declined dramatically to 104 CFU/g soil per plant at 5 days after treatment. And then the
populations slowly decreased to 103 CFU/g soil per plant at 14 days after treatment.
Survival of IN937b in Squash Roots
Internal root colonization by IN937b was evaluated by serial dilutions and plating
on rifampicin-amended NA plates. No IN937b cells were detected on plates from the
non-treated squash roots after surface-sterilization. IN937b was recovered in squash
roots at 1 day after treatment. However, the endophytic population of IN937b recovered
from the roots fluctuated from 101 to 103 CFU/g fresh root (Figure 3-7). At 2 weeks after
IN937b treatment, an increasing trend in the population of IN937b was observed with
level up to 103 CFU/g fresh root. Bacterial densities of IN937b colonized in the roots
were as high as 103 CFU/g fresh root 14 days after treatment.
IN937b colonized the rhizosphere and also occupied the internal tissues of
squash roots after application. At the early stage of colonization, the population of
IN937b survived in the rhizosphere. Over a 14 days period, the bacterial population in
the rhizosphere decreased, while endophytic population in root tissue survived longer
and accumulated gradually.
Discussion
Bacillus strains had the ability to form biofilms in liquid or solid medium. Roots of
some plants contain tightly bound Bacillus strains that form abundant biofilms. Both in
vitro and in vivo assays have showed that the rapid formation of biofilm under laboratory
conditions reflect the ability of biofilm formation in the natural environment (Bais et al.,
2004b). Interestingly, all strains which morphologically vary in colony morphology, are
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able to form biofilms, implying that Bacillus strains developed the same major adaptive
strategy to survive in the soil environment.
Under laboratory conditions, the bacterial cells produced extracellular polymers
that bind cells together and provide structural support for biofilm that resided at the
liquid-air interface of the medium in glass beakers. The lack of solid surface at this
interface imposes a greater requirement for self-organization on the constituent cells.
When grown in beakers filled partly with a liquid medium, bacterial strains form large
groups of cells on the liquid surface. During this process, bacterial strains produce
extracellular polysaccharide (EPS), which affords better access to oxygen (Flemming
and Wingender, 2010). EPS is effective in binding cells together and in anchoring them
to the beaker walls.
The architecture of the colony and the thickness of the pellicle formed at the air-
liquid interface are both influenced by surfactin produced from different Bacillus strains
(Leclere et al., 2006). Among these, B. subtilis is believed to form a robust biofilm when
it attains a certain population density. The motile cells of B. subtilis migrate to the air-
medium interface and biofilm formation rate is associated with cell populations. B.
subtilis cells assimilate sufficient oxygen to produce submerged fermentation that is
subjected to colony morphology and growth rate. Faster growth of bacterial cells causes
nutrient depletion in the culture, resulting in faster sporulation of Bacillus spp.
Sporulation of B. subtilis is delayed in biofilm when compared to that of B. subtilis at
submerged fermentation (Lindsay et al., 2005). Therefore, Bacillus cells in the biofilm
remain in their metabolically active state for a longer period of time compared to those
in their sporulation stage.
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Bacillus subtilis often found in the rhizosphere might be closely associated with
plant roots. Recently, the ability of Bacillus in the rhizosphere to form beneficial biofilm
has been well documented (O'Toole et al., 2000; Vlamakis et al., 2013). The behavior of
bacterial cells tending to attach and aggregate near plant root surfaces or internal roots
by self-organization can be considered a type of biofilm (Lugtenberg et al., 2001). These
communities have spatial and temporal organization due to cellular differentiation
(Ramey et al., 2004).
Biofilm helps microbial populations to swarm over the root surface and reach new
niches to attain more nutrients (Branda et al., 2001). On the well-colonized root surface,
approximately 10 - 40% of root surfaces can be occupied by Bacillus that formed biofilm
(Vlamakis et al., 2013). Motility of the bacterial cells is important for biofilm formation
and colonization by the bacteria. The motility that performed as swimming and
swarming was achieved through the cell movement of colonies. The strains with fast
motility usually spread or occupy large area in the medium, indicating that motility is an
important mechanism for Bacillus colonization, especially for those introduced to a new
environment (Kinsinger et al., 2003). As a first step of biofilm formation, the motility that
helps bacterial strains to access favorable colonization sites in the rhizosphere involves
rapid and coordinated population migration. As a second step, bacterial cells must
attach to highly structured biofilms on solid surfaces or pellicles and adhere to roots and
soil particle surfaces (Danhorn and Fuqua, 2007).
The population of IN937b recovered from potting mix was 103 CFU/g of fresh root
10 days after IN937b introduced into the rhizosphere of squash plants. The results from
the survival assay of IN937b showed poor colonization in the rhizosphere, although
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IN937b formed robust biofilms in defined medium (and likely on plant roots as well).
Also, results from the microtiter assays showed that biofilm formation strongly depended
on the initial population of IN937b inoculated in the medium, indicating that initial
population might be critical for biofilm formation.
In this experiment, the initial populations of IN937b were high and dramatically
decreased in the potting mix 2 weeks after treatment. Potting mix is a relatively simple
environment compared to the soil, which is a mixture of mineral compounds and
microorganisms. Bacterial behavior may be influenced by environmental factors
including the nutrients, water content in the soil and bacterial movement in the root
systems. On the other hand, the rapid colonization of IN937b in the internal roots
indicated that IN937b had potential to predominate in the root competition. These also
enable Bacillus strains to find colonization sites and acquire nutrients for multiplication
over a long period.
Different types of distribution were found in Bacillus strains. Some bacteria were
localized at the root cap while others were evenly distributed along the root surface.
Fluorescence in situ hybridization (FISH) revealed that the favorable site for Bacillus
strains to form biofilm could be extended 2-30 µm in the rhizosphere outward from root
surface of wheat (Watt et al., 2006). Effective colonization leads to plant growth
promotion (Ongena et al., 2005). Bacillus strains secrete a large number of compounds
into the rhizosphere which are beneficial to plant roots and shoot growth. Bacillus works
as biofertilizer for many plant species due to its interaction with plant roots through
biofilms on root surfaces (Seneviratne et al., 2010).
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Bacillus strains in this study were selected from different crops in diverse
environmental niches. These bacteria showed distinct features, especially in colony
morphology, biofilm formation and biocontrol activity. However, the ability to form
biofilms is still highly conserved in these test strains. These results imply that different
Bacillus spp. have developed the same strategy for adapting to different environments
(Chen et al., 2013).
B. subtilis IN937b produced surfactins. Surfactins are well known to stimulate
biofilm formation (Bais et al., 2004a). For example, surfactins are detected in the
rhizosphere of cucumber and tomato inoculated with B. subtilis strains (Ongena et al.,
2007; Kinsella et al., 2009). The movement of bacterial cells is largely dependent on the
production of surfactins, which help cells to spread over the root surfaces (Raaijmakers
et al., 2006). Surfactin production is widespread in Bacillus genus (Price et al., 2007).
Surfactin-producing Bacillus species can be easily isolated from soils. However,
surfactins are susceptible to degradation by other microorganisms in nature or in the
laboratory. In sterile soils without interference from other microorganisms, levels of
surfactins remained stable for 25 days (Asaka and Shoda, 1996).
Surfactins are important for microorganisms because they lower the surface
tension of water. In addition, surfactins have antimicrobial activities due to their abilities
to penetrate cellular membranes (Heerklotz and Seelig, 2007). Moreover, surfactins act
as a signal in the quorum-sensing to regulate bacterial population density in response to
environmental changes (Lopez et al., 2009). Surfactins are reported to act as a
stimulus in biofilm formation. High levels of surfactins were observed during colonization
and biofilm formation by B. amyloliquefaciens S499 on tomato roots. Recovery of
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surfactins from the rhizosphere indicated that bacterial colonization on the root leads to
high levels of surfactins that stimulate biofilm formation (Nihorimbere et al., 2012).
Surfactin biosynthesis requires enzymes called surfactin synthetases. The gene
sfp serves a regulatory role as well as a more direct role in surfactin synthesis. In this
study, PCR primers specific to the regions within sfp were used to screen a number of
Bacillus spp. strains for presence of the sfp. Only IN937b was confirmed to carry the sfp
gene responsible for the production of surfactins.
To confirm the PCR result for surfactin production, HPLC and quantitative
analysis were adopted to detect the amount of surfactins produced from B. subtilis
strain IN937b. Surfactins were extracted from No. 3S culture broth of IN937b by classic
methods including acid precipitation with HCl, recrystallization and extraction by organic
solvents such as methanol. Results of HPLC were consistent with the PCR detection.
Naturally occurring surfactins are a mixture of different molecules based on chain
length, β-hydroxy fatty acid, and amino-acid sequence (Lin and Jiang, 1997). In the
present study, several isomers of surfactins with different molecular weights were
detected.
Biofilm is important for bacteria to colonize in the rhizosphere and to act as a
biocontrol agent (Bais et al., 2004b). The biocontrol efficacy of B. subtilis against
Rhizoctonia solani has been shown to be achieved by the production of surfactins and
iturin A. It is possible that biofilm and surfactin production allowed B. subtilis and its
close relatives, such as B. amyloliquefaciens, to efficiently colonize plant roots and also
to provide protection to their hosts. A knockout of the sfp gene resulted in flat and small
colonies of the bacterium. This knockout mutant spread very little on the agar surface
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(Branda et al., 2001) revealing that the mutant failed to form mature aerial structures
related to biofilm formation. Loss of surfactin biosynthesis led to a deficiency in motility
and reduced colonization of roots and surfaces. Biocontrol of P. syringae infection of
Arabidopsis roots by B. subtilis 6051 is related to surfactin production on the surface of
the root (Bais et al., 2004b). B. subtilis 6051 can produce a variety of lipopeptides
including surfactins that are important for maintaining the aerial structure of biofilms.
Upon root colonization, B. subtilis 6051 formed a stable, extensive biofilm and secreted
surfactins, which act together to protect plants against infection by pathogenic bacteria
(Morikawa, 2006). It is strongly suggested that the production of surfactins is essential
for biofilm formation and colonization of plant roots.
In conclusion, our results suggest that many Bacillus PGPR strains are capable
of forming biofilms. It will be important to construct knock-out mutants of surfactin
synthesis genes. Future work is needed to determine the physiological relevance of the
regulation of surfactin synthesis genes and biofilm formation. The surfactin mutants will
continue to be useful in future studies on colonization models in the squash
rhizosphere, and this capability may be important for disease suppression. Biofilm
formation by IN937b may increase its colonization to the squash root. However, the
exact mechanisms in which biofilm formation contributes to plant biocontrol still remain
unknown. Successful colonization on root tissue is a first step critical in eliciting plant
biocontrol activities against soil borne pathogens. In the future, it will be imperative to
characterize the signaling molecules released by the plant host that improve Bacillus
spp. ability to colonize and form biofilm on the root surfaces.
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Table 3-1. List of primer sequences used for biosynthetic genes of surfactins, bacillomycin, iturin A, Fengycin, and Zwittermicin A.
Antibiotic Primer sequence
Surfactin F: ATGAAGATTTACGGAATTTA
(Hsieh et al., 2004) R: TTATAAAAGCTCTTCCTACG
Bacillomycin D
F:GAAGGACACGGGCAGAGAGTC
(Athukorala et al., 2009) R: CGCTGATGACTGTTCATGCT
Iturin A F: GATGCGATCTCCTTGGATGT R: ATCGCATGTGCTGCTTGAG
Fengycin F: TTTGGCAGCAGGACAAGTTT
(Ramarathnam et al., 2007)
RGCTGTCCGTTCTGCTTTTTC
Zwittermicin A F: TTGGGAGTTTATACAGCTCT
R: GACCTTTTGAAATGGGCGTA
Table 3-2. Biofilm formation by Bacillus strains with initial bacterial populations
Concentration (CFU/mL)a Biofilm formation time (Days)b
IN937b (104) 2 IN937b (103) 3 IN937b (102) 5 IN937b (101) None IN-19 (104, positive strain) 3 SE52 (104, negative strain) None
a The inocula for the microtiter plate assays were obtained by diluting the Bacillus cells to approximately 104, 103, 102 and 10 CFU/mL. One hundred µL of inocula were grown in 6-well PVC microtiter plates containing 10 mL of liquid MSgg medium at room temperature for 5 days before imaging. b Biofilm growth were observed by visual inspection. The biofilm formation was checked daily for the timing of biofilm formation. Table 3-3. Detection of the genes related to biofilm formation
Antibiotica
PCR results
IN937b SE76 SE52
Surfactin +b - - Zwittermicin A - - - Bacillomycin D - - - Iturin A - - - Fengycin - - -
a Five selected antibiotics are antimicrobial peptides that are associated with biosurfactins and in the fitness of Bacillus strains in the rhizosphere. b Polymerase chain reaction (PCR) screening using specific primers (Table 3-1) for antibiotic biosynthetic genes in strains IN937b, SE76 and SE52. + represents detection of the genes. – represents no detection of the genes.
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Table 3-4. Identification of surfactin ions by HPLC/ESI-MS
Surfactin ionsa
(+)ESI-MSb (-)ESI-MS
R mass R, u
MW, u
[M+H]+ [M+Na]+ [M-H]- [M-2H+Na]-
C9H19 127.1 993.6 994.3 1016.5 992.5 1014.6
C10H21 141.2 1007.7 1008.4 1030.6 1006.6 1028.7
C11H23 155.2 1021.7 1022.5 1044.7 1020.7 1042.8
C12H25 169.2 1035.7 1036.4 1058.6 1034.6 1056.7
C13H27 183.2 1049.7 1050.4 1072.6 1048.6 1070.7
C14H29 197.2 1063.7 1064.5 1086.7 1062.7 1084.8 a The surfactin consists of a seven-amino acid cyclopeptide with a saturated fatty acid chain (R) attached. The chain length of R varies from C9 to C14 to provide the major molecular weights (u) detected. Mass R represents the molecular weight of saturated fatty acid chain. MW represents the molecular weight of surfactins. b HPLC-ESI-MS ions assay for surfactins. Both positive and negative ion modes were given in the table. (+)ESI-MS represents positive ion mode; (-)ESI-MS represents negative ion mode. Table 3-5. HPLC/(+)ESI-MS peak areas and relative areas for the detected surfactins in
the crude extracts of IN937b.
Isomer 1 Isomer 2
MWa RT-1 minb
Area-1c %Total RT-2 min
Area-2 %Total
993 55.94 1,081,996,051 1.63 nd 0 0.00
1007 56.91 1,966,233,219 2.96 58.67 363,655,897 0.55
1021 58.07 26,434,118,167 39.81 59.29 1,430,159,095 2.15
1035 58.80 30,383,407,593 45.76 60.05 1,612,113,268 2.43
1049 59.92 2,308,838,698 3.48 61.02 219,785,102 0.33
1063 60.60 601,132,399 0.91 nd 0 0.00
SubTotal 62,775,726,127 3,625,713,362
Total 66,401,439,489 a (+)ESI-MS mass chromatograms of the [M+Na]+ ions of the surfactins produced by IN937b, which match those of the surfactin standard. b RT represents retention time from 54 to 63 during which surfactins elute through the HPLC column. c The sum of the intensities of the [M+H]+ and [M+Na]+ were used to produce the ion-peaks for the surfactins. The peak area was integrated in the appropriate relative abundance ion chromatograms for determining the abundance of surfactins.
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A
B Figure 3-1. Architecture of pellicles and colony morphology of PGPR strains. A. Pellicle
formation at the air-liquid interface in liquid MSgg medium of 12 Bacillus strains. Cells of Bacillus strains were inoculated in the liquid medium in the standing tubes at 28 °C for 3 days. The band at the top of the liquid medium represents the floating pellicle. B. Colony formation of 12 Bacillus strains on the surface of solid MSgg medium. Bacteria were streaked to form single colony on the medium and incubated at 28 °C for 24 hours.
99
Figure 3-2. Pellicle development of Bacillus strains. Pellicle formation at the air-liquid
interface under a dissecting microscope (10 ×). Strains that formed biofilm in pellicle formation assays (Fig 3.1 A) were selected to observe pellicle morphology under a microscope. The floating pellicles at the top were carefully placed on top of a microscopic glass slide, and photographed (10 ×) for surface structure.
Figure 3-3. Biofilm development properties of Bacillus strains. Biofilm formation from
selected Bacillus strains on the surface of a liquid MSgg medium in a beaker. Bacteria were inoculated in liquid MSgg medium in a beaker for floating pellicle development. The images were taken at room temperature 5 days after inoculation. All four strains (IN937b, IPC-19, 1216S, GB03) formed a biofilm with a wrinkled (rough surface appearance) pattern on the surface as shown in the top set of photo. The strains shown in the bottom set of photos were biofilm-negative phenotype.
100
Figure 3-4. Biofilm formation rate of Bacillus strains. Biofilm growth was observed by
visual inspection. The inocula for the microtiter plates were obtained by diluting the Bacillus cells to approximately 104, 103, and 102 CFU/mL. One hundred µL of inocula were grown in 6-well PVC microtiter plates containing 10 mL MSgg liquid medium at room temperature for 5 days before imaging. 1: IN937b (104 CFU/mL), 2: IN937b (103 CFU/mL), 3: IN937b (102 CFU/mL), 4: Control, 5: IPC-19 (+) (104 CFU/mL), 6: SE52 (104 CFU/mL) (-).
101
Figure 3-5. HPLC/MS analysis of the fraction from crude extraction from B. subtilis IN937b culture.
A
B
102
Figure 3-5. Continued
C
D
103
Figure 3-5. Continued
E
F
104
Figure 3-5. Continued
G
A) Chromatogram of surfactins detected by HPLC with (+)ESI-MS. MW 1016, 1030, 1044, and 1058 with black underline, 1072 and 1086 are not shown in the figure, B) Surfactins with MW 993: HPLC/(+)ESI-MS/MS of m/z 1016 [M+Na]+: At least four isomers: two defined by m/z 707 and 772 and two defined by m/z 693 and 786, C) Surfactins with MW 1007: HPLC/(+)ESI-MS/MS of m/z 1030 [M+Na]+: At least four isomers: two defined by m/z 707 and 786 and two defined by m/z 693 and 800 product ions, D) Surfactins with MW 1021: HPLC/(+)ESI-MS/MS of the m/z 1044 [M+Na]+ ions shows the presence of at least four (4) isomers: m/z 707, 800, 693, and 814 product ions, E) Surfactins with MW 1035: HPCL/(+)ESI-MS/MS of m/z 1058 [M+Na]+ At least four isomers with two defined by m/z 707 and 814 and two defined by m/z 693 and 828, F) Surfactins with MW 1049: HPLC/(+)ESI-MS/MS of m/z 1072 [M+Na]+ ions shows more isomers: three defined by m/z 707 and 828, on the top, the m/z 707 shifted to m/z 721, G) Surfactins with MW 1063: HPLC/(+)ESI-MS/MS of m/z 1086 [M+Na]+ ions: The two major isomers were those defined by m/z 707 and 842 and on the bottom, the m/z 707 shifted to m/z 721.
105
Figure 3-6. Colonization of the rhizosphere of squash plants by a mutant of IN937b with
rifampicin resistance. The data are expressed as log10 CFU per gram of potting mix. Data points are the means and standard errors of four replicates. Data were collected at the 1, 3, 5, 7 and 14 days after treatment. Values with same letter within a column are not significantly different according to Fisher’s protected LSD test (p = 0.05). Vertical bars represent mean ± S.D. (n = 5).
6.75 a
5.56 b
4.44 c
3.18 d 3.17 d
0
2
4
6
8
1 3 5 7 14
Lo
g c
fu/g
so
il
Time (Days)
106
Figure 3-7. Colonization of the squash roots by a mutant of IN937b with rifampicin
resistance. The data are expressed as log10 CFU per gram of fresh squash root. Data points are the means and standard errors of four replicates. Data were collected at the 1, 3, 5, 7 and 14 days after treatment. Populations of IN937b colonizing squash seedling roots. Values with same letter within a column are not significantly different according to Fisher’s protected LSD test (p = 0.05). Vertical bars represent mean ± S.D. (n = 5).
2.26 d
1.58 e
3.35 b
2.65 c
3.81 a
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
1 3 5 7 14
Lo
g c
fu/g
ro
ots
Time (Days)
107
Figure 3-8. PCR product profiles of the sfp gene. Gel displaying bands created by using primers for the sfp gene. Lane 1: Ladder 100-1000 bp makers, 2: SE76, 3: IN937a, 4: T4, 5: IN937b, 6: Negative control.
1 2 3 4 5 6
700bp
500bp
108
24FZB42
36E33L
0QMB1551
40SC2
27ATCC7061
24168
7IN937b
25NBRC100599
24ATCC14580
Consensus
.
.
.
M
.
.
.
.
.
.
.
.
V
.
.
.
.
.
.
.
.
L
.
.
.
.
.
.
.
.
Y
.
.
.
.
.
.
M
.
T
.
.
.
.
.
.
I
.
E
.
.
.
.
.
.
E
.
Q
.
.
.
.
.
.
S
.
L
.
.
.
.
.
.
K
.
I
.
.
.
.
.
.
V
.
L
.
.
.
.
.
.
V
.
T
.
.
.
.
.
.
D
.
R
.
.
.
.
.
.
S
.
E
.
.
.
.
.
.
I
.
D
M
.
.
.
.
.
P
.
I
K
.
.
.
.
.
T
.
V
I
.
.
M
.
M
L
.
M
F
M
.
I
M
K
N
.
K
A
K
.
A
K
I
E
.
A
I
I
.
I
I
Y
N
.
A
Q
Y
.
Y
Y
G
D
.
V
L
G
.
A
A
V
C
.
S
Q
I
.
L
V
Y
Q
.
F
H
Y
.
K
Y
M
I
.
V
L
M
.
C
M
D
W
.
H
D
D
.
P
D
R
W
.
A
D
R
.
A
Q
P
G
.
V
I
P
.
V
L
L
R
.
C
H
L
.
M
P
S
I
.
H
A
S
.
E
E
A
S
.
A
R
Q
.
K
A
G
D
.
G
K
E
.
D
G
E
L
.
A
Q
E
.
S
Q
E
Q
.
S
I
N
.
F
F
D
S
.
L
D
E
M
N
N
R
W
.
D
Q
R
N
R
R
M
H
.
P
L
F
G
F
M
M
Y
.
E
K
M
S
L
M
A
N
.
I
P
T
C
Q
A
A
L
.
F
F
F
L
A
M
V
L
.
H
V
I
S
L
V
64FZB42
76E33L
0QMB1551
80SC2
67ATCC7061
64168
47IN937b
65NBRC100599
64ATCC14580
Consensus
S
N
.
S
S
S
I
P
S
A
D
.
D
A
P
P
E
E
E
V
.
E
E
E
E
E
E
K
E
.
C
K
K
K
K
K
R
R
.
S
R
R
R
R
R
E
E
.
Y
A
E
E
E
K
K
K
.
F
A
K
K
R
K
C
A
.
E
A
C
C
V
V
R
N
.
S
E
R
R
N
G
R
S
.
L
R
R
R
R
R
F
Y
.
L
F
F
F
F
Y
Y
H
.
F
R
Y
Y
R
R
H
H
.
Q
F
H
H
N
Y
K
S
.
R
L
K
K
P
P
E
A
.
R
I
E
E
A
E
D
D
.
K
D
D
D
D
D
A
R
.
S
A
A
A
S
A
H
A
.
N
R
H
H
Y
S
R
R
.
F
R
R
R
R
R
T
F
.
L
T
T
T
A
T
L
I
.
L
L
L
L
L
L
I
I
.
G
L
L
L
L
I
G
G
.
R
G
G
G
A
G
D
C
.
L
E
D
D
D
E
M
V
.
S
V
V
V
V
V
L
I
.
A
L
L
L
L
L
I
S
.
K
V
V
V
V
A
R
R
.
Q
R
R
R
R
R
T
L
.
A
Q
S
S
S
H
A
V
.
A
M
V
V
L
I
A
L
.
S
I
I
I
I
I
A
G
.
L
H
S
S
C
S
K
K
.
L
D
R
R
E
E
A
I
.
L
M
Q
Q
A
T
Y
L
.
S
Y
Y
Y
Y
F
G
S
.
E
D
Q
Q
E
H
L
M
.
S
L
L
L
I
L
D
S
.
N
P
D
D
S
P
P
P
.
L
F
K
K
N
N
A
V
.
S
D
S
S
D
E
97FZB42
114E33L
0QMB1551
117SC2
100ATCC7061
97168
80IN937b
98NBRC100599
97ATCC14580
Consensus
G
Q
.
N
E
D
D
E
Q
I
V
.
I
I
I
I
I
I
S
P
.
S
V
R
R
E
R
F
I
.
V
F
F
F
Y
F
G
D
.
E
E
S
S
D
T
V
R
.
T
T
T
T
Y
E
Q
M
.
G
E
Q
Q
N
G
E
C
.
V
G
E
E
A
R
Y
P
.
F
N
Y
Y
Y
Y
G
V
.
G
G
G
G
G
G
K
C
.
Q
K
K
K
K
K
P
K
.
P
P
P
P
P
P
Y
L
.
L
V
C
C
F
A
I
Q
.
L
V
I
I
L
A
P
H
.
R
R
P
P
K
E
A
G
.
T
H
D
D
S
G
L
R
.
T
I
L
L
F
L
P
P
.
G
P
P
P
P
N
D
Q
.
A
S
D
D
N
D
M
L
.
P
F
A
A
F
F
H
P
.
G
H
H
H
C
H
F
E
.
Y
F
F
F
F
F
N
G
.
Q
N
N
N
N
N
.
M
.
.
.
.
.
.
.
.
P
.
.
.
.
.
.
.
.
Q
.
.
.
.
.
.
.
.
L
.
V
.
.
.
.
.
.
S
.
S
.
.
.
.
.
I
V
.
I
L
I
I
V
I
S
S
.
A
S
S
S
S
S
H
H
.
H
H
H
H
H
H
S
S
.
C
S
S
S
S
S
G
G
.
D
G
G
G
G
G
R
E
.
D
D
R
R
E
K
W
W
.
I
W
W
W
W
W
I
V
.
G
V
V
V
V
I
.
.
.
T
.
.
.
.
.
.
.
.
A
.
.
.
.
.
V
V
.
V
V
I
I
V
V
C
V
.
V
C
G
C
C
C
134FZB42
154E33L
34QMB1551
154SC2
137ATCC7061
134168
117IN937b
135NBRC100599
134ATCC14580
Consensus
A
A
.
F
A
A
A
A
G
.
F
.
.
.
.
.
.
.
V
T
.
P
I
F
F
T
A
D
K
.
E
D
D
D
H
G
S
F
M
A
D
S
S
D
A
K
A
I
H
A
Q
Q
S
Q
P
P
S
P
P
P
P
Q
P
I
V
V
M
V
I
I
V
I
g
G
G
G
G
G
G
G
G
G
I
V
I
V
I
I
I
I
V
d
D
D
D
D
D
D
D
D
D
I
V
L
I
I
I
I
V
V
E
E
V
E
E
E
E
E
E
K
Q
D
R
E
K
K
Q
K
M
M
I
I
I
T
T
I
I
K
N
E
S
K
K
K
C
K
P
P
R
P
P
P
P
P
P
G
N
F
N
I
I
I
I
I
T
V
K
R
D
S
S
E
N
I
D
L
N
L
L
L
L
F
D
V
A
K
A
E
E
D
D
I
M
L
T
I
I
I
I
I
.
K
.
.
.
.
.
.
.
.
M
.
.
.
.
.
.
.
A
A
S
M
A
A
A
A
A
K
D
R
E
K
K
K
T
K
R
G
S
S
R
R
R
H
R
F
V
G
Q
F
F
F
Y
F
F
L
E
L
F
F
F
F
F
S
T
L
T
S
S
S
A
S
P
D
F
M
A
K
K
P
P
T
S
V
E
D
T
T
A
S
E
E
S
E
E
E
E
E
E
Y
K
R
R
Y
Y
Y
V
H
S
A
I
E
E
S
S
E
D
D
Q
L
R
D
D
D
D
D
L
V
N
I
L
L
L
L
L
Q
M
N
V
L
L
L
L
M
A
K
Y
A
S
A
A
A
E
K
L
E
V
Q
K
K
K
K
173FZB42
193E33L
73QMB1551
194SC2
176ATCC7061
173168
156IN937b
174NBRC100599
173ATCC14580
Consensus
H
P
I
R
P
D
D
H
D
P
N
A
G
A
K
K
P
E
D
E
K
V
E
D
D
N
S
Q
Q
Y
E
R
E
E
E
E
Q
K
D
E
Q
Q
Q
R
R
T
I
S
Y
E
T
T
V
L
D
E
Y
S
A
D
D
S
S
Y
G
Q
V
Y
Y
Y
Y
Y
F
F
W
L
F
F
F
F
F
Y
L
A
L
F
Y
Y
Y
Y
H
T
I
T
H
H
H
D
H
L
Y
L
V
L
L
L
L
L
.
.
.
S
.
.
.
.
.
W
W
F
W
W
W
W
W
W
S
T
S
T
S
S
S
T
T
M
R
A
V
M
M
M
L
M
k
K
K
K
K
K
K
K
K
K
e
E
E
E
E
E
E
E
E
E
S
A
C
A
A
S
S
S
S
F
V
A
I
F
F
F
Y
F
I
L
I
S
I
I
I
I
I
k
K
K
K
K
K
K
K
K
K
Q
A
M
I
L
Q
Q
A
Q
A
T
I
L
T
E
E
R
A
G
G
K
R
G
G
G
G
G
K
E
Q
T
K
K
K
M
K
G
G
I
G
G
G
G
G
G
L
L
P
F
L
L
L
L
L
S
M
K
A
S
S
S
S
S
L
I
D
A
Y
L
L
I
L
P
S
F
S
G
P
P
P
P
L
P
R
L
L
L
L
L
L
D
V
L
Q
S
D
D
Q
D
S
D
R
V
S
S
S
S
S
F
I
D
L
F
F
F
F
F
S
T
I
E
T
S
S
A
S
V
I
Q
I
A
V
V
I
V
R
S
V
N
R
R
R
R
K
L
A
D
E
L
L
L
K
L
K
P
I
I
S
H
H
N
N
208FZB42
233E33L
103QMB1551
229SC2
211ATCC7061
208168
180IN937b
209NBRC100599
208ATCC14580
Consensus
D
N
L
R
E
Q
Q
L
E
D
D
S
F
D
D
D
D
Q
G
S
K
K
G
G
G
Q
G
H
P
D
S
Q
Q
Q
S
R
V
N
E
G
A
V
V
I
A
S
L
L
C
T
S
S
T
S
I
L
L
F
L
I
I
I
V
E
V
V
Q
A
E
E
S
E
L
F
T
S
L
L
L
Q
T
P
K
F
T
P
P
P
S
P
D
D
D
F
D
D
D
D
P
G
R
E
S
H
S
S
S
G
H
Q
S
H
E
H
H
R
Y
E
E
L
F
A
S
S
E
P
P
L
Y
P
P
P
P
A
P
C
V
K
Q
C
C
C
W
C
F
E
Q
Y
F
Y
Y
A
Y
I
N
V
A
V
I
I
F
I
R
T
D
A
Q
K
K
Q
Q
T
V
L
Q
P
T
T
Q
A
Y
M
P
S
Y
Y
Y
Y
Y
E
R
T
Y
S
E
E
E
E
A
D
N
R
L
V
V
I
I
.
L
S
.
.
.
.
.
.
.
R
I
.
.
.
.
.
.
.
P
K
.
.
.
.
.
.
.
S
G
.
.
.
.
.
.
.
V
.
.
.
.
.
.
.
D
D
.
L
D
D
D
D
D
E
Y
.
G
P
P
.
P
P
E
M
.
N
T
G
.
G
G
Y
A
.
Y
Y
Y
.
Y
Y
K
S
.
I
Q
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180IN937b
230NBRC100599
226ATCC14580
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109
Figure 3-9. Comparison of the predicted amino acid sequence for the sfp gene present in several Bacillus strains. Multiple sequence alignment of sfp homologs from B. amyloliquefaciens FZB42 (FZB42), B. cereus E33L (E33L), B. megaterium QM B1551 (QMB1551), B. polymyxa SC2 (SC2), B. pumilus ATCC 7061 (ATCC7061), B. subtlis 168 (168), B. subtilis IN937b (IN937b), Brevibacillus brevis NBRC 100599 (NBRC100599), B. licheniformis ATCC14580 (ATCC14580). Conserved amino acid residues were shaded in the following manner: black = 100% identity or conserved substitution, grey ≥ 75% identity, light grey ≥ 50% identity, white < 50% identity.
Figure 3-10. Phylogram of sfp homologs from several Bacillus strains. Multiple sequence alignment of sfp homologs from B. amyloliquefaciens FZB42 (FZB42), B. cereus E33L (E33L), B. megaterium QM B1551 (QMB1551), B. polymyxa SC2 (SC2), B. pumilus ATCC 7061 (ATCC7061), B. subtlis 168 (168), B. subtilis IN937b (IN937b), Brevibacillus brevis NBRC 100599 (NBRC100599), B. licheniformis ATCC14580 (ATCC14580). An unrooted neighbor-joining phylogenetic tree using Poisson-correction (Booststrp values: 1000 replications) distances was constructed based on the amino acid sequences alignment.
110
CHAPTER 4 THE ROLE OF SUBTILOSIN A-LIKE ANTIMICROBIAL PEPTIDE IN SUPPRESSION
OF PHYTOPHTHORA CAPSICI BY BACILLUS SUBTILIS IN937B
Introduction
Bacillus spp. are widely recognized as producers of secondary metabolites of
low-molecular-weight compounds that are deleterious to other microorganisms
(Thomashow et al., 2007). Approximately 4% of the B. subtilis 168 genome is devoted
to the biosynthesis of secondary metabolites (Kunst et al., 1997). Similarly,
approximately 8.5% of the whole genome in B. amyloliquefaciens FZB42 is dedicated to
synthesize antibiotics which have significant potential for plant disease management
(Chen et al., 2007).
The classification of antimicrobial metabolites produced by Bacillus spp. is largely
based on non-ribosomal or ribosomal biosynthesis (Montesinos, 2007). Non-ribosomal
peptide antibiotics are often modified posttranslationally, such as lipopeptides (surfactin,
Chapter 3) and polyketides (Stein, 2005). This group of antibiotics possess antimicrobial
capabilities, also involved in biofilm, swarming development, and induction of plant
resistance (Ongena et al., 2005; Ongena et al., 2007). Secondary metabolites through
ribosomal biosynthesis act as pheromones in quorum-sensing, or lead to programmed
cell death. For example, bacteriocins have fungicidal and bactericidal capabilities for
suppressing Alternaria solani and Erwinia carotovora (Hammami et al., 2012).
Antibiotic production by Bacillus spp. may play a major role in maintaining
population density when they are introduced into the soil. The nutrition resources for
microbial proliferation and survival are limited in the rhizosphere. Over time,
consumption of the limiting nutrients will influence the dynamics between Bacillus and
other microbes. Bacillus spp. compete with their neighbors for space and nutrients by
111
impairing or killing other microbes (Hibbing et al., 2010). As microbial growth inhibitors,
antibiotics result in changes of microbial community structure by inhibiting growth of
different species or related strains of the same species. In general, Bacillus spp.
produce a wide range of antibiotics that are highly specialized for a given habitat may
target different potential competitors (Stein, 2005). On the other hand, the stimulation or
repression of biosynthesis of antimicrobial metabolites helps Bacillus respond
appropriately to the environment such as changes in minerals and carbon levels
(Mannanov and Sattarova, 2001).
Antifungal antibiotic production is involved in disease suppression. Bacillus spp.
produce many antibiotics which directly suppress plant fungal pathogens, such as
Rhizoctonia solani, Botrytis cinerea, Verticillium dahliae, and Sclerotinia sclerotiorum
(Raaijmakers et al., 2002; Ongena and Jacques, 2008). Some commercial Bacillus
strains such as FZB42, GB03, QST713, and MBI600 produce cyclic lipopeptides (CLPs)
including fengycin, surfactin, or iturin. These CLPs have been associated with
suppression of plant pathogens (Joshi and Gardener, 2006). For example, iturins and
surfactin were active against R. solani, and pure fengycin inhibited Pyricularia oryzae
(Asaka and Shoda, 1996; Joshi and Gardener, 2006). Studies on surfactin lipopeptides
showed that surfactins caused hyphal swelling and fungal growth inhibition (Vitullo et
al., 2012). When growing on an agar surface supplemented with surfactins, F.
oxysporum mycelia exhibited retarded growth accompanied by increased branching and
rosette formation as well as hyphal swelling. The production of bacillomycin and
fengycin by B. subtilis UMAF6614, UMAF6639 and UMAF8561 were implicated as the
mechanism of control of powdery mildew on melon caused by Podosphaera fusca
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(Romero et al., 2007). Mycosubtilin-overproducing mutants of B. subtilis ATCC6633
were more effective than the wild-type strain in controlling Pythium damping-off on
tomatoes (Leclere et al., 2005).
Disintegration of membranes is well known as the primary function of antibiotics
against bacteria, fungi and oomycetes. Subtilosin A produced by several B. subtilis
strains (Stein et al., 2002) has a macrocyclic structure with three inter-residual linkages
(Marx et al., 2001), which involve permeabilized cell membrane by insertion of the
peptide after interaction with a cell-surface receptor (Moll et al., 1999). The damage
results in irreversible modifications leading to cell death (Aly et al., 2013). For
oomycetes, zoospore germination and sporangial formation represent the important
steps in the asexual life cycle and the spreading of disease, and as a result,
suppressing zoospore and sporangial germination may be an efficient strategy for the
management of diseases caused by oomycetes (Yoshida et al., 2001). A close
correlation between the production of antifungal compounds and the prevention of
zoospore germination has been reported in biocontrol of Pythium by B. cereus (Shang
et al., 1999).
To date, only a small proportion of the antimicrobial compounds produced by soil
bacteria have been studied (El-Hassan and Gowen, 2006). One challenge is that only 1-
5% of soil bacteria can be cultured. Moreover, biosynthesis of antibiotics by
microorganisms is often regulated by nutrition resources (carbon level) and greatly
influenced by environmental factors (pH and temperature) in the soil.
In the context of biocontrol, a successful exploratory attempt to repress
Phytophthora blight by using antagonistic B. subtilis has been reported on pepper (Lim
113
and Kim, 2010). The mechanisms involving Bacillus spp. against Phytophthora blight on
squash have not yet been adequately explored. The Bacillus PGPR strain IN973b
significantly suppressed Phytophthora blight on squash in the greenhouse and
displayed in vitro inhibitory activities against P. capsici.
The specific objectives of this study were to investigate the mechanisms involved
in biocontrol of P. capsici on squash with B. subtilis strain IN937b. To evaluate the
inhibitory effect of antifungal compounds from the cell-free culture of IN937b, P. capsici
zoospore germination, hyphal growth, and its effect in detached leaf assays were
determined. To evaluate the role of antifungal compounds secreted by B. subtilis
IN937b in the inhibition of P. capsici, we set out to determine the gene that is
responsible for biosynthesis of Subtilosin A like litibiotics and its structural
characteristics.
Materials and methods
Dual Culture Assay
All the bacterial isolates were screened for antagonism against P. capsici on
potato dextrose agar (PDA) plates using the dual culture technique (Yoshida et al.,
2001). One plug of P. capsici isolate #151 mycelium (6-mm diameter) was placed in the
center of PDA medium. The bacterial strain was streaked on the other side of medium 3
cm away from the center. The plates were incubated at 25 °C for 7 days after which the
inhibition zone was measured and recorded.
Antibiotic Extraction and Analysis
For isolation of the antagonistic compounds responsible for inhibiting fungal
growth, strain IN937b was cultured in No.3S medium (Tsuge et al., 2005) and incubated
at 28°C on a rotary shaker incubator at 150 rpm for 2 days. The culture was centrifuged
114
at 3,000 rpm for 30 min and the supernatant was collected and acid precipitated as
described in Chapter 3. The methanol (HPLC grade) extracts were further analyzed by
HPLC for preliminary characterization.
Antifungal Activity of Methanol Extracts Against Phytophthora capsici
Mycelial Growth. Crude extract of IN937b cell-free culture was dried in a clean
fume hood (Labconco Corporation, Kansas City, Mo). The dry matter was dissolved in
the methanol (40 mg/mL). The effective concentration (EC50) of the extracts was
determined for P. capsici by serial dilution. The EC50 was considered to be the lowest
concentration of the methanol extract that caused 50% inhibition of fungal growth. The
corn meal (CM) agar was amended with methanol extracts at a final concentration of 0,
0.2, 0.4, 0.8, 1.0 mg/mL. An agar plug (6 mm in diameter) was taken from the colony of
P. capsici on PARPH medium and placed at the center of CM agar amended with
methanol extracts. Each treatment had 3 replicates. All the plates were incubated at 25
°C for 9 days. The diameter of the colony was measured every 3 days after incubation.
The inhibition rate was defined as 𝐈𝐧𝐡𝐢𝐛𝐢𝐭𝐢𝐨𝐧 𝐫𝐚𝐭𝐞(%) = [(𝐍𝟎 − 𝐍𝟏) / 𝐍𝟎] × 𝟏𝟎𝟎%,
where N0 is the mycelium diameter at a final concentration of 0 mg/mL, and N1
represents the mycelial diameter of colonies on the medium amended with methanol
extracts at different concentrations.
Zoospore Germination. Zoospore suspensions were prepared using the
method described in Chapter 2. One hundred microliters of zoospore suspension were
mixed with 100 µL of methanol extracts at the final concentration of 0, 10, 20, 30, or
40mg/mL (total volume is 200 µL) for the extracts. The mixture was flooded on the CM
agar and placed in the hood to allow to air dry. The plates were incubated at 25 °C for 3
h and the percentage of zoospore germination on each plate was determined by
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viewing under a microscope at 400× magnification. The relative proportion of
germinated zoospores was calculated (Keinath, 2007) by dividing the number of
germinated zoospores by the total number (50) of zoospores counted on each plate.
Two plates were used for each concentration as replicates and the experiment was
conducted twice under the same conditions.
Sporangial Formation. Phytophthora capsici was grown on plugs in the DI
water with the methanol extracts added at final concentrations of 0 and 40 mg/mL of
crude extract. Four Petri dishes were used for each concentration and were placed
under the light (one 20-watt General Electric F20T12-CW linear fluorescent lamp) at
room temperature to induce sporangial formation. After 48 h, the plug was examined for
sporangial formation under a dissecting microscope at 70× magnification. The number
of sporangia formed on the plug was counted.
Detached Leaf Assays
Detached leaf assays described by Chiou and Wu (2001) were modified to
investigate the direct and indirect effects of methanol extracts on P. capsici on the
leaves. Young, fully developed leaves were collected from 2-week-old squash plants
and the leaf surfaces were rinsed three times with sterile DI water. Twenty microliters of
methanol extracts were dropped onto the detached leaves, and leaves were placed in
the Petri dishes supplemented with moist filter paper. Twenty microliters of zoospore
suspensions (104 zoospore/mL) were placed on leaves 3 cm away from the site where
methanol extracts were applied. The leaves were placed under the light at room
temperature for 7 days. Disease severity was visually assessed using a scale of 0-5,
where 0 = no symptoms, 1 = less than 10%, 2 = 10-25%, 3 = 50%, 4 = 51-75%, and 5 =
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75-100% of leaves covered with brown lesions (Chiou and Wu, 2001). Leaves treated
with methanol served as a non-treated control. Each treatment had three replications.
HPLC/MS Assays of Surfactin Production
Subtilosin A was quantified using HPLC/ESI-MSn by Dr. Jodie V. Johnson as
described in Chapter 3.
DNA Extraction and PCR Analysis
Bacillus PGPR strain IN937b was inoculated into 10 mL of LB medium and
incubated on a rotary shaker incubator at 150 rpm overnight at 28 °C. DNA extraction
was carried out using the Qiagen QIAamp DNA mini kit according to the manufacturer’s
instructions. PCR reactions were carried out based on the methodology and specific
primers established by Velho et al. (2013). The following primers were used for
amplifying the subtilosin A (sboA) gene that are critical for production of the
antimicrobial peptides subtilosin A:
sboA-f: 5’-CATCCTCGATCACAGACTTCACATG-3’
sboA-r: 5’- CGCGCAAGTAGTCGATTTCTAACAC-3’.
The PCR reaction was performed in a final volume of 25 µL containing 2.5 µL of
10X PCR buffer, 0.5 µL of dNTP mix, 1.0 µL each of PCR primers, 0.5 µL of Taq DNA
polymerases, and 1 µL each of template genomic DNA. Amplification was done with a
conventional thermal cycler (BioRad, Laboratories, Hercules, CA) with an initial
denaturing at 94 ℃ for 1 min, annealing at 50 ℃ for 30s and extension at 72 ℃ for 1
min, for a total of 35 cycles, and followed by a final extension of 72 ℃ for 10 min.
Following amplification, the PCR reaction mix was separated through 1% agarose gel
electrophoresis.
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Results
Dual Culture Assays
To evaluate the antifungal activities of all selected Bacillus PGPR, bacterial
strains were tested using a dual culture assay. Inhibition zones were clearly observed
between the mycelial growth and bacterial colonies (Figure 4-1). Results of the dual
culture assays for these strains are given in Table 4-1. Strain 1PN-19 exhibited the
greatest inhibition to growth of P. capsici with an inhibition zone of 11 mm. Bacillus
strains Companion, GB03, IN937a and IN937b showed moderately inhibitory activities
with an inhibition zone less than 10 mm, whereas strains SE34, SE49, SE52, SE76,
INR7, 1PC-11 and Actinovate AG had no effects on the growth of P. capsici.
Methanol Extracts of IN937b Culture
The active substances were collected from the cell-free culture of IN937b
(surfactin production No. 3s medium) described in Chapter 3. At pH 2.0, the substances
of antibiotic activity were precipitated from the cell-free supernatant and stabilized in
acidic solution at 4 °C. The active substances were then extracted by methanol under
neutral conditions (adjusted to pH=7.0). After evaporation, the dry weight of the active
substances was approximately 40 mg from 1 liter of liquid medium culture.
Chromatographic Separation by HPLC
The extracts contained numerous compounds. A compound with MW 3400 Da in
the extracts was abundant. At a retention time of 45-49 min, an m/z ratio for an ion had
been selected for fragmentation. The compound produced m/z 1699 [M-2H]2- and m/z
1710 [(M-H+Na)-2H]2- ions in (-)ESI-MS and m/z 1701 [M+2H]2+ and m/z 1712 [(M-
H+Na)+2H]2+ ions in (+)ESI-MS. The molecular mass of the corresponding metabolite
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has the same molecular weight as subtilosin A which has been found to be produced by
a great variety of B. subtilis strains (Stein, 2005).
PCR Detection of sbo Genes in Bacillus PGPR IN937b
A 666-bp PCR product was amplified from IN937b using the primers sboA-f and
sboA-r specific to fragment corresponding to the B. subtilis subtilosin gene. DNA
sequences analyzed by BLAST software revealed that this PCR fragment in IN937b had
100% similarity to B. subtilis 168 (Accession No. AL009126) whose genome has been
fully sequenced. The results indicated that IN937b is positive for the presence of the
gene sbo for cyclic peptide antibiotic subtilosin A synthesis. Amplification of the PCR
products indicates that IN937b may produce subtilosin A. The result is consistent with
the observation in the samples tested by HPLC.
Assays of Hyphal Growth Inhibition
The antifungal activity of the methanol extracts was confirmed from the culture of
IN937b. One hundred microliters of methanol extracts (40 mg/mL) were pipetted into
holes (6 mm diameter) 2 cm away from the edge of the CM agar. The methanol extracts
of IN937b strongly inhibited mycelial growth of P. capsici on CM agar 7 days after being
added into the hole (Figure 4-2 A.). The average growth of the colony toward the hole
was less than 5 mm, representing an inhibition of 75% compared to the unaffected
colonies. When the concentration of the methanol extract solution was reduced to 10
mg/mL, the hyphae of P. capsici were able to continue growing toward the inhibition
zone, but the layer of hyphae were thinner and more branches were observed
compared to the unaffected hyphae (Figure 4-2 B.).
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Effect of Methanol Extracts on Mycelium Morphology
The hyphal morphology affected by the methanol extracts was examined in detail
by microscopy. The tips of P. capsici hyphae grown on CM agar plants were normal
(Figure 4-3 A.) from the edge of the mycelia, whereas the extract challenged hyphae of
P. capsici were typically induced branching, and the hyphal tips became swollen as
shown in Fig 4-3.
The EC50 of the Extracts
The 0.2, and 0.4 mg/mL methanol extracts had no effects on mycelial growth of
P. capsici (Table 4-1). None of the extracts completely inhibited mycelial growth. The
extracts at 0.8 and 1.0 mg/mL had a stronger effect on the mycelial growth than the
other concentrations. The estimated EC50 of the extracts was 1.0 mg/mL.
Effect of Methanol Extracts on Zoospore Germination
Zoospores began to germinate 2 h after incubation at 28 °C. As the concentration
of methanol extracts increased, a prominent reduction in the percentage of germinated
zoospores was observed (Figure 4-4). The greatest inhibition (46.2%) was achieved
with the 40 mg/mL extract, and none of the tested concentrations completely inhibited
P. capsici.
Effect of Methanol Extracts on Sporangial Formation
For the assessment of the inhibitory effects of methanol extracts on sporangial
formation, P. capsici hyphae were induced under the light for sporangial formation for 2
days at room temperature. Normal sporangia with an intact shape were present in DI
water. The hyphae treated with methanol extracts had deformed and swollen tips, the
same as the observations for mycelial growth inhibition (Figure 4-5). The 40 mg/mL
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methanol extract completely inhibited sporangia formation (Table 4-1), and adversely
affected the production of sporangia.
Effect of Methanol Extracts on Detached Leaf Infection
The methanol extracts were evaluated for their potential to suppress
Phytophthora blight using a detached leaf bioassay (Figure 4-6). Fully expanded young
leaves of squash plants from the greenhouse were used in this experiment. The 40
mg/mL extract significantly (p≤0.05) inhibited the development of Phytophthora blight
compared to the methanol control (Table 4-3). No disease developed on leaves treated
with the 40 mg/mL extract.
Discussion
Bacillus subtilis strain IN937b isolated from tomato rhizosphere (Murphy et al.,
2000) showed inhibition activities against P. capsici on PDA plates (Zhang et al., 2010).
Subtilosin A, an antimicrobial peptide produced by B. subtilis IN937b was found to be
involved in reduction in zoospore germination, sporangial formation and hyphal growth
of P. capsici, and in vitro the suppression of lesion expansion on the detached leaves
after being challenged with P. capsici.
B. subtilis is well known for producing various antimicrobial substances as
secondary metabolites to inhibit plant pathogens (Mannanov and Sattarova, 2001).
Antibiotics secreted by B. subtilis include non-ribosomally synthesized peptide
antibiotics (e.g. surfactin and mycobacillin) and ribosomally synthesized precursor
lantibiotics (e.g. subtilin and subtilosin A) (Stein et al., 2002; Bais et al., 2004b; Leclere
et al., 2005; Arguelles-Arias et al., 2009).
The antifungal activity of bacteria might be due to competition for nutrients with
other microorganisms (Janisiewicz et al., 2000). Bacillus strains which contribute to
121
biologically controlling plant diseases synthesize antibiotic compounds that suppress or
kill other microbes, modify microbial attachment to plant tissue surfaces, and improve
survival of the Bacillus cells in their habitat. The findings in our studies, together with the
fact that biocontrol efficiency of Bacillus strains were closely correlated with antibiotics
production, strongly support the relevance of antibiosis as an important factor involved
in the protection of squash against P. capsici by these PGPR strains.
Hyphal Growth Inhibition
Sixteen Bacillus strains were tested for their ability to inhibit hyphal growth of P.
capsici on PDA plates. These strains varied in their capacities against P. capsici. Strain
IN937b showed moderate levels of fungal inhibition (inhibition zone was approximately
5 mm). Thus, the antibiotics produced by IN937b were presumably partially responsible
for suppressing the hyphal growth of P. capsici (Ahmed et al., 2003; Zhang et al., 2010).
Methanol extract of strain IN937b was increased inhibition of hyphal growth
compared to IN937b cells did on PDA plates. In the present study, antibiotics produced
by IN 937b were condensed by precipitation with HCl from the culture supernatants and
were extracted from the pellet with methanol (Vater et al., 2002). The condensed
extracts had a 40-fold increase in inhibition of hyphal growth compared with the culture
of IN937b. The very weak antifungal activity displayed by the culture of IN937b
suggested that antibiotics are produced in very small quantities. The methanol extracts
could have resulted in the enrichment of antibiotics and therefore led to the increased
antifungal activity. Application of antibiotics rather than bacterial cells into agricultural
systems may improve disease control (McManus et al., 2002).
122
Zoospore Germination and Sporangial Formation
We demonstrated that zoospore germination and sporangial formation of P.
capsici were significantly inhibited by the methanol extracts of IN937b. Zoospore
germination is believed to be the most important phase in the asexual life cycle and
spread of P. capsici (Judelson and Blanco, 2005). In the present study, the extracts
were able to reduce P. capsici zoospore germination with results similar to those
previously reported (Kone et al., 2009). Microscopic observations revealed that
sporangial formation of P. capsici was completely inhibited in DI water pretreated with
the extracts. These findings support the hypothesis that secretion of antifungal
compounds is the primary mechanism contributing to the suppression of B. subtilis
strain IN937b against P. capsici. Antibiotics are capable of modifying the fungal
membrane permeability causing enlargement of the mycelium and distortion of
sporangia and zoospores (Leelasuphakul et al., 2008).
Detached Leaf Assays
In this study, we used detached leaves to assess disease severity on squash
leaves treated with the extracts produced by IN937b. Although conditions in this assay
were highly favorable to P. capsici infection, the extracts of IN937b, applied before
pathogen challenge, completely suppressed the disease development on the attached
leaves. However, if treated with the extracts simultaneously or after the pathogen
inoculation, the extracts failed to suppress the disease development. Therefore,
antibiosis by the strain IN937b plays a major role in the suppression of Phytophthora
blight in squash. Inhibition of P. capsici on squash leaves by the extracts observed in
this study suggests that the extracts do not have a therapeutic effect but exert
preventive activities against the Phytophthora blight disease (Curtis et al., 2004). Once
123
the pathogen is established in the plant tissues, the effect of suppression on mycelial
growth diminished.
Identification of Subtilosin A
Subtilosin A was isolated from IN937b culture, and identified by HPLC/ESI-MS.
Subtilosin A, with a molecular mass of 3399.7 Da (Marx et al., 2001), is a cyclic
polypeptide that is ribosomally synthesized in Bacillus spp.. HPLC analysis showed that
there were large quantities of subtilosin A in the methanol extracts. The initial pH and
culture temperature are critical factors for microbial growth and metabolic biosynthesis,
and they substantially affect antibiotic production (Gong et al., 2006).
Subtilosin A targets cell membranes to form transient pores inducing lipid-
perturbation and contributing to its antibiotic activity. It has been reported that subtilosin
A involves permeabilization of target cell membranes (Thennarasu et al., 2005).
Subtilosin A promotes internal osmotic imbalance and wide cytoplasmic disorganization
through abundant vacuoles formation. The aggregation of cytoplasm increases along
with a loss of characteristic organelles. These changes result in the plasmolysis of
fungal cells and ultimately cause morphological damage visible in sporangia and
zoospore germination.
The exploitation of antibiotics as active ingredients of pesticides is promising in
developing novel fungicides to control crop diseases. Development of compounds
suitable for agricultural use as pesticide ingredients requires intrinsic toxicity and
stability. Therefore, exploring less toxic and more stable compounds may be one of the
most promising strategies for developing novel biocontrol agents. B. subtilis strains
could be important candidates for biocontrol agents because they are known to produce
more than two dozen antibiotics (Stein, 2005).
124
In conclusion, we have demonstrated that secondary metabolites secreted by B.
subtilis IN937b strongly inhibited the growth of P. capsici. Although antibiosis seems to
be an important mechanism involved in biocontrol activity of the active compounds.
Further studies of the modes of action of these secondary metabolites are currently
underway in order to improve their efficacy.
125
Table 4-1. Antagonism of Bacillus PGPR strains against P. capsici on PDA plates
Bacillus strains Isolates of P. capsici
#121 #146 #151
SE34 - - -
SE49 - - -
SE52 - - -
SE56 - - -
SE76 - - -
INR7 - - -
1PC-11 - - -
Actinovate AG - - -
1PN-19 +++ +++ +++
BUEX 1216S ++ ++ ++
BUEX 1216C ++ ++ ++
Companion + + +
GB03 + + ++
IN937b + + +
IN937a ++ ++ ++
- Represents no inhibition zone. + Represents 1–5 mm wide inhibition zone, ++ represents 6–10 mm wide zone, +++ represents more than 10 mm wide zone. Values are average of three replications.
126
Table 4-2. Effect of Methanol Extracts of IN937b on hyphal growth of P. capsici
a All the plates were incubated at 25 °C for 9 days. The diameter of mycelial colony was measured 3, 6 and 9 days after incubation. b Each value represents the mean of three replicates. Means followed by the same letter within each column were not significantly different according to Fisher’s protected LSD test (p = 0.05).
Concentration
(mg/mL)
Diameter of mycelial colony (cm) and inhibition rate (%) a
Day 3 (%) Day 6 (%) Day 9 (%)
0.0 3.9 a b 0.0 e 6.7 a 0.0 c 8.0 a 0.0 e
0.2 3.7 b 5.3 d 6.6 a 1.3 c 7.5 b 6.3 d
0.4 2.9 c 25.8 c 4.4 b 34.3 b 7.1 c 11.3 c
0.8 2.7 d 30.8 b 4.4 b 34.3 b 5.5 d 31.2 b
1.0 2.0 e 48.8 a 3.1 c 53.8 a 5.1 e 36.3 a
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Table 4-3. Effect of Methanol Extracts of IN937b in Detached Leaf assays
Treatment
Disease severity a
Day 5 Day 7 Day 9
Methanol (CK) 1.1±0.3 b a 2.7±0.5 a 4.8±0.4 a
Methanol extracts
(40 mg/mL) 0 b 0 b 0 b
a Disease severity was evaluated based on the scale of 0-5: where 0 = no symptoms; 1 = less than 10%; 2 = 10-25%; 3 = 50%; 4 = 51-75%; 5 = 75-100% of leaves covered with lesions. b Each value represents the mean of four replicates. Means followed by the same letter within each column were not significantly different according to Fisher’s protected LSD test (p = 0.05).
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A
B
Figure 4-1. Antagonism of Bacillus spp. against P. capsici on PDA plates. In vitro assays of Bacillus strains to suppress P. capsici were conducted on PDA plates. A 6 mm-diameter mycelial plug of P. capsici was placed in the center of PDA. The bacterial strain was streaked a 4 cm long line 3 cm away from the center of plug. The plates were incubated at 25 °C for 7 days. A) strains IN937b and IN937a, CK: only mycelial plug of P. capsici on the PDA, B) strains SE76 and IPC-11, CK: only mycelial plug of P. capsici on the PDA.
129
A
B
Figure 4-2. Antagonism of methanol extracts of IN937b against P. capsici on CM agar plates. One hundred microliters of methanol extracts were pipetted into each of the holes (6 mm diameter) 2 cm away from the edge of the CM agar. A 6 mm-diameter mycelial plug of P. capsici was placed in the center of CM agar plates. A) 100 µL methanol extracts (40 mg/mL), B) 100 µL methanol extracts (10 mg/mL). A thin layer and more branches were observed close to the hole (black arrow).
130
A
B
Figure 4-3. Effect of methanol extracts of IN937b on morphology of P. capsici. Effect of methanol extracts on morphology of P. capsici under a light microscope. A) normal mycelia of P. capsici observed at the edge of the plate away from the extracts, B) abnormal mycelial swollen and branching of P. capsici towards to the extracts. Distorted morphology in the presence of methanol extracts (white arrows).
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Figure 4-4. Effect of methanol extract concentrations of IN937b on zoospore germination. One hundred microliters of zoospore suspensions (105 zoospores/mL) mixed with 100 µL methanol extracts at the final concentration of 0, 10, 20, 30, or 40mg/mL. Data are the percentage of germinated zoospores after 3 h incubation at 25 °C. The equation of regression analysis represents a dose response of germinated zoospores to the extract concentration, where x is the extract concentration and y is the zoospore germination rate. Each value represents the mean of four replicates. Means followed by the same letter within each column were not significantly different according to Fisher’s protected LSD test (p = 0.05).
83.8 a
73.1 a
54.6 b47.3 bc
45.1 bc
35.4 c
y = -1.1041x + 75.871R² = 0.86
0
10
20
30
40
50
60
70
80
90
0 10 20 30 40
Ge
rmin
ati
on
Rate
(%
)
Concentrations of methanol extracts (mg/mL)
132
A
B
Figure 4-5. Effect of methanol extracts of IN937b on sporangial formation. The mycelial plug was placed in 15 mL DI water with 100 µL methanol extracts (40 mg/mL). The plates were placed under the light at room temperature to induce sporangial formation. After 48 h, the number of sporangia was examined under a dissecting microscope. A) Sporangia were observed without methanol extracts, B) No sporangia were found and swollen tips of hypha existed in treatment with methanol extracts.
133
A
B
C
D
134
Figure 4-6. Effect of methanol extracts on detached leaf infection. Squash leaves (less than 9 cm) were collected from 2 week-old plants. Twenty microliters of methanol extracts were dropped on to the detached leaves and placed in the Petri dishes supplemented with moist filter paper. One day later, 20 µL zoospore suspensions (104 zoospore/mL) were dropped on to the leaves 3 cm away from the site that treat with methanol extracts. A) Rating scale of disease severity. Disease severity was evaluated using a scale of 0-5: where 0 = no symptoms; 1 = less than 10%; 2 = 10-25%; 3 = 50%; 4 = 51-75%; 5 = 75-100% of leaves covered with brown lesions, B) Symptom development recorded 5 days after pathogen inoculation, C) Symptom development recorded 7 days after inoculation, D) Symptom development recorded 9 days after inoculation.
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CHAPTER 5 ELICITATION OF PLANT SYSTEMIC DEFENSE BY BACILLUS PGPR STRAINS
Introduction
Plants are constantly exposed to various pathogens. In order to prevent potential
plant infection by pathogens, plants possess an immune system that recognizes the
pathogens and activate defenses that protect plants from infectious fungi, oomycetes,
bacteria, and nematodes, etc. (Kuc, 1982). A first line of defense is called pathogen-
associated molecular pattern (PAMP) - triggered immunity (PTI), resulting in non-host
resistance that prevents further pathogen invade (Jones and Dangl, 2006). PTI leads to
fast defense responses via synthesis of toxins, antifungal proteins, and chemical signals
during initial stages of pathogen infection (Nicaise et al., 2009). However, some
pathogens develop strategies to suppress PTI. Therefore, plants have to develop a
second line of defense called effector-triggered immunity (ETI), which relies on systemic
signals produced at and dispersed from infection sites (Amil-Ruiz et al., 2011). In
general, ETI is associated with a hypersensitive response (HR) and a systemic
activation of plant defenses at the site of signal perception with pathogenesis-related
(PR) proteins, phytoalexins and reactive oxygen species.
Plant defenses are not only activated in response to pathogen infection, but also
can be stimulated by beneficial microbes. After exposure to biotic stimuli provided by
PGPR, plants can acquire enhanced levels of resistance to subsequent pathogen
attack. This phenomenon is commonly referred to as induced systemic resistance (ISR)
(Kloepper et al., 2004). Biological control using introduced PGPR with the capacity to
elicit ISR against plant pathogens has been extensively studied over the past two
decades in greenhouse and field trials (Yan et al., 2002). In many cases, ISR elicits
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plant immunity against multiple plant pathogens (Kloepper et al., 2004). For example,
select mixtures of compatible Bacillus spp. with the capacity to elicit ISR protect the
hosts against infection by pathogens Ralstonia solanacearum on tamato, Colletotrichum
gloeosporioides on pepper, and Rhizoctonia solani on green kuang futsoi (Brassica
chinensis var. parachinensis) (Jetiyanon and Kloepper, 2002).
A common feature of resistance responses induced by PGPR is “priming”, a
state in which plants treated with PGPR display fast and strong activation of defense
responses upon pathogen challenge. A priming effect is when PGPR strains cause the
plant to be in an “alert” state to detect pathogens with the response occurring faster
and/or stronger compared to plants not previously exposed to the priming stimulus
(Yang et al., 2009). Primed plants display a unique physiological status that can
respond rapidly to pathogen attacks (Conrath et al., 2006). For example, Bacillus sp.
CHEP5 and Pseudomonas sp BREN6 strains reduced root and stem wilt disease
severity caused by Sclerotium rolfsii on peanut (Arachis hypogaea L) (Laura Tonelli et
al., 2011). PGPR-mediated ISR caused a faster increase in phenylalanine ammonia-
lyase and peroxidase levels in peanut plants compared to non-treated control plants
after pathogen challenge, indicating that a state of priming was activated by treatment
with CHEP5 and BREN6. However, ISR usually does not confer adequate control, but
provides long-lasting increased resistance against a broad range of pathogens (Akram
et al., 2008).
ISR occurs when plants respond to an appropriate elicitor from the tissues
surrounding the initial infection site, which spreads throughout the plant via the emission
of molecular signals that reach distant tissues triggering increased resistance to the
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pathogens. The signaling of ISR is controlled by salicylic acid (SA), jasmonic acid (JA)
and ethylene (ET) (Pieterse et al., 2002). Paenibacillus alvei K165 primed for enhanced
SA-dependent defenses (Tjamos et al., 2005), while some other Bacillus spp. such as
Bacillus cereus AR156, were able to prime both the SA and the JA/ET pathways (Niu et
al., 2011). Recently, lipopolysaccharides, 2,4-diacetylphloroglucinol, and siderophores
produced by PGPR have been found to be responsible for ISR (Henry et al., 2011). All
these signaling molecules can distribute from local to far sites.
To defend against pathogen attack, there are two defense pathways that are
generally associated with an enhanced level of resistance conferred by ISR: the
phenylpropanoid pathway and the oxylipin pathway. Phytoalexin synthesis induction
through stimulation of the expression of genes involve phenylalanine ammonia-lyase
(PAL). The first step of the oxylipin pathway is catalyzed by PAL, which activates and
catalyzes phenylalanine to cinnamic acid, the precursor of lignin, salicyclic acid, and
phytoalexin phenylpropanoids, leading to secondary metabolites such as phytoalexins
that are toxic to fungi (Silva et al., 2004a). PAL is also involved in lignin biosynthesis
and accumulation of phenolics in response to fungal infection. Enzyme stimulation as
part of ISR is generally activated after pathogen infection (Choudhary et al., 2007).
ISR is associated with an oxidative burst (Bargabus et al., 2003) and cell wall
reinforcement (van Loon, 2007) by accumulation of a defense related enzyme
peroxidase (PO). PO shows affinity to cellular lignification and has direct antimicrobial
activity in the presence of hydrogen peroxide (Silva et al., 2004b). The oxidative burst is
the earliest event in plant defense response in both compatible and incompatible plant-
pathogen interactions as well as in rhizobacteria - plant interactions (Jourdan et al.,
138
2009). In general, the transient primary burst is nonspecific and has no effect on
disease progression. Only during incompatible interactions, more prolonged peaks of
hydrogen peroxide production lead to specific recognition (Van Breusegem et al., 2001).
The enforcement of cell walls by lignification involves blocking of fungal pathogen
penetration. Upon challenge infection with the fungus, the root cell walls are rapidly
strengthened at the sites of attempted fungal penetration through appositions that
contain callose as well as phenolic materials and fungal ingress is effectively prevented
(Yoshida et al., 2005). Tobacco plants treated with B. pumilus SE34 exhibited newly
formed barriers beyond the site of fungal infection. These barriers were cell wall
appositions that contained large amounts of callose and were infiltrated with phenolic
compounds (Zhang et al., 2002). Phenolic compounds accumulated in host cell walls,
intercellular spaces, and on the surface and inside of the invading pathogen’s hyphae
(Nakkeeran et al., 2006).
ISR induced by Bacillus PGPR was shown to be generated in whole squash
plants and a split-root system. In studies of protection of cucumber against diseases
caused by soil-borne fungi, a split-root assay was developed in which the inducing
bacteria and the pathogen were simultaneously inoculated on separate halves of roots,
and then planted in separate pots (Liu et al., 1995). ISR was expressed as a delay in
symptom development, reduced disease severity, and reduced disease incidence
compared to the non-bacterized controls. In a previous study, the compatibility between
P. capsici and susceptible pepper plants (Capsicum chinense Jacq) could be a non-host
compatible interaction due to a consistent increase activity of defense enzymes a few
hours after inoculation with P. capsici (Goretty Caamal-Chan et al., 2011). It appears
139
that P. capsici has the ability to block the defense response and to manipulate the
metabolism of the host by its effectors for successful infection (van Loon et al., 2006).
The objectives of this study were: (1) to understand the nature of ISR by Bacillus
PGPR IN937b and SE76 in squash plants with split-root assays by measuring the levels
of PAL and PO, (2) to evaluate the potential host defense response in squash plants
against P. capsici by monitoring PAL and PO activities, and (3) to analyze the changes
in enzyme activity of PAL and PO by treatment with Bacillus PGPR applied at different
squash growth stages.
Materials and Methods
Inoculum Preparation and Inoculation
Inoculum of P. capsici isolate #151 was prepared according to Chapter 2. The
concentration of zoospores was determined under a microscope using a
hemocytometer.
For foliar inoculation, the concentration of P. capsici inoculum was adjusted to
103 zoospores/mL using DI water. Plant leaves were sprayed with 15 mL of the
inoculum until run off. Each inoculated plant was covered with a plastic bag to maintain
high moisture for 2 days. Inoculated plants were maintained on benches in the
greenhouse with high soil moisture by hand watering daily.
Application of Bacillus PGPR Treatment in the Greenhouse
PGPR strains IN937a, IN937b and SE76 were evaluated for their ability to
systemically induce resistance against P. capsici in the greenhouse. Two-week-old
squash plants were treated with 20 mL of PGPR suspensions prepared according to
Chapter 2 at 1×108 cfu/mL as a soil drench. One week later, the same treatment with
PGPR were applied to the seedlings for a second time.
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Split-Root Assay
To set up the split-root systems, germinated seeds were planted in 60-well
plastic trays filled with potting mix. After 10 days, the roots were washed with running
tap water. The roots were then split evenly with a scalpel blade and each half of the root
system of each seedling was quickly moved into two adjacent plastic pots, each
containing potting mix. Each pot was separated to guarantee physical separation of
each half root. The plants were placed on the bench in a greenhouse.
One week after roots were spilt, one half of the root system of each plant was
divided into groups, each treated with a different inoculum. Group 1: one half of the root
system of each plant was treated with 20 mL of IN937b suspensions at 1×108 cfu/mL
applied as a soil drench labeled as IN937b-2. The other half of the root system of each
plant were treated with same amount of DI water labeled as IN937b-1. Group 2: One
half of the root system of each plant was treated with 20 mL of ASM at 30 mg/L applied
as a soil drench labeled as ASM-2. The other half of the root system of each plant were
treated with same amount of DI water labeled as ASM-1. Group 3: Both halves of the
root system of each plant were treated with DI water labeled as non-treated control (CK-
1 and CK-2).
Enzyme Activity Assays
Samples of fresh squash leaves and roots were taken from each of the pots 1, 3,
5, 7, and 10 days after IN937b treatment. The samples were washed with running tap
water, dried and 0.5 g of each was ground in liquid nitrogen in a mortar with a pestle.
The ground tissue was then mixed with 0.1 M sodium phosphate buffer (pH 6.5) and
centrifuged at 12,000 rpm for 10 min at 4 °C. The supernatant used for the enzymatic
activity assay was stored in a 1.5 mL Eppendorf tube at -20 °C. Healthy plants with no
141
PGPR or pathogen challenge were used as a background. Background PAL and PO
activity was found to be same during experimental period.
Enzyme Activity Assays
Peroxidase (PO) Fifty microliters of roots or leave extracts were added to 2.95
mL of PO reaction buffer consisting of 50 mM sodium phosphate buffer (pH 6.0), 60 mM
guaiacol and 0.6 M H2O2 (Chen et al., 2000). The reaction was measured for 90 s
(recording the value every 15 s) at wavelength of 470 nm with a spectrophotometer
(UNICO SpectroQuest TM 4802 UV/VIS Double Beam, United Products & Instruments
Inc., Dayton, NJ). One unit of enzyme activity was defined by a change in absorbance
of 0.01 for 1 g fresh weight per minute.
Phenylalanine ammonia-lyase (PAL) One hundred µL of crude plant tissue
(roots or leave) extracts were mixed with 1.9 mL of 0.015 M L- phenylalanine and 0.1 M
Tris-HCl buffer solution (pH 8.5) (Chen et al., 2000). The mixture was placed in a water
bath at 40 °C for 30 min. PAL activity was measured spectrophotometrically at a
wavelength of 290 nm. One unit represents the conversion of 1 µmol L-phenylalanine to
cinnamic acid for 1 g fresh weight per minute at 40 °C.
Results
Induction of Systemic Resistance by PGPR with P. capsici Challenge
Soil treatment with IN937b resulted in a significant (P≤0.05) visible reduction in
lesion development (both number and size) compared with the non-treated control 7
days after inoculation (Table 5-1). SE76 failed to elicit ISR because the number and
size of lesions increased as much as in the non-treated control. ASM was the most
effective treatment in this experiment to induce ISR, as it completely stopped the
occurrence and spread of symptoms.
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Treatment with IN937b at 108 and 107 CFU/mL significantly (P≤0.05) reduced the
size of lesions compared to the non-treated control 7 days after pathogen inoculation
(Table 5-2). The greatest reduction in lesion size by IN937b was at 108 CFU/mL, where
there was more than 70% reduction compared to the non-treated control plants. IN937b
at 107 and 106 CFU/mL had no effect on disease development compared to the non-
treated control. ASM completely inhibited the disease during the period of this
experiment.
Increased Activities of PO and PAL Induced Locally by PGPR Treatment with P. capsici Challenge
Compared to the non-treated plants, SE76 induced significantly greater levels of
PAL and PO activities in plants roots 3 days after PGPR treatment (Figure 5-1 and
Figure 5-2). When squash roots were challenged with P. capsici, significantly greater
PAL activities were observed in all PGPR treatments and ASM 3 DAI compared to the
non-treated control (Figure 5-3). PAL activities were significantly greater in treatments of
ASM and IN937b than the non-treated control 5 DAI. In general, levels of PAL increased
drastically 1 DAI in all treatments due to the rapid response of the squash plants to the
pathogen. However, PAL activities 3 DAI significantly decreased in all treatments except
for the background control compared to those at 1 DAI.
Bacillus PGPR IN937b and SE76 induced significantly greater levels of PO
activities 5 DAI compared to the non-treated control (Figure 5-4). PO activities reached
the highest levels 3 DAI in all treatments and then decreased 5 DAI. In the non-treated
control plants, PO activities significantly increased 1 and 3 DAI when the pathogen
started to invade squash plants, but dramatically declined 5 DAI when stem wilting
started to occur. Treatment with IN937a had no effect on PO activities, while ASM
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treatment resulted in significantly greater levels of PO activities 3 and 5 DAI compared
to the non-treated control plants.
Systemically Induced Enzyme Activity as Determined by Split-Root System without P. capsici Challenge
Data from the split-root system assays showed that IN937b not only locally
increased levels of PO and PAL activities in the root half that was bacterized, but also
systematically increased PO and PAL activities in the other half (distant side) of the root
system which was only treated with water at 10 days after IN937b treatment (Figure 5-5
and 5-6).
Increased PAL and PO Activities Systemically in Response to P. capsici in Different Seedling Stages
Treatment with IN937b and SE76 induced significantly higher PAL activity in
plant leaves compared to the non-treated control 5 DAI for young and older plants (Fig
5-7). When first PGPR treatment was applied 17 days after emergence, significantly
greater PAL activity were detected 5 DAI in all PGPR treatments and ASM compared to
the non-treated control. Similarly, significantly greater PAL activity were detected 5 DAI
in all PGPR treatments compared to the non-treated control when the first PGPR
treatment was applied 10 days after emergence.
Squash plants treated with IN937b, SE76 and ASM induced significantly greater
levels of PO activity in the leaves compared to the non-treated control 5 DAI in the
young plants with the first PGPR treatment 10 days after emergence (Fig 5-8). In older
plants for which the first PGPR treatment was performed 17 days after emergence,
there was no difference of PO activity were observed in all treatment 5 DAI which the
first PGPR treatment was applied 10 days after emergence. PO activity was greater in
leaves treated with ASM for both young and older plants.
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Discussion
The purpose of this study was to demonstrate that Bacillus PGPR strains are
able to trigger ISR in squash plants against P. capsici, and to reveal the underlying
mechanisms of PGPR suppression of Phytophthora blight disease. Bacillus PGPR
IN937b applied in the rhizosphere reduced the number and size of lesions on the leaves
infected by P. capsici. Since PGPR and the pathogen were spatially separated, the
protection is not associated with the ability of the PGPR strains to inhibit fungal growth
and, therefore, it may be, at least partially, attributed to ISR-mediated protection. The
results from this study demonstrated that both Bacillus PGPR and the pathogen were
able to affect activities of defense-related enzymes, i.e. PAL and PO.
Actigard® (ASM) is a well-known activator of host resistance in many plants
(Louws et al., 2001; Kone et al., 2009). In this study, ASM induced significantly systemic
resistance in squash, resulting in elimination of P. capsici infection. It suggests that
ASM may induce resistance more effectively than Bacillus PGPR (Barilli et al., 2010).
Defense-Related Enzymes
Plants suppress pathogens by building lines of defense and develop an array of
cellular mechanisms to defend themselves against invading pathogens (Ramamoorthy
et al., 2001). Plants can recognize a pathogen and limit it to the site of the infection
(Jones and Dangl, 2006), and stop pathogen infection and movement (Takemoto et al.,
2003).
In general, increases in enzyme activities related to plant defenses are important
indicators in response to disease development. PO is an oxido-reductive enzyme
involved in the wall binding process (Viswanathan and Samiyappan, 2002). PO has
been implicated in the last enzymatic step of lignin biosynthesis, which is the oxidation
145
of hydroxyl which is subsequently coupled to lignin polymer. PO is also believed to be
involved in a number of physiological processes that may contribute to resistance
including cross linking of extensin monomers and lignification and they are also
associated with deposition of phenolic compounds into plant cell walls during resistance
reactions (Passardi et al., 2004). To be effective against a pathogen, defense
mechanisms, including increased PO activity, should be rapidly elicited in response to
infection. One of the immediate host responses to infection is the accumulation of
phenolics at the infection site. Similar results were also obtained in a previous study,
where microbial mixture of Bacillus strains IN937a and IN937b were found to induce
maximum SOD and PO activities compared with the non-treated control (Jetiyanon,
2007).
PAL is the first enzyme in the phenylpropanoid biosynthesis pathway leading to
synthesis of phytoalexins or phenols, which have defense functions in plants, such as
reinforcement of plant cell walls, antimicrobial activity and synthesis of signaling
compounds such as salicylic acid (Wen et al., 2005). In the present study, after
challenge with the pathogen P. capsici, PAL activities were positively correlated the
plant’s resistance against the pathogen as indicated by reduced disease severity. PAL
is essential for the synthesis of all protective substances through the phenylpropanoid
pathway. Generally, induction of PAL is correlated with increased resistance in the host
to pathogenic infection. PAL leads to the biosynthesis of phenols, phytoalexins, and
lignins with the involvement of PO.
Increased PAL and PO activities in plants by PGPR enhance the production of
phenolic compounds, and hence induce development of physical and chemical barriers
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to pathogen attack. Increased enzyme activities in PGPR-mediated ISR might be one of
the defense mechanisms involved in suppression of P. capsici. Phenolics generally
fungitoxic and increase the mechanical strength of the host cell walls. Lignin is a
phenolic polymer which is difficult for pathogens to breach and has been implicated in
plant defense against pathogens.
In the present study, induced responses in squash plants occurred in a short time
period. Resistance activated by PGPR did affect pathogen infection. However, following
the early and increased expression of defense enzymes, PAL activity dramatically
decreased after P.capsici infection, suggesting that the pathogen had the ability to
suppress induced defense responses in plants by interfering with signaling cascades
and detoxification of phytoalexins (El-Hadrami et al., 2009).
Locally Induced Enzyme Activity
When the squash roots were treated with Bacillus PGPR strains and inoculated
with P. capsici, the differential induction of resistance may have been activated
depending on the induction site. When roots were treated with Bacillus PGPR,
metabolic processes may have produced reactive oxidative oxygen species (ROS) such
as H2O2, O2 and OH (Demidchik, 2012). The production of toxic oxygen derivatives
increased as a result of infection by any foreign microbes. PGPR strains are able to
alleviate ROS via increased activities of antioxidant enzymes such as PO in plants, and
establish a relationship with plant roots without being harmful to roots (Zamioudis and
Pieterse, 2012). In the present study, the observed increased localized PO levels in
squash roots by bacterization are in agreement with the reports that cucumber roots
with PGPR induced localized resistance (Chen et al., 2000; Liang et al., 2011).
147
Priming: Getting Ready for Pathogen Invasion
PGPR may alter the cellular mechanisms and reprogram metabolism after they
establish a relationship with plants, and induce plant resistance upon subsequent
challenge with a pathogen (Van der Ent et al., 2009). Plants treated with PGPR become
primed to respond faster and show stronger activation of cellular defense responses
upon pathogen challenge compared with unprimed plants (Conrath et al., 2002).
Treatment with Bacillus strains in roots also increased the activities of PO and PAL
enzymes in squash leaves. This is also supported by the observations in the split-root
assays that certain Bacillus strains induced increased PO and PAL activities in the other
half root which was treated with water and distant from the treated half roots.
Older plants tended to develop disease symptoms later compared to younger
plants after they were inoculated with the same amount of P. capsici. PAL and PO
activities were greater in older plants than younger plants in the non-treated control,
IN937b and ASM treatments. In general, the activities of PAL and PO increased in
these treatments with reduced disease severity in squash plants.
In the present study, PAL activity was activated by Bacillus PGPR IN937b and
SE76. It appears that the pathogen blocked the plant defense response development as
previously reported (Cooper and Rao, 2006). PO and PAL activities were promptly
triggered by PGPR treatments, and the levels of PO and PAL activities were faster than
the chemical inducer (ASM) did, and this seems to be a consensus for PGPR treatment.
PO activity is activated faster to contain the reactive oxygen species produced by host
plants that respond to inhibit pathogen growth (Ishida et al., 2008).
In conclusion, higher levels of PO and PAL activities may have contributed
collectively to induced resistance in squash plants against P. capsici, the pathogen of
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Phytophthora blight, which eventually resulted in lower disease severity compared to
the non-treated control.
149
Table 5-1. Effect of Bacillus PGPR on number and size of lesions in the detached leave assay under the lab conditions
lesion size in cm2
Day 5 Day 7
IN937b a 9.0 a c 13.0 b
SE76 12.0 a 32.5 a
ASM 0.0 b 3.9 b
Non-treated control 11.9 a 44.6 a a PGPR were applied twice (1.0 ×108 CFU/mL; 20mL) as a soil drench. ASM (Actigard® 30 mg/L; 20 mL) was applied as a positive SAR inducer for suppressing Phytophthora blight. b P. capsici inoculum was diluted to 1 × 103 zoospores/mL. Plants were sprayed with 20 mL of P. capsici inoculum until ran off. Data of the number and size of lesions were collected at 5 and 7 days after pathogen inoculation. c Each value represents the mean of four replicates. Means followed by the same letter within a column are not significantly different according to Fisher’s protected LSD test (p = 0.05). Table 5-2. Effect of different concentrations of IN937b on the number and size of
lesions in the detached leave assay under the lab conditions
Treatment lesion size in cm2
Day 3 Day 5 Day 7
937b-108 2.5 a 4.0 a 5.0 b
937b-107 3.9 a 5.5 a 8.0 b
937b-106 6.6 a 8.5 a 10.5 a
Non-treated control
6.5 a 7.6 a 16.8 a
a PGPR were applied twice as a soil drench. The concentration of IN937b was adjusted to 106, 107, and 108 CFU/mL, and 20 mL was applied to each plant. ASM (Actigard® 30 mg/L; 20mL/plant) were applied as SAR control for suppressing Phytophthora blight. b P. capsici inoculum was diluted to 1 × 103 zoospores/mL. One leaf of each plant was sprayed with 5 mL of P. capsici inoculum until ran off. Data of the number and size of lesions were collected at 3, 5, and 7 days after pathogen inoculation. c Each value represents the mean of ten replicates. Means followed by the same letter within a column are not significantly different according to Fisher’s protected LSD test (p = 0.05).
150
Figure 5-1. Induction of PAL by PGPR in squash roots. PAL activities in squash roots treated by PGPR only. Squash roots were sampled 1 day and 3 days after PGPR treatment. Bars represent the standard error of the mean from three replications. Means followed by the same letter are not significantly different according to Fisher’s protected LSD test (p = 0.05).
Time (Day)
PA
L (
U/g
FW
)
0
5
10
15
20
25
30
Non-treated Control
IN937b
SE76
IN937a
ASM
Background Control
1 3
bb
a
ab
ab
b
151
Figure 5-2. Induction of PO by PGPR in squash roots. PO activities in squash roots treated by PGPR only. Squash roots were sampled 1 day and 3 days after PGPR treatment. Bars represent the standard error of the mean from three replications. Means followed by the same letter are not significantly different according to Fisher’s protected LSD test (p = 0.05)..
152
Figure 5-3. Induction of PAL by PGPR in roots against P. capsici. PAL activities in squash roots treated by PGPR and challenged with P.capsici (3 days after PGPR treatment). Squash roots were sampled 0, 1, 3 and 5 day after P. capsici inoculation. Bars represent the standard error of the mean from three replications. Means followed by the same letter are not significantly different according to Fisher’s protected LSD test (p = 0.05).
153
Figure 5-4. Induction of PO by PGPR in roots against P. capsici. PO activities in squash roots treated by PGPR and challenged with P.capsici (3 days after PGPR treatment). Squash roots were sampled 0, 1, 3 and 5 day after P. capsici inoculation. Bars represent the standard error of the mean from three replications. Means followed by the same letter are not significantly different according to Fisher’s protected LSD test (p = 0.05).
154
Figure 5-5. Induction of PAL by IN937b in split root system. PAL activities on squash roots treated by IN937b only. CK-1: root with water treatment, CK-2: root with water treatment, IN937b-1: root with water treatment, IN937b-2: root with IN937b treatment, ASM-1: root with water treatment, ASM-2: root with ASM treatment. Squash roots were sampled 1, 3, 5 and 10 day after PGPR treatment. Bars represent the standard error of the mean from three replications. Means followed by the same letter are not significantly different according to Fisher’s protected LSD test (p = 0.05).
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Figure 5-6. Induction of PO by IN937b in split root system. PO activities in squash roots treated by IN937b only. CK-1: root with water treatment, CK-2: root with water treatment, IN937b-1: root with water treatment, IN937b-2: root with IN937b treatment, ASM-1: root with water treatment, ASM-2: root with ASM treatment. Squash roots were sampled 1, 3, 5 and 10 day after PGPR treatment. Bars represent the standard error of the mean from three replications. Means followed by the same letter are not significantly different according to Fisher’s protected LSD test (p = 0.05).
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A
B
Figure 5-7. Induction of PAL by PGPR against P. capsici in different growth stages. PAL activities in squash roots treated by PGPR and challenged with P.capsici (3 days after PGPR treatment). Squash roots were sampled 0, 1, 3 and 5 day after P. capsici inoculation. A) First PGPR treatment started at 10 days after emergence, B) First PGPR treatment started at 17 days after emergence. Bars represent the standard error of the mean from three replications. Means followed by the same letter are not significantly different according to Fisher’s protected LSD test (p = 0.05).
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A
B
Figure 5-8. Induction of PO by PGPR against P. capsici in different growth stages. PO activities in squash roots treated by PGPR and challenged with P.capsici (3 days after PGPR treatment). Squash roots were sampled 0, 1, 3 and 5 day after P. capsici inoculation. A) First PGPR treatment started at 10 days after emergence, B) First PGPR treatment started at 17 days after emergence. Bars represent the standard error of the mean from three replications. Means followed by the same letter are not significantly different according to Fisher’s protected LSD test (p = 0.05).
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CHAPTER 6 SUMMARIES AND FUTURE PERSPECTIVES
Squash is one of widely grown and consumed vegetable crops worldwide. The
large scale of squash plants suffers heavy losses with pests and diseases. One of the
most economically important diseases on squash is Phytophthora blight, caused by
Phytophthora capsici. Control of this disease, so far, has largely depended on
fungicides. Intensive use of fungicides leads to the appearance of less sensitive strains
and resistant strains. The risk of fungicide resistance causes environmental hazards
and decrease of fungicide efficacy. To prevent the excessive use of fungicides, an
integrated approach is needed for the control of Phytophthora blight, including biological
control along with limited fungicide use. PGPR strains have been demonstrated against
a broad spectrum of pathogens on many vegetable crops. The overall aim of this
dissertation was to investigate Bacillus PGPR strains for controlling Phytophthora blight
on squash and plant defense induced by PGPR.
Summaries
In this dissertation, I was able to show that the application of selected Bacillus
PGPR strains significantly increased plant growth and decreased Phytophthora blight
disease severity on squash plants (Chapter 2). Of the efficient strains tested, Bacillus
subtilis strain IN937b delayed disease development, enhanced the plant growth
parameters, and increased leaf chlorophyll content in leaves under greenhouse
conditions. Also, I established appropriate timing of IN937b applications with different
plant ages to obtain remarkable efficacy of disease control. IN937b applications prior to
the pathogen inoculation, offer adequate time or IN937b to multiply and establish on the
root surface, which prevents early infection by P. capsici.
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Another important result of this dissertation is that I have described biofilm
formation of selected Bacillus PGPR strains in the liquid or solid medium, and the
production of surfactins by IN937b (Chapter 3). Use of specific PCR-primers was
applied for the detection of genes involved in the biosynthesis of surfactins in Bacillus
spp. The PCR-based screening has been a useful and efficient approach for the
identification of potential surfactin producing bacteria, especially when the surfactin
primers were designed to detect genes involved in the biosynthesis. The production of
surfactins and biofilm suggests that biofilm may be important for rhizosphere
colonization by IN937b. The population dynamics of IN937b were investigated in the
commercial soilless media using serial dilutions and plating on selective media.
Population densities stayed high at the early stage of inoculation e.g. 1 day after
treatment, but decreased dramatically two weeks after treatment. IN937b also found in
the interior tissues of the roots as they have naturally evolved to compete and survive in
the harsh conditions and compete in space with the pathogen.
The antagonistic effects are mainly due to production of antimicrobial antibiotics,
which play a major role in biological control of plant pathogens. The results from present
study revealed the production of antifungal antibiotic subtilosin A from IN937b. The
efficacy of antibiotics extracted from the culture of IN937b was evaluated for in vitro
suppressing P. capsici in vitro (Chapter 4). The methanol extracts of IN937b inhibited
the germination of the zoospores and sporangia formation. It may stop or delay
zoospores entry into the host tissue at the site of infection, also influenced the
development of P. capsici hypha and accumulated in squash roots. Inhibition of the
pathogen is through the localized direct antifungal activity of IN937b and their cell-free
160
culture extracts, which points towards the activity of antibiotics as the potential
mechanism of disease control.
Plant resistance against diseases can be systemically induced by plant
pathogens or non-pathogenic rhizobacteria. IN937b was selected for further studies for
its potential to induce systemic resistance in squash against P. capsici (Chapter 5).
Systemic and local induction of resistance by PGPR was assessed through increased
activities of plant defense related enzymes peroxidase (PO) and phenylalanine
ammonia lyase (PAL) in squash plants treated with PGPR and challenged with P.
capsici. To determine the existence of systemic resistance on squash plants, the
pathogen P. capsici was inoculated as inducers on leaves spatially separated from the
PGPR strain IN937b applied as a soil drench. The activities of PAL and PO were
determined in bacterized roots and distant induced squash leaves. The results
demonstrated that systemic resistance against the squash root disease was not only
induced by plant pathogens, but also by non-pathogenic PGPR. When disease
symptoms started to appear on the tissues, the enzyme activates reached a peak. This
reduced pathogen infection, and impaired the disease development on squash roots.
But the pathogen-induced enzyme activities was stronger than the PGPR-mediated ISR
on squash plants. A split-root technique was utilized on squash plants grown in soilless
potting mixes. IN937b stimulated PO activates in the bacterized side of roots locally and
in the distant side of roots systematically. Without P. capsici infection, PAL activities
were not influenced by IN937b locally and systematically.
Future Perspectives
Our data strongly support the hypothesis that PGPR strains provide remarkable
disease suppression as an integrated approach in vegetable crops production. Further
161
studies need to be performed to evaluate the effect of PGPR in the field and to explain
how plants communicate with PGPR in the soil.
In particular, the following issues might be taken into account in the future:
To extend our results of Chapter 2, I will try to optimize the biocontrol efficacy in
the field including the rate of application, timing, number of applications, and bacterial
survival at specific level of pH, temperature and soil fertility under field conditions.
Furthermore, I will investigate the effect of PGPR combined with commonly used
fungicides in the field to reduce the use of chemicals, which could be integrated into
existing management programs for Phytophthora blight in squash.
Detection and visualization of biofilm formation in situ will be studied (Chapter 3).
Visualization in situ act as a powerful tool that allows us to investigate the spatial
colonization pattern of the pathogen and the biocontrol agent on the root surface, and
also to determine biofilm production pattern of the biocontrol agent coincide with the
spatial colonization of the pathogen. It will be interesting to characterize those signaling
molecules released by the plant host that trigger Bacillus PGPR to colonize and form
biofilm on the root surfaces, and to understand how the PGPR senses and responds to
the signals in stimulating biofilm formation.
To expand the in vitro results in Chapter 4 and to study the hypothesized effects
of subtilosin A, the mode of action of antifungal activity will be studied. As model
systems to study the principles of antibiotics biosynthesis, Bacillus PGPR strains are
natural producers of antibiotics that can be used to enhance antifungal activity. Based
on the role of antibiotics and their action in the biocontrol of pathogens, antibiotics
162
produced by PGPR are excellent candidates for design and development of novel
fungicides.
PGPR partly induced defense response in the squash plants. It suggested that
signal substances initially form within inoculated sites, and translocate to the entire plant
when plants were induced (Chapter 5). Certainly the signal would play an important role
in connecting bacterized sites and distant sites of the plants. Surfactin, PAL and PO
need to be investigated for their role at the molecular level in regulation of defense
response by using real-time PCR.
It is known that diseases have deleterious effects on crop production that
increasing the cost for growers and food safety. This study provides future researchers
with important tools for understanding biocontrol mode of actions that can be used to
develop powerful and effective biocontrol agents and to manipulate rhizosphere
ecosystems for crop production.
163
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BIOGRAPHICAL SKETCH
Xiaodan Mo was born in Jinzu, Guangxi, China. She obtained her bachelor’s
degree in the Department of Plant Protection from China Agricultural University in 2006,
and she was admitted to the master of science program in the Department of Plant
Pathology at China Agricultural University the same year. From 2006 to 2009, she was
involved in the research project mainly focused on the function of β-glucanase and its
heterologous expression in the Bacillus PGPR. In August 2009, Xiaodan was awarded a
fellowship from China Scholarship Council and joined the Ph. D program in the
Department of Plant Pathology at the University of Florida. In 2011, she began working
with Dr. Shouan Zhang on biological control of Phytophthora blight by Bacillus PGPR
strains on squash. As a graduate student, Xiaodan has presented her work in 2012 and
2013 American Phytopathological Society Annual Meetings. She received 2012 Warren
Wood, Sr. Memorial Fellowship from Miami Dade County, FL. Xiaodan received her Ph.
D. from the University of Florida in December 2013.