OPTIMIZING THE EFFICACY AND ENVIRONMENTAL FITNESS OF A COMMERCIAL
PSEUDOMONAS BACTERIAL BIOCONTROL PRODUCT FOR THE CONTROL OF
TURFGRASS DISEASE.
By
Liewei Yan

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
MASTERS OF SCIENCE
Plant Pathology
2011

ABSTRACT
OPTIMIZING THE EFFICACY AND ENVIRONMENTAL FITNESS OF A COMMERCIAL
PSEUDOMONAS BACTERIAL BIOCONTROL PRODUCT FOR THE CONTROL OF
TURFGRASS DISEASE.
By
Liewei Yan
A fungicide/antibiotic-producing bacterial strain of Pseudomonas aureofaciens Tx-1 (Tx1) has been commercialized for golf course use with some success in recent years. To achieve
adequate control it must be applied daily. This may be partially explained by UV susceptibility
of Tx-1, which is a common limitation of biocontrol agents. Results from various wavelengths of
ultraviolet radiation (A, B, C) exposure tests showed a significant drop in Tx-1 survivors
following increased dosage. Significant improvement of UV tolerance was achieved and
demonstrated in all UV exposure tests by mixing a sunscreen. In an attempt to identify the most
UV-resistant individual cells, Tx-1 was exposed to intensive UV light (UV-C) for various
lengths of time (in vitro). Cells (10TC30) with significantly improved survival rate after 7 J/m

2

2

UV-C irradiation was found followed by 10 cycles of 42 J/m of UVC exposure. Field data of
dollar spot and anthracnose control studies showed that applied at high concentration
7

2

(10 CFU/cm ), Tx-1 sunscreen mixture and the strain 10TC30 provided significant disease
control compared with non-treated control in the field study. Population sizes of the treatments
were monitored, and results demonstrate the improved percentage of survival of Tx-1 with the
protection of sunscreen.

To all the lovely people in Vargas’s Lab, Paul, Nancy, Ron, and Joe

iii

ACKNOWLEDGMENTS

I offer my sincerest gratitude and thanks to Dr. Joe Vargas, who has supported me
throughout my degree with his patience and knowledge. I attribute my degree to his effort and
encouragement and without him this thesis could not have been completed. One simply would
not wish for a better professor; To Dr. George Sundin for his guidance on my committee,
especially in the field of microbiology and UV radiation impact on microbes, this comprised a
great deal of my project; To Dr. Kevin Frank for his advice and insight for my research.
During my two years of graduate study, I have been blessed with a friendly and cheerful
group of fellow lab members. I would like to thank Nancy Dykema and Ron Detweiler for their
support and indoctrination throughout my project. Special thanks to Paul Giordano for the
wonderful friendship and memory we shared for these two years.
Lastly, I would like to thank my patents for their love and support.

iv

TABLE OF CONTENTS
Page
LIST OF TABLES

v

LIST OF FIGURES

vii

INTROUCTION
LITERATURE CITED

1
4

LITERATURE REVIEW
Dollar spot
Anthracnose
Biological control
LITERATURE CITED

7
7
9
10
15

CHAPTER ONE
UVR SENSITIVITY TEST FOR TX-1 IN VITRO AND MEANS TO IMPROVE ITS UV
TOLERANCE
Abstract
21
Introduction
22
Materials and Methods
23
Results
25
Discussion
44
LITERATURE CITED
47
CHAPTER TWO
FIELD STUDY FOR TX-1, 10TC30, AND HUMIC AICD
Abstract
Introduction
Materials and Methods
Results
Discussion
LITERATURE CITED

v

50
51
52
57
67
72

LIST OF TABLES
Page
Table 1.01

Survival of P. aeruginosa (phr-, uvrA-) or P. aureofaciens Tx-1 humic acid
-2 -1
mixture after exposed to 1.69 J m s UV-B radiation.
33

Table 1.02

Survival of P. aeruginosa (phr-, uvrA-) or P. aeruginosa (phr-, uvrA-) mixed with
-2 -1
different concentrations of humic acid after exposed to 1.49 J m s UV-B
radiation.
34

Table 1.03

Average fungal colony area (cm ) of R. floccosum on PDA medium inoculated
with P. aureofaciens Tx-1 or P. aureofaciens Tx-1 humic acid mixture that
2
received 90-180 kJ/m UV-A radiation.
35

Table 1.04

Average fungal colony area (cm ) of R. floccosum on PDA medium inoculated
with P. aureofaciens Tx-1 or P. aureofaciens Tx-1 humic acid mixture that
2
received 20.85-41.7 J/m UV-C radiation.
36

Table 1.05

Average fungal colony area (cm ) of R. floccosum on PDA medium inoculated
with P. aureofaciens Tx-1 or P. aureofaciens Tx-1 humic acid mixture that
2
received 8.26-13.77 kJ/m UV-B radiation.
37

Table 1.06

Survival of P. aureofaciens Tx-1 or 10TC30 after exposed to 60-180 J/m UV-B
radiation.
40

Table 2.01

List and application rates of treatments tested in 2009 field study for the
suppression of dollar spot and anthracnose in East Lansing, Michigan.
54

Table 2.02

List and application rates of treatments tested in 2010 field study for the
suppression of dollar spot and anthracnose in East Lansing, Michigan.
55

Table 2.03

Means and LSD comparisons for treatment effects on dollar spot disease
incidence on different dates in 2009.

2

2

2

2

vi

58

Table 2.04

Means and LSD comparisons for treatment effects on anthracnose disease
incidence on different dates in 2009.

59

Table 2.05

Means and LSD comparisons for treatment effects on turf quality on different
dates in 2009.
60

Table 2.06

Means and LSD comparisons for treatment effects on dollar spot disease
incidence on different dates in 2010.

62

Means and LSD comparisons for treatment effects on anthracnose disease
incidence on different dates in 2010.

63

Table 2.07

Table 2.08

Means and LSD comparisons for treatment effects on turf quality on different
dates in 2010.
64

vii

LIST OF FIGURES
Page
Figure 1.01

Survival of P. aureofaciens Tx-1 after exposed to UV-A radiation.

28

Figure 1.02

Survival of P. aureofaciens Tx-1 after exposed to UV-B radiation.

29

Figure 1.03

Survival of P. aureofaciens Tx-1 with or without the addition of humic acid after
exposed to UV-A.
30

Figure 1.04

Figure 1.04. Survival of P. aureofaciens Tx-1 with or without the addition of
humic acid after exposed to UV-B

32

Figure 1.05

Figure 1.05. Log survival of P. aureofaciens Tx-1 with or without the addition of
humic acid after exposed to UV-C radiation.
33

Figure 1.06

Log survival of P. aureofacious Tx-1 and 10TC30 after exposed to UV-C
radiation.

39

Figure 1.07

Survival of P. aureofacious Tx-1 incubated for 24 or 72 hr. after exposed to UVA radiation.
42

Figure 1.08

Log survival of P. aureofacious Tx-1 incubated for 24 or 72 hr after exposed to
UV-C radiation.
43

Figure 2.01

Population size of P. aureofaciens Tx-1 in foliage and thatch.

viii

65

INTRODUCTION
Dollar spot, caused by Rutstroemia floccosum syn. Sclerotinia homoeocarpa F. T. Bennet,
and anthracnose, caused by Colletotrichum cereale Manns sensulato Crouch, Clarke & Hillman,
are two of the most common, destructive and costly fungal diseases of high maintenance
turfgrasses (Vargas, 2005). Although successful management of these two diseases has been
achieved throughout the years by the traditional usage of chemical fungicides, the development
of pathogen resistance to multiple classes of systemic fungicides (Vargas, 2005; Murphy, 2008;
Putman, 2010), the public’s concern over chemical use on golf courses, and the increased
number of EPA restrictions on chemical fungicide development and application, have prompted
research on alternative disease control strategies.
Remarkable progress has been made at Michigan State University toward development of an
effective biological control agent, the bacterium Pseudomonas aureofaciens strain Tx-1 (Tx-1), a
soilborne bacterium capable of producing multiple antifungal compounds (Powell, 2000). In
vitro studies have shown promising growth inhibition of both R. floccosum and C. cereale
(Powell, 2000); Tx-1 was labeled by the EPA as a bio pesticide in 2000. A commercialized
fermentation and delivery system was developed for Tx-1 (Dwyer, 1999). The system utilizes the
irrigation system on golf courses for the distribution of Tx-1. This successful introduction of Tx1 to golf courses has provided an alternative management for turfgrass diseases.
Due to the frequent mowing of turfgrass on golf courses, foliage is the primary infection site
for many fungal diseases, including R. floccosum and C. cereale. Practical experience in the field
has raised questions about the persistence of Tx-1. To further understand the persistence of Tx-1
on the foliage, Dwyer (1999) tested Tx-1 under field condition and showed that satisfactory

1

disease suppression of Tx-1 largely depends on high inoculum concentration and high
application frequency. Lower concentration and application frequency of Tx-1 can result in
significantly reduced disease control. This finding was further supported by his thesis work
showing that field population of Tx-1 decline dramatically one month after the last application in
the fall. Under high disease pressure during summer, Hardebeck (2004) found that diluted
inoculum applied through the irrigation system failed to achieve sufficient dollar spot inhibition
on fairways. In order to optimize the performance of Tx-1 in the field, it is necessary to improve
the persistence of Tx-1 on turfgrass foliage.
Inhibition by ultra-violet radiation (UVR) is a common limitation of biological control use in
the field (Harper, 2006). It has been reported that photo-degradation caused by two different
wavelengths of UV within sun light , UV-A (320-400 nm) and UV-B (280-320 nm), inactivates
most microbial insecticides and fungicides in field conditions (Tamez-Guerra et al., 2005;
Hadapad, 2009). Based on preliminary research in the laboratory, the lack of persistence of P.
aureofacious Tx-1 on turf foliage may be partially explained by its inhibition by UVR (Powell,
pers. comm.). No related data have been published regarding solar UVR sensitivity of Tx-1.
Numerous studies have been done in attempt to screen effective UVR sunscreen to improve
residual tolerance to UV after application due to the lethal impacts of UVR on microbial
biocontrol agents in the field, (Tamez-Guerra et al., 2005). Several promising UVR sunscreen
for biological pesticides have been identified. A humic substances components, humic acid (HA),
ubiquitously present in the environment (Trump, 2006), was previously found to be able to
protect microbial organisms from UV radiation (Corin, 1998; Templeton, 2006; Cantwell, 2008;
Lee, 2009), has low toxicity to microbial organism (Corin, 1998; Lee, 2009), and has been

2

tested on turfgrass before (Cooper, 1998; Dyke, 2009), could be a potential UVR sunscreen for
Tx-1.
Therefore, we hypothesized that Tx-1 might be sensitive to solar UVR; by promoting the
UVR tolerance of Tx-1, it is possible to improve its disease control efficacy in the field. The
objective of this study was to investigate potential methods to improve UV tolerance of Tx-1. To
achieve this goal, UV sensitivity of Tx-1 was first confirmed by challenging it with UV-A and
UV-B. In an attempt to improve UV tolerance of Tx-1, three different approaches were taken:
the first approach was to test the efficacy of HA as a UV protectant for Tx-1. The second
approach was promoting pigmentation of Tx-1 by prolonging the incubation time. The third
approach was to select a spontaneously UV-resistant Tx-1 cells by using intensive UV radiation
(UV-C). In vitro bioassays were performed on isolates of TX-1 form the different treatments for
their biocontrol efficacy after UV exposure. The efficacy tests were performed both in vitro an in
the field.

3

LITERATURE CITED

4

Cantwell, R., Hofmann, R. and Templeton, M. (2008). Interactions between humic matter and
bacteria when disinfecting water with UV light. Journal of Applied Microbiology 105,
25-35.
Cooper, R.J., Liu, C., Fisher, D.S. (1998). Influence of Humic Substances on Rooting and
Nutrient Content of Creeping Bentgrass. Crop Science 38, 1639.
Corin, N., Backlund, P., Wiklund, T. (1998). BACTERIAL GROWTH IN HUMIC WATERS
EXPOSED TO UV-RADIATION AND SIMULATED SUNLIGHT. Chemosphere,
1947-1958.
Dwyer, P.J., Jr., . (1999). Field Efficacy, Persistence, and Antibiotic Production of
Pseudomonas auerofaciens. In Department of Botany and Plant Pathology (East Lansing:
Michigan State University).
Dyke, A.V., Johnson, P.G., Grossl, P.R. (2009). Influence of humic acid on water retention and
nutrient acquisition in simulated golf putting greens. Soil Use and Management 25, 255261.
Hadapad, A.B., Hire, R.S., Vijayalakshmi, N., Dongre, T.K. (2009). UV protectants for the
biopesticide based on Bacillus sphaericus Neide and their role in protecting the binary
toxins from UV radiation. Journal of Invertebrate Pathology 100, 147-152.
Hardebeck, G.A., R. F. Turco, R. Latin, and Z. J. Reicher. (2004). Application of
Pseudomonas aureofaciens Tx-1 through irrigation for control of dollar spot and brown
patch on fairway height turf. HortScience 39(7), 1750-1753.
Harper, D.R. (2006). Biological contorl by microoorganisms. In Encyclopedia of Life Sciences
(John Wiley & Sons.
Lee, E., Lee, H., Jung, W., Park, S., Yang, D., Lee, K. (2009). Influences of humic acids and
photoreactivation on the disinfection of Escherichia coli by a high-power pulsed UV
irradiation. Korean Journal of Chemical Engineering 26, 1301-1307.
Murphy, J., F. Wong, L. Tredway, et al. (2008). Best management practices for anthracnose
on annual bluegrass turf. Golf Course Management 76(8), 93-104.
5

Powell, J.F., Vargas, J. M., Jr., et al. (2000). Management of dollar spot on creeping bentgrass
with metabolites of Pseudomonas aureofaciens (TX-1). Plant Disease 84, 19-24.
Putman, A.I., Jung, G., and Kaminski, J. E. (2010). Geographic distribution of
fungicideinsensitive Sclerotinia homoeocarpa isolates from golf courses in the
northeastern United States. Plant Disease 94, 186-195.
Tamez-Guerra, P., Rodriguez-Padilla, C., and Galá
n-Wong Luis, J. (2005). Solar Radiation
(UV) Protectants for Microbial Insecticides. In Semiochemicals in Pest and Weed
Control (American Chemical Society), pp. 127-147.
Templeton, M.R., Hofmann, R., Andrews, R.C. (2006). UV inactivation of humic-coated
bacteriohages MS2 and T4 in water. Environmental Engineering Science 5, 537-543.
Trump, J.I.V., Sun, Y., Coates, J.D. (2006). Microbial Interactions with Humic Substances.
Advances in Applied Microbiology 60, 55-96.
Vargas, J.M.J. (2005). Management of turfgrass diseases. . (Hoboken, New Jersey: John Wiley
& Sons, Inc.).

6

LITERATURE REVIEW
Dollar spot
Dollar spot disease, caused by Rutstroemia floccosum syn. Sclerotinia homoeocarpa F. T.
Bennet, affects almost all species of turfgrass on golf courses (Vargas, 2005). In order to meet
the industrial quality and playability of the turfgrass, management of this disease is so expensive
that it became the most economically important turfgrass disease in North America (Smiley,
2005).
Symptoms and Epidemiology
On low mowed turf, the disease can be seen as silver-dollar-sized spots with bleached-out
color. Spots may coalesce into irregular shaped patches as the disease progresses and destroys
large areas of turf. Bleached lesions with brown or reddish bands can be seen on longer blades.
The brown bands do not develop on annual bluegrass [Poa annua L. f. reptans (Hausskn.) T.
Koyama] (Vargas, 2005). Under high humidity conditions during early morning, the active
growth of the pathogen is present as white fluffy mycelium on the foliage (Vargas, 2005). Dollar
spot occurs on turf from late spring to the end of fall, but is typically most active from July
through early September (Smiley, 2005). Foliar wetness and guttation fluid are crucial elements
for disease development. Humid days at temperatures of 15 to 32°C followed by cool nights are
the conditions that favor the formation of guttation water and also the infection by R. floccosum.
When suffering from low fertility and water stress, turfgrasses are more prone to dollar spot
infection. The pathogen can be spread by mowers through infected turf clippings, and by
irrigation water and foot traffic (Vargas, 2005).
Control and Management
7

Management strategies for dollar spot include cultural, chemical, and biological control
methods. Cultural practices reduce the impact of dollar spot toward turfgrass and decrease the
need for chemical fungicides. Maintaining adequate fertility during the disease season, especially
via light and frequent applications of nitrogen, decreases the severity of dollar spot and increases
the efficacy of chemical programs (Vargas, 2005). Keeping soil moisture at field capacity level
of also facilitates disease control. Removing the dew during the morning is a common cultural
method that has been adopted by golf course superintendents to prevent dollar spot (Vargas,
2005). In addition, Giordano (2010) and Nikolai (2001) have shown that rolling on golf course
greens can inhibit dollar spot development.
Chemical control
Chemical fungicides are necessary in many cases to achieve acceptable disease control
(Vargas, 2005). Both contact (multisite) and systemic (site-specific) fungicides can be used to
manage dollar spot. The efficacy of contact fungicides, such as chlorothalonil, for long term
disease control is limited by their short persistence on the turf. Their multisite mode of action
largely reduces the risk of fungal resistance. Resistance to contact fungicides among R.
floccosum has not been observed so far. Contact fungicides will be more efficient if applied as a
preventive treatment. Based on chemical structures or biochemical modes of action, systemic
fungicides commonly used on turfgrass can be categorized into four groups: benzimidazoles,
dicarboximides, demethylation inhibitors (DMI), and QoI fungicides. Compared to contact
fungicides, systemic fungicides are more effective for long term dollar spot suppression.
However, the site-specific mode of action encourages the selection of R. floccosum strains with
reduced sensitivity or even resistance to fungicides (Vargas, 2005). Repeated application of
systemic fungicides has been shown to promote the populations of resistant strains and
8

compromise the efficacy of existing chemical treatments for managing dollar spot (Putman,
2010).
Anthracnose
Anthracnose, caused by Colletotrichum cereale Manns sensulato Crouch, Clarke & Hillman,
is one of the most important and destructive diseases on golf course greens and fairways (Vargas,
2005). During warm weather when the pathogen is most active, C. cereale can cause both foliar
anthracnose (foliar blight) and crown rot anthracnose (basal rot) (Smiley, 2005; Vargas, 2005).
Unlike foliar anthracnose, crown rot anthracnose mainly destroys the crowns, the meristematic
tissue of the annual bluegrass plants and kills the host (Smiley, 2005).
Symptoms and Epidemiology
On infected annual bluegrass putting greens or fairways, symptoms initially appear as
yellow to bronze colored spots, from 0.25 to 0.50 inch (0.64-1.30 centimeters) in size (Vargas,
2005; Murphy, 2008). These spots may coalesce and form large, irregularly shaped patches as
the disease progresses (Murphy, 2008). Infection on an individual leaf may first appear as
reddish brown spots which may eventually spread to the entire blade (Vargas, 2005). On infected
blades and stems, unique black reproductive structures (acervuli) with black spines (setae) that
been produced by C. cereale can serve as a diagnostic tool for C. cereale (Vargas, 2005; Murphy,
2008). In the case of crown rot anthracnose, acervuli can be seen in darkened crowns (Vargas,
2005). Anthracnose is a major disease on annual bluegrass worldwide. It is most destructive
during heat stress periods (Murphy, 2008). Foliar anthracnose normally occurs under high
temperatures during summer and fall (Murphy, 2008); (Vargas, 2005). Crown rot anthracnose,
on the other hand, occurs in both hot and cool seasons (Vargas, 2005; Murphy, 2008).
9

Cultural Management
Cultural management to improve host health during stress periods includes maintaining
2

nitrogen fertility level around 0.5 lb. N /1000ft per month, increasing the mowing height,
irrigating lightly and frequently during the day and providing sufficient drainage to avoid soil
saturation. These practices will not only facilitate the control of this disease and minimize the
damage on greens and fairways, but also increase the effectiveness of chemical fungicide
treatments (Vargas, 2005; Murphy, 2008). In addition to traditional cultural approaches to
control anthracnose, recent studies demonstrate the potential usefulness of lightweight rolling
and light frequent topdressing (Murphy, 2008).
Chemical control
Due to the epidemiology and destructiveness of anthracnose, preventive fungicide treatments
control the disease more effectively than curative applications (Murphy, 2008). Among eight
classes of fungicides that are available for anthracnose management, only the benzimidazole,
DMI and QoI classes achieve satisfactory curative control (Murphy, 2008). The other classes of
fungicides are more effective when applied preventively (Crouch, 2003; Towers, 2003). Similar
to R. floccosum, C. cereale has became resistant to systemic fungicides for many years, which
includes QoIs, benzimidazoles and DMI classes (Wong, 2004).
Biological control
Biological control has been used as a successful alternative strategy for managing soilborne
or foliar diseases in a diverse range of crop systems (Baker, 1987; Cook, 1993). Aside from the
efficacy of a suitable biological control organism, the strategies for maintaining population levels

10

and viability of the biological control agent are also needed to achieve satisfaction disease
inhibition (Stack, 1988).
Pseudomonas aureofaciens was first discovered by Kluver (1956) from clay soil.
Pseudomonas is a gram-negative bacterium, aerobic, abundant in agricultural rhizosphere, and
belongs to the category of fluorescent Pseudomonas (Weller, 2007). The word “aureofaciens”
literally means “made golden”, which refers to its ability to produce orange pigments and turn
the growth medium golden (Palleroni, 1984). The golden pigmented antibiotics have been
categorized as phenazine antibiotics (Palleroni, 1984), which are N-containing heterocyclic
molecules with a range of fungal suppression properties (Turner, 1986). Produced through a
secondary metabolite pathway called the shikimic acid pathway (Turner, 1986), these antibiotic
metabolites contribute 90% in fungal inhibition (Pierson, 1992). Studies on P. aureofaciens
strain 30-84 revealed three phenazine components: phenazine 1-carboxylic acid (PCA), 2hydroxy-phenazine-1-carboxylic acid (2-OH-PCA), and 2-hydroxy-phenazine (2-OH-PZ)
(Pierson, 1992). Several hypotheses have been proposed regarding the biochemical mechanisms
by which phenazine antibiotics suppress fungal growth. They include the generation of toxic
oxygen species in intracellular space, inhibition of DNA replication, and disruption of the
electron transport and energy cycling pathway (Pierson III, 1996). By using a phenazine-

deficient mutant (Phz ) of P. aureofaciens strain 30-84, Thomashow et al. (1990) demonstrated
that phenazine metabolites are not only essential in fungal pathogen inhibition, but also vital to
its competitive fitness in the soil environment.
Pseudomonas aureofaciens strain Tx-1 (Tx-1) was identified and developed at Michigan
State University. It is known to produce at least two phenazine antibiotics: PCA and pyrrolnitrin

11

(Dwyer, 1999).

Results of antifungal bioassays demonstrated in vitro and vivo, PCA, a

phenazine metabolite secreted by Tx-1, exhibits antifungal activity against a wide range of
turfgrass pathogens identical to that of a chemical fungicide, chlorothalonil (Powell, 2000). This
antifungal activity is consistent with the satisfactory control of dollar spot (R. floccosum),
summer patch (Magnaporthe poae), and pink snow mold (Macrodochrum nivale) in field studies
(Powell, 1993; Dwyer, 1999). After Tx-1 received an EPA label in 2000, a commercial
fermentation and irrigation delivery system was developed for it. Adopted by numerous golf
courses nationwide, this system allows golf course managers to grow Tx-1 on the course while
apply it to the entire golf course through the irrigation system (Dwyer, 1999; Sigler, 2001;
Vargas, 2005). Despite its successful introduction to turfgrass disease management, issues with
the persistence of Tx-1 on the foliage and its antifungal efficacy under higher disease pressure
have arisen. One of the issues related to its persistence and disease suppression in the field is that
high inoculum concentrations and high application frequency are needed (Dwyer, 1999). Lower
concentration and application frequency result in dramatically decreased disease control (Dwyer,
1999). Inoculums applied through the irrigation system failed to achieve sufficient dollar spot
suppression on fairways under intensive disease pressure during summer (Hardebeck, 2004).
Effect of solar ultraviolet radiation (UVR) on microbial biocontrol agents
In most cases, biological control agents are delivered to a ecological unsuitable environment
(Deacon, 1991), thus the survival and biological antagonistic activity of biocontrol agents are
highly influenced by environmental conditions such as humidity, pH, nutritional availability,
temperature, and UV intensity (Lahlali et al., 2011). It has been reported that photo-degradation
caused by two different wavelengths of UV in sunlight , UV-A (320-400 nm) and UV-B (280320 nm), inactivates most microbial insecticides and fungicides under field conditions (Tamez12

Guerra et al., 2005; Hadapad, 2009). Mechanisms of phototoxicity caused by UV-A and UV-B
are different. High energy photons that carried by UV-B cause direct damage to DNA by
forming cyclobutane pyrimidine dimmers, which disrupt DNA structure and introduce mutation
(Pfeifer, 1997; Griffiths, 1998; Jacobs, 2001). Lethal effects triggered by UV-A are mainly due
to generation of intracellular reactive oxygen species (Eisenstark, 1987; Griffiths, 1998). The
sensitivity to UV has limited the commercial development of certain bioinsecticides (Pusztai M,
1991; Hadapad, 2009), it is reasonable to believe that UV can be more destructive for foliar
applied biological control agents (Bull, 1976).
The survival and colonization of bacteria or microbial biocontrol agents in UV intensive
habitats, such as the leaf surface, largely depend on tolerance to radiation (Sundin, 2004).
Phyllosphere bacteria have developed various strategies to improve UV tolerance (Sundin, 1999),
including pigmentation (Sundin, 1999, 2004) and DNA-repair operon (Sundin, 1996). Unlike
eukaryotic microbes, whose pigments significantly improve survival under UV-B radiation
(Wang, 1994), the small size of bacterial cells limits the utilization of pigment-dependent selfshading mechanisms (Garcia-Pichel, 1994) and may only provide protection against the lowerenergy UV-A (Sundin, 2004).
UV sunscreen for microbial biology control agent
Due to the lethal impacts of UV on microbial biocontrol agents in the field, numerous
studies have been done in attempt to screen effective UV protectants to improve residual
tolerance to UV after application (Tamez-Guerra et al., 2005).

Researchers working with

biological pesticides, such as Bacillus sphaericus Neide, have identified several promising UV
protectants (Hadapad, 2009; Lahlali et al., 2011). No UV protectants have ever been tested with

13

Tx-1 on turf systems. Nevertheless, the discovery of UV protectants for biopesticides prompted a
searching for UV protectant candidates for Tx-1.
UV protectant candidate: Humic acid
Humic substances (HS) are produced through the decomposition of organic substances
released by dead organisms, a process also called humification (Steinberg, 2004; Trump,
2006).HS is ubiquitous in the environment, and is present in soil, water and sediment (Trump,
2006). Humic acid (HA) is one of the three basic components of HS (Steinberg, 2004; Trump,
2006). Traditionally, HA can be isolated from HS based on its solubility at pH values below pH
2.0 (Steinberg, 2004; Trump, 2006). The potential of using HA as a UV protectant has been
supported in multiple papers, in which HA showed the ability to reduce the sensitivity of
numerous bacteria and bacteriophages to UV radiation (Corin, 1998; Templeton, 2006; Cantwell,
2008; Lee, 2009). Investigation of the macromolecular structure of HA revealed an abundance of
functional groups that contain conjugated unsaturated double bonds capable of absorbing UV
radiation (Corin, 1998; Uyguner and Bekbolet, 2005). No inhibition or toxicity was observed
when bacteria were grown in HA media (Corin, 1998; Lee, 2009). Effects of HA on turfgrass
were studied by Cooper (1998) and Dyke (2009). Their data showed when applied to bentgrass,
HA increases root length of bentgrass or root mass.

14

LITERATURE CITED

15

Baker, K.F. (1987). Evoloving concepts of biological control of plant pathogens. Annual
Review of Phytopathology 25, 67-85.
Bull, D.L.R., R. L.; House, V. S.; Pryor, N. W. (1976). Improved formulations of the Heliothis
nuclear polyhedrosis virus. Journal of Economic Entomology 69, 731-736.
Cantwell, R., Hofmann, R. and Templeton, M. (2008). Interactions between humic matter and
bacteria when disinfecting water with UV light. Journal of Applied Microbiology 105,
25-35.
Cook, R.J. (1993). Making greater use of introduced microorganisms for biological control of
plant pathogens. Annual Review of Phytopathology 31, 53-80.
Cooper, R.J., Liu, C., Fisher, D.S. (1998). Influence of Humic Substances on Rooting and
Nutrient Content of Creeping Bentgrass. Crop Science 38, 1639.
Corin, N., Backlund, P., Wiklund, T. (1998). BACTERIAL GROWTH IN HUMIC WATERS
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Sigler, W.V., Nakatsu, C. H., Reicher, Z. J., Turco, R. F. (2001). Fate of the Biological
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19

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management. Golf Course Management 72(6), 75-80.

20

CHAPTER ONE
UVR SENSITIVITY TEST FOR TX-1 IN VITRO AND MEANS TO IMPROVE ITS UV
TOLERANCE

ABSTRACT

A fungicide/antibiotic-producing bacterial strain of Pseudomonas aureofaciens Tx-1 (Tx1) has been commercialized for golf course use with some success in recent years. To achieve
adequate control it must be applied daily. This may be partially explained by UV susceptibility
of Tx-1, which is a common limitation of biocontrol agents. In this study, results from various
wavelengths of ultraviolet radiation exposure tests confirmed this hypothesis by showing a
significant drop in Tx-1 survivors following increased dosage. Three approaches were tested to
improve UV tolerance of Tx-1: 1) protection with a promising sunscreen, humic acid (HA), was
demonstrated in all UV exposure tests. Moreover, addition of HA the highly UV-B sensitive
mutant Pseudomonas aeruginosa (phl-, uvrA-) provide statistically significant improvement in
UV-B tolerance. 2) In an attempt to identify the most UV-resistant individual cells, Tx-1 was
exposed to intensive UV light (UV-C) for various lengths of time (in vitro). Cells (10TC30) with
2

significantly improved survival rate after 7.3 J/m UV-C irradiation was found followed by 10
2

cycles of 42 J/m of UVC exposure. 3) When extend the incubation time to 72 hours, Tx-1
2

demonstrated better UV-A tolerance at 60 and 90 kJ/m . Results from bioassays against R.
floccosum showed the antifungal activity of Tx-1 did not change after UV exposures.

21

Introduction
Biocontrol of turfgrass diseases is an important tool for golf course managers.
Pseudomonas aureofaciens Tx-1 (Tx-1) is a commercially available biocontrol agent used for the
control of turfgrass diseases. The persistence of Tx-1 on the foliage and its antifungal efficacy
under high disease pressure have been addressed (Dwyer, 1999; Hardebeck, 2004). Preliminary
research indicates that the bacterium might be susceptible to inhibition by solar ultraviolet
radiation (UVR) (Powell, pers. comm.). Inactivation by UV is a common limitation of biological
control agent in the field (Harper, 2006). Photo-degradation caused by two different wavelengths
of UVR, UV-A (320-400 nm) and UV-B (280-320 nm), inactivates most microbial insecticides
and fungicides in field conditions (Tamez-Guerra et al., 2005; Hadapad, 2009). However, no
related data have been reported on UVR sensitivity of Tx-1. Studies have been done in attempt to
screen effective UVR sunscreen to improve residual tolerance to UVR after application (TamezGuerra et al., 2005). Several promising UVR sunscreen for biological pesticides were identified.
A component of humic substances, Humic acid (HA), is a potential UVR sunscreen for Tx-1. It
is ubiquitous in the environment (Trump, 2006), has been found to protect microbial organisms
from UV radiation (Corin, 1998; Templeton, 2006; Cantwell, 2008), and has low in vitro
toxicity to microbes. It has been tested on turfgrass before, and no negative effects were
observed (Cooper, 1998; Dyke, 2009).
Therefore, we hypothesized that Tx-1 might be sensitive to solar UVR; by promoting the
UVR tolerance of Tx-1, it is possible to improve its disease control efficacy in the field. The
objectives of this study were to: characterize the sensitivity of Tx-1 to UV-A and UV-B in vitro
and investigate potential methods for improving UV tolerance of Tx-1. To achieve this goal, UV
sensitivity of Tx-1 was first confirmed by challenging it with UV-A and UV-B. In an attempt to
22

improve UV tolerance of Tx-1, three different approaches were taken: 1) Testing the efficacy of
HA as a UV protectant. 2) Promoting pigmentation of Tx-1. 3) Selecting a spontaneously UVresistant Tx-1 cells. To investigate biocontrol efficacy of Tx-1 with different approaches after
UV exposure, in vitro bioassays against R. floccosum were performed.
Materials and Methods
Preparation of Bacterial Cultures
Tx-1 cultures were grown in 50 mL of Tryptic soy broth (Becton, Dickinson and
Company. USA), and P. aeruginosa cultures were grown in Luria broth base (LIFE
0

TECHNOLOGIES, Paisley, Scotland) at 25 C on a rotary shaker at 100 rpm for 24 to 72 hours
prior to UV exposure. Before each UV exposure, 1 mL aliquots of the cultures were pelleted and
washed twice in 1 mL sterile phosphate buffered saline solution (PBS). These 1 mL cultures
were resuspended in 9 mL PBS, or 9 mL PBS plus 2 mL HA stock solution (10 mg/mL)
(SIGMA-ALDRICH, Co., Spruce Street, St. Louis, MO) in a sterile glass petri dish and stored on
ice until irradiation.
UV irradiation
Cultures were exposed to different UV lamps: UV-B wavelengths were generated by an
XX-15M UV lamp (UVP Products, San Gabriel, CA.) and filtered through polystyrene which
blocks UV-C wavelengths (<290 nm) (Sundin, 2001); UV-A wavelengths was generated by an
XX-15L; UV-C radiation was produced by an XX-15C UV lamp (UVP Products). The lamps
were fixed at constant height above the bacterial cultures. UV output was measured by a UV-X
radiometer (UVP Products) once lamps were warmed up and stabilized for 15 min. Energy
intensity of all UV wavelengths was determined based on UV output levels, as used in previous
23

studies (Jacobs, 2001; Sundin, 2004). Bacterial cultures were constantly agitated by a rocking
shaker (seven revolutions per minute) under the UV-A lamp or by hand under UV-B or UV-C
lamps to prevent shading. To avoid light repair mechanisms, all bacterial cultures were kept in a
dark condition during UV-B and UV-C irradiations.
After each UV irradiation, 200-µl samples were transferred from the dish to sterile micro-5

tubes for dilution. Cultures were serially diluted in sterile PBS to 10 , 10

-6

or 10

-7

concentrations by repeatedly transferring 40-µl samples into 360 µl PBS until the targeted
dilution concentration was reached. Three to four replicates of 50 µl of diluted samples were
then plated on 0.5 X potato dextrose agar (Becton, Dickinson and Company. Sparks, MD) plates
for Tx-1, or Luria agar for P. aeruginosa (Becton, Dickinson and Company. Sparks, MD). All
0

plates from UV-B and UV-C irradiations were then incubated for 72 h at 25 C under dark
conditions.
After incubation, plates were examined and bacterial colonies counted. The mean
CFU/0.05 ml was calculated by multiplying the number of CFUs by the dilution factor.
Effects of Humic acid on population size of Tx-1
To exam the affects of HA on Tx-1, bacterial cultures were grown in 100 mL of Tryptic
soy broth (Becton, Dickinson and Company. Sparks, MD) mixed with 0.2 g of HA sodium salt
(SIGMA-ALDRICH, Co. St. Louis, MO). pH of the growth media and population size were
monitored up to 24 hours.
Bioassay against dollar spot fungus

24

Bacteria were grown on potato dextrose agar (Becton, Dickinson and Company. Sparks,
0

MD) for 48 hours at 25 C. One transfer loop was streaked down the center of each PDA plate.
0

The plates were incubated at 25 C for 24 hours prior to fungal inoculation. Rutstroemia
floccosum (dollar spot), was grown on PDA. Four 2-mm agar plugs containing fungal mycelia
were transferred to two PDA plates without bacteria (control) and two plates with P.
0

aureofaciens. Each plug represented one replication. Plates were incubated at 25 C. Fungal
growth was measured after two days of incubation. Colony diameter was measured parallel to
and perpendicular to the bacterial streak, and the average radius was calculated from these
2

measurements. Colony area was calculated as πr , where r = average radius.

Data analysis
Data was analyzed by using the SAS (Statistical Analysis Software, Cary, N.C.) ANOVA
least significant difference (LSD) test (p=0.05). All tests were replicated three times.
Results
Characterization of the solar UVR sensitivity for Tx-1
To estimate the solar UV sensitivity of Tx-1, the bacterium was exposed to UV-A or UVB. The exposure length of UV irradiations was per-determinate based on average of four hour
(1100 to 1500) solar UV irradiance during summer time (Sundin, 2004). The sensitivity to UVA and UV-B was compared with unexposed cells. Results showed a marked reduction in UV-A
2

2

survival following doses of 60 kJ/m and 90 kJ/m (Figure 1). Percentage of survival decreased
2

2

significantly to 41.6% when the dose increased to 120 kJ/m . After received 180 kJ/m UV-A,
up to 94% cells were killed (Figure 1.01). Results from UV-B exposure showed survival
25

2

significantly dropped to 8.9 % after 8.26 kJ/m dose of UV-B (Figure 1.02). Only 4% survived
2

13.77 kJ/m UV-B exposure, which is close to the average UV-B output within 4 h during
summer time (Sundin, 2004) (Figure 1.02). Together, this observation suggests that Tx-1 is
sensitive to solar UV radiation.
UVR protection from humic acid (HA)
The objective of these experiments was to determine the effect of HA on UVR
degradation on Tx-1. In this study, HA was added with bacterial cultures as described in
material and methods. The culture + HA mixture was then challenged with UV-A, UV-B or UVC. UVR protection from HA was demonstrated in both UVR exposure tests (Figure 1.03-1.05).
Compared to unprotected Tx-1, which suffered a significant drop in survivors following
increased UVR doses as described in previous section, up to 100% and 77% of Tx-1 were
2

2

protected by addition of HA when the dose increased to 180kJ/m of UV-A and 13.77 kJ/m of
UV-B, respectively (Figure 1.03, 1.04). When exposed to high-energy UV-C wavelengths, 1- to
4-log more inactivation of unprotected Tx-1 was observed for UVR doses ranged from 7.1 to
2

42.6 J/m (Figure 1.05), which is consistent with the sensitivity to UV-A and UV-B, while 85%
Tx-1 survived with the protection of HA.
In previous tests on HA, 120 mg/L HA demonstrated the best protection for UV sensitive
2

2

bacteria under 14 mJ/cm (0.14kJ/m ) UV exposure (Cantwell, 2008). Due to the intensiveness
of UV radiation level in our tests, concentration of HA was set to 1.67g/L, approximately 100
times more than 120 mg/L. To determine the optimal concentration of HA for UV protection, a
highly UV-B sensitive bacterium, Pseudomonas aeruginosa (phl-, uvrA-), was tested. The
26

addition of HA conferred UV-B tolerance to this highly susceptible P. aeruginosa mutant (Table
2

1.01). Unprotected cells died with 0.2 kJ/m of exposure while 66.3% of cultures that were
2

mixed with HA survived. When the UV dose increased to 0.9 J/m , 23% of P. aeruginosa
culture survived with the addition of HA. Four different concentrations of HA (1.67g/L, 0.89g/L,
0.42g/L and 0.17g/L) were tested with P. aeruginosa. Results show that the addition of 1.67g/L
HA significantly improved UV tolerance of Tx-1 during the 10 min UV-B exposure, while
0.89g/L HA was unable to protect the cells when the energy of UV-B increased to 0.9 kJ/m

2

(Table 1.02). Collectively, the addition of HA significantly increased UV tolerance of Tx-1 and
the P. aeruginosa UV-B sensitive mutant.

27

Figure 1.01 Survival of P. aureofaciens Tx-1 after exposed to UV-A radiation.

100
90

100

82.3

82.3

80
Survival %

70
60
50

41.6 *

40
30

14.3 *

20

5.9 *

10
0
0

60

90

120

UV-A dose (kJ/m2)
*, Significant at the 0.05 statistical probability level.

28

150

180

Figure 1.02 Survival of P. aureofaciens Tx-1 after exposed to UV-B radiation.

100

100

90
80
Survival %

70
60
50
40
30
20

7.9 *

8.9 *

10

4*

0
0

8.26

11.02

UV-B dose (kJ/m2)

*, Significant at the 0.05 statistical probability level.

29

13.77

Figure 1.03. Survival of P. aureofaciens Tx-1 with or without the addition of humic acid after
exposed to UV-A.
100
90

82.3

82.3

80

P. aureofaciens Tx-1

Survival %

70
60
50

P. aureofaciens Tx-1
+ HA

41 *

40
30
20

14 *

5.9 *

10
0
0

60

90

120

150

UV-A dose (kJ/m2)
*, Significant at the 0.05 statistical probability level.

30

180

Figure 1.04. Survival of P. aureofaciens Tx-1 with or without the addition of humic acid after
exposed to UV-B.

100

100

90
80
82

Survival %

70

77

77

P. aureofaciens
Tx-1

60
50
40

P. aureofaciens
Tx-1 + HA

30
20

8.9 *

7.9 *

10

4*

0
0

8.26
11.02
UV-B dose (kJ/m2)

*, Significant at the 0.05 statistical probability level.

31

13.77

Figure 1.05. Log survival of P. aureofaciens Tx-1 with or without the addition of humic acid
after exposed to UV-C radiation.

2
*

Log (Survival %)

1

*

0
-1

*

-2

*

P. aureofaciens Tx-1

*

-3
P. aureofaciens Tx-1 + HA

*

-4
0

7.1

14.2

21.3

28.4

UV-C dose (J/m2)
*, Significant at the 0.05 statistical probability level.

32

35.5

42.6

Table 1.01. Survival of P. aeruginosa (phr-, uvrA-) or P. aureofaciens Tx-1 humic acid
-2 -1
mixture after exposed to 1.69 J m s UV-B radiation.
Treatment

Time (min)

Percentage of
survival

LSD

P. aeruginosa (phr-, uvrA-)

0

100

a

P. aeruginosa (phr-, uvrA-) +1.67g/L HA

0

100

a

P. aeruginosa (phr-, uvrA-) +1.67g/L HA

2.5

82.6

b

P. aeruginosa (phr-, uvrA-) +1.67g/L HA

5

34.8

c

P. aeruginosa (phr-, uvrA-) +1.67g/L HA

10

26.1

cd

P. aeruginosa (phr-, uvrA-)

2.5

0

d

P. aeruginosa (phr-, uvrA-)

5

0

d

P. aeruginosa (phr-, uvrA-)

10

0

d

a

, Treatment means followed by the same letter do not significantly differ (LSD, p=0.05).

33

a

Table 1.02. Survival of P. aeruginosa (phr-, uvrA-) or P. aeruginosa (phr-, uvrA-) mixed
-2 -1
with different concentrations of humic acid after exposed to 1.49 J m s UV-B radiation.
Treatment

UV-B dose (kJ/m )

Percentage of
survival

LSD

P. aeruginosa (phr-, uvrA-)+ 0.89g/L HA

0

100.00

a

P. aeruginosa (phr-, uvrA-)+ 0.42g/L HA

0

100.00

a

P. aeruginosa (phr-, uvrA-)

0

100.00

a

P. aeruginosa (phr-, uvrA-)+ 0.17g/L HA

0

100.00

a

P. aeruginosa (phr-, uvrA-)+ 1.67g/L HA

0

100.00

a

P. aeruginosa (phr-, uvrA-)+ 1.67g/L HA

0.25

66.34

b

P. aeruginosa (phr-, uvrA-)+ 1.67g/L HA

0.5

49.71

c

P. aeruginosa (phr-, uvrA-)+ 0.89g/L HA

0.25

31.11

d

P. aeruginosa (phr-, uvrA-)+ 1.67g/L HA

1.04

22.97

e

P. aeruginosa (phr-, uvrA-)+ 0.89g/L HA

0.5

11.89

f

P. aeruginosa (phr-, uvrA-)+ 0.89g/L HA

1.04

1.79

g

P. aeruginosa (phr-, uvrA-)+ 0.42g/L HA

0.25

1.17

h

P. aeruginosa (phr-, uvrA-)

0.25

0.00

h

P. aeruginosa (phr-, uvrA-)+ 0.17g/L HA

0.25

0.00

h

P. aeruginosa (phr-, uvrA-)

0.5

0.00

h

P. aeruginosa (phr-, uvrA-)+ 0.17g/L HA

0.5

0.00

h

P. aeruginosa (phr-, uvrA-)+ 0.42g/L HA

0.5

0.00

h

P. aeruginosa (phr-, uvrA-)

1.04

0.00

h

P. aeruginosa (phr-, uvrA-)+ 0.17g/L HA

1.04

0.00

h

P. aeruginosa (phr-, uvrA-)+ 0.42g/L HA

1.04

0.00

h

2

a

, Treatment means followed by the same letter do not significantly differ (LSD, p=0.05).

34

a

2

Table 1.03. Average fungal colony area (cm ) of R. floccosum on PDA medium
inoculated with P. aureofaciens Tx-1 or P. aureofaciens Tx-1 humic acid mixture that received
2
90-180 kJ/m UV-A radiation.
2 DAT
UV-A Dose
2
(kJ/m )
P. aureofaciens Tx-1

0.00

P. aureofaciens Tx-1

LSD

Average fungal
colony
2 b
area(cm )

LSD

2.2

cd

6.2

b

90.00

1.9

cd

4.6

bc

P. aureofaciens Tx-1

180.00

6.5

b

5.2

b

P. aureofaciens Tx-1+
humic acid

0.00

1.7

cd

4.5

bc

P. aureofaciens Tx-1+
humic acid

90.00

1.2

d

2.4

c

P. aureofaciens Tx-1+
humic acid

180.00

1.7

cd

4.2

bc

Unexposed control

b

Average fungal
colony
2b
area(cm )

4 DAT

0.00

15.7

a

29.7

a

2

a

2

,Average fungal colony area (cm ) calculated using πr , where r=average radius.

a

, Treatment means followed by the same letter do not significantly differ (LSD, p=0.05).

35

a

2

Table 1.04. Average fungal colony area (cm ) of R. floccosum on PDA medium
inoculated with P. aureofaciens Tx-1 or P. aureofaciens Tx-1 humic acid mixture that received
2
20.85-41.7 J/m UV-C radiation.
2 DAT
UV-C Dose
2
(J/m )
P. aureofaciens Tx-1

0.00

P. aureofaciens Tx-1

LSD

Average
fungal colony
2 b
area(cm )

LSD

1.8

c

4.4

b-d

20.85

0.8

e

1.8

f

P. aureofaciens Tx-1

41.7

1.2

c-e

2.7

d-f

P. aureofaciens Tx-1+
humic acid

0.00

1.6

cd

4.8

bc

P. aureofaciens Tx-1+
humic acid

20.85

1.3

c-e

3.8

c-e

P. aureofaciens Tx-1+
humic acid

41.7

0.9

de

2.2

ef

Unexposed control

b

Average fungal
colony
2 b
area(cm )

4 DAT

0.00

15.7

a

29.7

a

2

a

2

, Average colony area (cm ) calculated using πr , where r=average radius.

a

, Treatment means followed by the same letter do not significantly differ (LSD, p=0.05).

36

a

2

Table 1.05. Average fungal colony area (cm ) of R. floccosum on PDA medium
inoculated with P. aureofaciens Tx-1 or P. aureofaciens Tx-1 humic acid mixture that received
2
8.26-13.77 kJ/m UV-B radiation.
1 DAT

Treatment

2 DAT

4 DAT

Average
Average
Average
fungal
UV-B dose fungal colony
fungal colony
colony
2
2 b
a
2 b
a
2 b
a
(kJ/m )
area(cm )
LSD area(cm )
LSD area(cm ) LSD

P. aureofaciens
Tx-1+ humic acid

0

1

c

2.52

b

5.7

b

P. aureofaciens
Tx-1+ humic acid

11.02

1.1

c

2.81

b

6.0

b

P. aureofaciens
Tx-1+ humic acid

13.77

1.2

bc

2.77

b

5.9

b

P. aureofaciens
Tx-1

8.26

1.2

bc

3.21

b

6.1

b

P. aureofaciens
Tx-1+ humic acid

8.26

1.3

bc

2.55

b

5.4

b

P. aureofaciens
Tx-1

11.02

1.4

bc

2.69

b

5.5

b

P. aureofaciens
Tx-1

0

1.5

bc

2.87

b

5.7

b

P. aureofaciens
Tx-1

13.77

1.7

b

3.17

b

6.2

b

Control

0

4.8

a

18.1

a

29.9

a

b

2

2

, Average colony area (cm ) calculated using πr , where r=average radius.

a

, Treatment means followed by the same letter do not significantly differ (LSD, p=0.05).

37

In vitro bioassay tests
To investigate the anti-fungal property of Tx-1 after it was exposed to UV or mixed of
HA, in vitro bioassays against R. floccosum (dollar spot) were performed as described in material
and methods. Results showed the biological control efficacy of Tx-1 cells that received UVR
exposure, with or without HA addition against R. floccosum pathogen was maintained compared
to unexposed Tx-1 (Table 1.03-1.05).
Identification of improved UVR tolerant bacterial strains of Tx-1
UV-C wavelengths introduce similar yet more intense DNA damage to microbes (Sundin,
1999). This wavelengths have been used to generate data to estimate the UV-B tolerance of close
related bacteria (Sundin, 2004). In an attempt to identify UVR tolerant strains, cells of Tx-1
2

were exposed to multiple cycles of 42 J/m of UV-C radiation as described in material and
2

methods. Survivors of Tx-1 after each radiation cycle were selected and exposed to 42 J/m of
UV-C radiation along with the unexposed Tx-1. The strain of Tx-1, which survived through 10
2

cycles of 42 J/m of UV-C exposures, was selected and named as 10TC30. 30% more 10TC30
2

cells survived after exposure to 7.3 J/m of UV-C compared to unexposed Tx-1 (Figure 1.06).
When exposed to UV-A, 10TC30 showed no difference in survival compared to unexposed Tx-1
(Table 1.06).

38

Figure 1.06. Log survival of P. aureofacious Tx-1 and 10TC30 after exposed to UV-C
radiation.

3
*

2

Log (Survival %)

1
0
-1
-2
-3
-4

10TC30
Tx-1

-5
-6
0

7.3

14.6

21.9

29.2

UV-C dose (J/m2)
*, Significant at the 0.05 statistical probability level.

39

43.8

58.4

Table 1.06. Survival of P. aureofaciens Tx-1 or 10TC30 strain after exposed to 60-180
J/m UV-B radiation.
2

Treatment

UV-A Dose
2
(kJ/m )

Percentage of
survival

LSD

P. aureofaciens
Tx-1

0

100

ab

P. aureofaciens
Tx-1

60

100

ab

P. aureofaciens
Tx-1

90

63.103

c

P. aureofaciens
Tx-1

120

51.724

cd

P. aureofaciens
Tx-1

150

46.207

de

P. aureofaciens
Tx-1

180

31.034

ef

10TC30

0

100

ab

10TC30

60

85.556

b

10TC30

90

67.222

c

10TC30

120

37.778

d-f

10TC30

150

41.667

d-f

10TC30

180

28.333

f

a

a

, Means followed by the same letter in a column are not significantly different according to
Fisher’s LSD (P>0.05).

40

Prolonging incubation time to increase UVR tolerance
Tx-1 cultures that incubated for 24 or 72 h were tested for sensitivity to UV. The effects
of increased incubation period which produce more pigment in the cells were tested to see if the
pigment increases TX-1 tolerance to UV exposure.

Tx-1 cultures incubated for 72 hours
2

performed significantly better when exposed to less than 90 kJ/m UV-A irradiation than did the
24 hours cultures (Figure 1.07). There were no differences in the survival cells from either
incubation periods when the energy output of UV-A was above 90 kJ/m2 (Figure 1.07). This
improved UV tolerance of Tx-1 due to prolonged incubation time was not consistent under UVC exposure, since no difference were observed between these two incubation time after UV-C
exposures (Figure 1.08).

41

Figure 1.07. Survival of P. aureofacious Tx-1 incubated for 24 or 72 hr. after exposed to UV-A
radiation.

100
90

a*

P. aureofaciens Tx-1 grown
for 24 hours

a*

80
P. aureofaciens Tx-1 grown
for 72 hours

Survival (%)

70
60
b

50

b

bc

40

b-d
b-d

30
20

cd

10
0
0

60

90

120

150

UV-A dose kJ/m2
*, Means followed by the same letter are not significantly different according to Fisher’s LSD
(P>0.05).

42

Figure 1.08. Log survival of P. aureofacious Tx-1 after UV-C exposure.
Tx-1 grown for 24 h

a

2

Tx-1 grown for 72 h
b

Log (Survival (%)

1
0

c
-1
c
c

-2

c
-3
c
-4
0

7.1

14.2

21.3

28.4

35.5

42.6

UV-C dose J/m2
Means followed by the same letter are not significantly different according to Fisher’s LSD
(P>0.05).

43

Discussion
This is the first analysis of UVR sensitivity test for P. aureofacious Tx-1, and the results
confirmed that the UV-A and UV-B wavelengths within sunlight can largely limited the survival
of Tx-1. More than 90% of Tx-1 cells were killed by UV output which is close to 4 hr of solar
UV during the summer time (Figure 1.01, 1.02). This is not surprising, since solar photoinhibition has been shown to inactivate most microbial biocontrol agents in the field (TamezGuerra et al., 2005; Hadapad, 2009).

Interestingly, although the survival of Tx-1 was

significantly decreased after UV radiation, results from bioassay show no change in anti-fungal
activity against R. floccosum (Table 1.03-1.05). It could be possible that the cellular damage
caused by UVR did not affect the process of antibiotic production of Tx-1. This observation
indicates that selection for UV tolerant isolates could be made. The UV sensitivity of Tx-1
addresses the necessity of developing strategies to improve UV tolerance of Tx-1.
One way to improve UV tolerance of biocontrol agent is through adding sunscreen
materials (Tamez-Guerra et al., 2005). The potential of using HA as a UV protectant has been
supported in multiple papers, in which HA showed the ability to reduce the sensitivity of
numerous bacteria and bacteriophages to UV radiation (Corin, 1998; Templeton, 2006; Cantwell,
2008). Different from these studies, in which HA was tested only under low-dose UV radiation
(0.05-0.14 kJ/m2), this is the first study to demonstrate the UV protection property of HA under
intensive UV-A (60-180 kJ/m2), UV-B (8.26-13.77 kJ/m2), and UV-C (7.1-42.6 J/m2) radiation
(Figure 1.03-1.05). It has been suggested that the degree of UV protection from HA is
concentration dependent. This was demonstrated when a highly UV-B sensitive Pseudomonas
aeruginosa (phl-, uvrA-) strain was mixed with various concentration of HA (Cantwell, 2008).
Our data are in the agreement with this hypothesis by demonstrating the level of UV protection
44

from HA dropped significantly as the concentration decreased (Table 1.02). The fact that antifungal activity of Tx-1 was not changed after mixing with HA further supports the role of HA as
a suitable UV sunscreen for Tx-1.
Based on the results from UV sensitivity tests of Tx-1, which demonstrate unchanged
anti-fungal activity of Tx-1 following UV-A, UV-B, and UV-C exposures (Table 1.03-1.05), we
hypothesized that by using intensive UV radiation, it is possible to isolate UV tolerant Tx-1
without affecting anti-fungal property. 10TC30, a strain of Tx-1 which survived 10 cycles of 42
2

J/m of UV-C exposures, showed significantly improved UV tolerance (Figure 1.06). This result
is similar to the recent studies which demonstrate UVR-inducible mutability can lead to gains in
UVR tolerance (Weigand, 2009, 2011). We hypothesize that the increase in UVR tolerance of
Tx-1 after receiving multiple cycles of UV-C exposure could be a result of the inducible
mutability which was trigged by UV-C. When exposed under UV-A, 10TC30 showed no
increasing in UV-A tolerance (Table 1.06). This is not a surprise, since the mechanisms of phototoxicity caused by UV-A are different from DNA-damaging UV-B/UV-C (Jacobs, 2001; Sundin,
2004). Moreover, it has been shown that UV-B/UV-C tolerance may not translate to UV-A
tolerance (Sundin, 2004). More tests are needed to understand the nature of UVR tolerance of
10TC30.
It has been proposed that cell pigmentation could increase UV-A tolerance (Sundin,
2004). Tx-1 is known to produce at least one pigmented antibiotic, PCA (Dwyer, 1999). The
production of the antibiotics of Tx-1 occur primarily during the stationary phase, which starts at
about 20 h of incubation (Powell, 1993). Hence, we hypothesized that prolonged incubation
period may promote the production of PCA, which may lead to an improvement in UV-A

45

tolerance. Indeed, Tx-1 incubated for 72 h was more tolerant to UV-A (Figure 1.07), but the
sensitivity to UV-C radiation of Tx-1 from both incubation times remained the same (Figure
1.08). Thus, the increased incubation period is most likely to increase only UV-A tolerance of
Tx-1. It is important to note that the population size of the longer incubation period was 1 log
larger than the 24 h one. More UV energy is needed to inactive bacteria as the population
increased (Gomes, 2008). The improvement in population size of Tx-1 may also contribute to its
UV-A tolerance.

46

LITERATURE CITED

47

Cantwell, R., Hofmann, R. and Templeton, M. (2008). Interactions between humic matter and
bacteria when disinfecting water with UV light. Journal of Applied Microbiology 105,
25-35.
Cooper, R.J., Liu, C., Fisher, D.S. (1998). Influence of Humic Substances on Rooting and
Nutrient Content of Creeping Bentgrass. Crop Science 38, 1639.
Corin, N., Backlund, P., Wiklund, T. (1998). BACTERIAL GROWTH IN HUMIC WATERS
EXPOSED TO UV-RADIATION AND SIMULATED SUNLIGHT. Chemosphere,
1947-1958.
Dwyer, P.J., Jr., . (1999). Field Efficacy, Persistence, and Antibiotic Production of
Pseudomonas auerofaciens. In Department of Botany and Plant Pathology (East Lansing:
Michigan State University).
Dyke, A.V., Johnson, P.G., Grossl, P.R. (2009). Influence of humic acid on water retention and
nutrient acquisition in simulated golf putting greens. Soil Use and Management 25, 255261.
Gomes, A.I., Santos, J.C., Vilar, V.J.P., Boaventura, R.A.R. (2008). Inactivation of Bacteria
E. coli and photodegradation of humic acids using natural sunlight. Applied Catalysis B:
Environmental 88, 283-291.
Hadapad, A.B., Hire, R.S., Vijayalakshmi, N., Dongre, T.K. (2009). UV protectants for the
biopesticide based on Bacillus sphaericus Neide and their role in protecting the binary
toxins from UV radiation. Journal of Invertebrate Pathology 100, 147-152.
Hardebeck, G.A., R. F. Turco, R. Latin, and Z. J. Reicher. (2004). Application of
Pseudomonas aureofaciens Tx-1 through irrigation for control of dollar spot and brown
patch on fairway height turf. HortScience 39(7), 1750-1753.
Harper, D.R. (2006). Biological contorl by microoorganisms. In Encyclopedia of Life Sciences
(John Wiley & Sons.
Jacobs, J.L., Sundin, G.W. (2001). Effect of Solar UV-B Radiation on a Phyllosphere Bacterial
Community. Applied and Environmental Microbiology 67, 5488-5496.

48

Jacobs, J.L., Carroll, T.L., Sundin, G.W. (2004). The Role of Pigmentation, Ultraviolet
Radiation Tolerance, and Leaf Colonization Strategies in the Epiphytic Survival of
Phyllosphere Bacteria. Microbial Ecology 49, 104-113.
Kim, J.J., Sundin, G.W. (2001). Construction and Analysis of Photolyase Mutants of
Pseudomonas aeruginosa and Pseudomonas syringae: Contribution of Photoreactivation,
Nucleotide Excision Repair, and Mutagenic DNA Repair to UV-B Radiation. Applied
and Environmental Microbiology 67, 1405-1411.
Powell, J.F. (1993). Utilization of Bacterial Metabolites for the Management of Fungal
Turfgrass Pathogens. In Department of Botany and Plant Pathology (East Lansing:
Michigan State University).
Sundin, G.W., Jacobs, J.L. (1999). Ultraviolet Radiation (UVR) Sensitivity Analysis and UVR
Survival Strategies of a Bacterial Community from the Phyllosphere of Field-Grown
Peanut (Arachis hypogeae L.). Microbial Ecology 38, 27-38.
Tamez-Guerra, P., Rodriguez-Padilla, C., and Galá
n-Wong Luis, J. (2005). Solar Radiation
(UV) Protectants for Microbial Insecticides. In Semiochemicals in Pest and Weed
Control (American Chemical Society), pp. 127-147.
Templeton, M.R., Hofmann, R., Andrews, R.C. (2006). UV inactivation of humic-coated
bacteriohages MS2 and T4 in water. Environmental Engineering Science 5, 537-543.
Trump, J.I.V., Sun, Y., Coates, J.D. (2006). Microbial Interactions with Humic Substances.
Advances in Applied Microbiology 60, 55-96.
Weigand, M.R., Sundin, G.W.,. (2009). Long-term effects of inducible mutagenic DNA repair
on relative fitness and phenotypic diversification in Pseudomonas cichorii 302959.
Genetics 181, 199-208.
Weigand, M.R., Tran, V.N., Sundin, G.W.,. (2011). Growth Paraeter Components of Adaptive
Specificity during Experimental Evolution of the UVR-Inducible Mutator Pseudomonas
cichorii 302959. PUBLIC LIBRARY of SCIENCE One 6, e15975.

49

CHAPTER TWO
FIELD STUDY FOR TX-1, 10TC30, AND HUMIC AICD

ABSTRACT

To understand the effect of improved UV tolerance on the disease control efficacy under
field condition, a study was done in July to October of 2009 and 2010. Various concentrations of
Tx-1 or 10TC30, along with combinations with and without HA were tested for disease control.
7

2

When applied at 10 CFU/cm , Tx-1 and Tx-1 + HA treatments resulted in similar dollar spot
5

2

and anthracnose suppression. When applied at 10 CFU/cm , Tx-1 alone treatment provided
significant dollar spot control at six rating dates in 2009, while Tx-1 + HA showed no effect on
dollar spot incidence. HA alone treatment showed no effect on disease severity and turf quality
7

2

for over two years. 10TC30 applied at 10 CFU/cm was the first biological treatment that
significantly controlled dollar spot on 21-Jul 2010. Results for population sampling showed that
the 10TC30 + HA treatment had significantly higher log population size than Tx-1 alone
treatment. All together, these results indicate that the addition of HA has limited effect on
disease control efficacy of Tx-1. The improvement in only UV tolerance might not be conclusive
for promoting the overall persistence and disease control efficacy of Tx-1.

50

Introduction
The ability to persist and colonize in the environment at sites when disease control is
needed is a requirement for an effective biological control in the field (Chang, 1981). In most
cases, the inability to persist or establish itself in a specific crop system leads to the failure of this
potential biological control agent (Wood, 1996). It has been shown that photo-degradation
caused by two different wavelengths of UV, UV-A (320-400 nm) and UV-B (280-320 nm),
inactivates most microbial insecticides and fungicides in field conditions (Tamez-Guerra et al.,
2005; Hadapad, 2009). The results from in vitro studies confirmed the UVR susceptibility of Tx1 by showing a significant decrease in Tx-1 survivors following increased UV dosage. To
improve the UVR tolerance of Tx-1, three methods were tested: 1) a potential UV sunscreen,
humic acid (HA) was found and showed significant protection for Tx-1 in all UV exposure tests.
2) 10TC30, the strain of Tx-1 which survived 10 cycles of 30 sec of UV-C exposure was
2

identified and showed improved tolerance to 7.3 J/m UV-C irradiation. 3) Prolonging the
2

incubation period from 24 to 72 hr demonstrated better UV-A tolerance at 60 and 90 kJ/m .
Moreover, results from bioassays against R. floccosum showed the production of efficacious
antibiotics did not change after UV exposures or the addition of HA.
Disease control efficacy under field condition is the essential test for any disease
management agent (Powell, 1993). Hence, in an effort to understand the effect of improved UV
tolerance on the disease control efficacy under field conditions, a study was performed from July
to October of 2009 and 2010. Various concentrations of Tx-1 or 10TC30, along with
combinations with and without HA were tested for disease control. To further investigate the
impact of HA on population size of the biocontrol agent in the field, populations in the foliage
51

7

2

and thatch of the treatments that were applied at 10 colony forming units (CFU) per cm were
monitored in 2010.
Materials and Methods
Field Condition
This study was conducted from July through October in 2009 and 2010 at the Hancock
Turfgrass Research Center on the campus of Michigan State University, East Lansing, Michigan.
The annual bluegrass (Poa annua) fairway was maintained at 1.27 cm. Fertility program was
2

maintained as 113.4g of N/92.9m biweekly during the study. Irrigation timing was adjusted to
provide adequate moisture to maintain the annual bluegrass. Randomized complete block
contained four replications was chosen as the statistical error control design. The plot sizes were
set up as 1.37 by 0.61 m, with one foot buffer strips on two sides.
Bacterial Fermentation
Tx-1 or 10TC30 were grown for 48 to 72 hr before applying. 1 ml of frozen seed culture
was transferred into 50 ml Tryptic soy broth (TSB) (Becton, Dickinson and Company. USA) and
incubated for 24 hr. The 50 ml cultures were then inoculated to 1 L of TSB media. To achieve
the optimal growth, the 1L cultures were incubated on a shaker (SK-71, Lab Companion,
JEIOTECH. Korea) at 100 rpm with constant air circulation. Temperature was maintained at 25
0

C.

52

Daily concentration of Tx-1 was measured by using a spectrophotometer. The absorbance
values were translated into concentration of Tx-1 using a formula from a standard curve that was
generated in previous studies (Powell, 1993).
Treatment application
The treatment list of 2009 and 2010 are shown in Table 2.01 and 2.02, respectively.
5

Based on previous studies with Tx-1(Dwyer, 1999), two concentrations of Tx-1, 10 CFU/cm
7

2

2

and 10 CFU/cm , were chosen for this study. The concentration of HA (SIGMA-ALDRICH,
2

Co. St. Louis, MO) was determined as 7.09 g/92.9m , which was calculated based on the results
from in vitro study. Bacterial cultures and HA were mixed prior to each application. Banner
Maxx (propiconazole), a chemical fungicide was applied biweekly as the positive control of the
study. A nitrogen powered backpack sprayer was used to deliver all the treatments. The sprayer
2

2

was calibrated to apply biological control agents at rate of 8.33 L/92.9m and 4.16 L/92.9m for
Banner Maxx and HA. In 2009, biological treatments were delivered after 4:30 p.m. to achieve
optimal condition for application and bacterial growth. This schedule was adjusted to 12:30 p.m.
in 2010.

53

Table 2.01. List and application rates of treatments tested in 2009 field study for the
suppression of dollar spot and anthracnose in East Lansing, Michigan.
Treatments
P. aureofaciens Tx-1
P. aureofaciens Tx-1
P. aureofaciens Tx-1+
humic acid
P. aureofaciens Tx-1+
humic acid
humic acid
Banner Maxx

Rate
2 x 10
2 x 10
2 x 10
2 x 10

Rate Unit
5

2

Five days a week

2

Five days a week

2

Five days a week

2

Five days a week

2

Five days a week

2

14 days

CFU/cm

7

CFU/cm

5

CFU/cm

7

CFU/cm

7.09

g/92.9m

0.03

L/92.9m

Untreated

54

Application Interval

Table 2.02. List and application rates of treatments tested in 2010 field study for the
suppression of dollar spot and anthracnose in East Lansing, Michigan.

Treatments
P. aureofaciens Tx-1
P. aureofaciens Tx-1
P. aureofaciens Tx-1+
humic acid
P. aureofaciens Tx-1+
humic acid
10TC30
10TC30
10TC30 + humic acid
10TC30 + humic acid
humic acid
Banner Maxx

Rate
2 x 10
2 x 10
2 x 10
2 x 10
2 x 10
2 x 10
2 x 10
2 x 10

Rate Unit
5

2

Daily

2

Daily

2

Daily

2

Daily

2

Daily

2

Daily

2

Daily

2

Daily

2

Daily

2

14 days

CFU/cm

7

CFU/cm

5

CFU/cm

7

CFU/cm

5

CFU/cm

7

CFU/cm

5

CFU/cm

7

CFU/cm

7.09

g/92.9m

0.03

L/92.9m

Untreated

Field population of Tx-1
55

Application Interval

To measure the population sizes of bacterial treatments applied at 10

7

CFU/cm

2

,

rifampicin resistant strains of Tx-1 and 10TC30 were used. The procedure of selecting rifampicin
resistant strains was described previously by Dwyer (1999). Three population samplings were
arranged though out the study in 2010. The first sampling was collected at 14-Jul 2010, which
was 24 hr after the first application. Second sampling was done at 27-Aug 2010, in the middle of
the season. The third one was conducted at 10-Oct 2010, at the end of the field season. During
7

every sampling date, three random samples were taken from each plot treated with 10 CFU/cm

2

All samples were taken using a soil probe (19.05 mm in diameter) that was surface-sterilized by
10% bleach and rinsed in dH2O between each sample collection.

The collected samples were diluted and plated as previously described (Dwyer, 1999). In
short, section from top 1 cm of the samples were dissected and transferred into 10 ml of PBS
solution, pH 7.0. After serial dilution for each sample, 40 ul aliquots were plated on rifampicin
amended media (50 mg/L) at various levels of dilutions in four replications. Colony counting
was conducted following 48-72 hr of incubation period.
Data collection and analysis
Disease ratings and turf quality were taken every 7-14 days. The occurrence of disease
was rated by visual estimation of the percent of each plot exhibiting disease symptoms. Turf
quality was rated on a scale of 1-9, where 1=completely dead, 7= acceptable, and 9=excellent. A
combination of features such as color, density, and uniformity were evaluated as the base of
quality. Data was analyzed using ANOVA LSD (p=0.05) procedure from SAS (Statistical
Analysis Software, Cary, N.C.).

56

.

Results
2009
In 2009, dollar spot ratings were taken on nine dates (Table 2.03). Tx-1, with and without
7

2

HA, applied daily at the rate of 10 CFU/cm provided significant control of dollar spot
compared to the untreated control (Table 2.03). The level of disease reduction achieved by 10

7

2

CFU/cm treatments was similar to the chemical fungicide Banner Maxx (Table 2.03).
7

2

Significant dollar spot control was first observed at 29-Aug for Tx-1 10 CFU/cm treatment,
5

2

which is one rating date before Tx-1 HA treatment. Tx-1 alone applied at 10 CFU/cm also
provided significant dollar spot control since 29-Aug, but the level of disease suppression
decreased followed by increased disease severity at 27-Sep and 14-Oct (Table 2.03). Tx-1+HA
5

2

treatment applied at 10 CFU/cm and HA alone treatments had no significant impact on dollar
spot control (Table 2.03).

57

Table 2.03 Means and LSD comparisons for treatment effects on dollar spot disease incidence on different dates in 2009.
Treatments

Rate

Rate Unit

Tx-1

2 x 10

Tx-1

2 x 10

Tx-1 + HA

2 x 10

Tx-1 + HA

2 x 10

HA

7.09

g/92.9m

Banner
Untreated

0.03

L/92.9m

5

7
5

7

CFU/cm
CFU/cm
CFU/cm
CFU/cm

2
2
2
2

2
2

7/31

8/12

8/18

8/29

9/6

9/13

9/20

9/27

10/14

0a

0a

2.8 a-c

2.9 bc

6.4 bc

11.3 b-d

8.8 bc

31.3 b

41.3 b

0a

0.5 a

4.5 a-c

2.5 bc

3 bc

6.3 d

10.5 c

10 c

0a

3a

7.5 a-c

9.8 ab

13 ab

21.3 a-c

3.5 c
16.3
ab

45 ab

55 ab

0a

3.1 a

7 a-c

5.5 a-c

7.5 cd

2.3 c

10.8 c

10.8 c

0a

0.5 a

7 a-c

9.3 a-c

6.3 bc
13.8
ab

25 ab

21.3 a

52.5 ab

60 a

0a
0a

2.5 a
1.9 a

0.1 c
10.8 a

0.1 c
12.5 a

0c
19.5 a

0.5 d
31.3 a

0c
23.8 a

0c
57.5 a

0c
63.8 a

Means followed by the same letter in a column are not significantly different according to Fisher’s LSD (P>0.05).

58

Table 2.04 Means and LSD comparisons for treatment effects on anthracnose disease incidence on different dates in 2009.

Rate
Unit

Treatments

Rate

Tx-1

2 x 10

Tx-1

2 x 10

Tx-1 + HA

2 x 10

Tx-1 + HA

2 x 10

HA

7.09

g/92.9m

Banner
Untreated

0.03

L/92.9m

5

7
5

7

7/31

CFU/cm
CFU/cm
CFU/cm
CFU/cm

2
2
2
2

2
2

8/12

8/18

8/29

9/6

9/13

9/20

9/27

16.3 a

16.3 bc

15.3 bc

21.3 a

8.8 ab

10.5 a

1.5 bc

15 a

11.8 bc
14.8 ac

10 c

7.5 bc

1.3 b

1.25 b

0c

18.8 a

23.8 a

31.3 a

9.8 bc
17.5 ac

18.8 ab

13 a

10.8 a

3.5 ab

20 a

9.5 c

14.5 bc

5.5 c

5.75 c

2.5 b

1.3 b

0c

20.5 a

13 a-c

30 a

30 a

20.5 a

10 ab

10 a

3.3 ab

20 a
15.5 a

22.5 ab
11.3 bc

13.8 bc
23.8 ab

6.5 c
22.5 ab

7 bc
18 a-c

9 ab
9.3 ab

0b
11.3 a

0c
4a

Means followed by the same letter in a column are not significantly different according to Fisher’s LSD (P>0.05).

59

Table 2.05 Means and LSD comparisons for treatment effects on turf quality on different dates in 2009.

Rate
Unit

7/31

Treatments

Rate

Tx-1

2 x 10

Tx-1

2 x 10

Tx-1 + HA

2 x 10

Tx-1 + HA

2 x 10

HA

7.09

g/92.9m

Banner
Untreated

0.03

L/92.9m

5

7
5

7

CFU/cm
CFU/cm
CFU/cm
CFU/cm

2
2
2
2

2
2

8/12

8/18

8/29

9/6

9/13

9/20

9/27

10/14

4.8 a-c

5a

5 ab

4.8 bc

4.5 bc

4.5 bc

4.8 bc

4.3 bc

3.5 a

4.8 a-c

4.8 ab

5 ab

5.3 ab

5.3 ab

5b

5.3 ab

5.3 ab

3.5 a

4.3 bc

4.5 ab

4.3 b

4.3 bc

3.8 c

3.8 c

4.3 b-d

3.8 c

3.3 a

5 ab

4.5 ab

5.3 ab

5.3 ab

5.3 ab

5.3 ab

6a

5.3 ab

3.5 a

4c

4.3 b

4b

3.8 c

3.8 c

3.8 c

4 cd

3.3 c

3a

5.3 a
4.8 a-c

4.5 ab
4.5 ab

6a
4b

6.5 a
3.5 c

5.8 a
3.8 c

6.3 a
3.5 c

5 a-c
3.5 d

6a
3.5 c

5a
3a

Means followed by the same letter in a column are not significantly different according to Fisher’s LSD (P>0.05).

60

Anthracnose disease incidence in 2009 were taken and reported in Table 2.04. Significant
7

2

suppression on anthracnose was achieved by 10 CFU/cm treatments and Banner Maxx in 2009
at three rating dates (18-Aug, 20-Sep, 27-Sep for Tx-1; 29-Aug, 20-Sep, 27-Sep for Tx-1+HA
and Banner Maxx) (Table 2.04). Tx-1 applied at 10

7

2

CFU/cm was the first treatment that

significantly controlled anthracnose on 18-Aug. No difference was observed among 10

5

2

CFU/cm treatments, HA alone treatment, and the untreated control (Table 2.04).

Nine turf quality ratings were recorded in 2009 (Table 2.05). Compared to the untreated
7

2

control, 10 CFU/cm treatments showed significant improvement of turf quality from 29-Aug to
27-Sep (Table 2.05). The significant increase in turf quality for the chemical treatment (Banner
7

Maxx) lasted from 18-Aug to 27-Sep, which was one rating date longer than the 10 CFU/cm
5

2

2

treatments (Table 2.05). Tx-1 applied at 10 CFU/cm showed significant effect in turf quality at
20-Sep (Table 2.05). No difference in turf quality were observed among the Tx-1 + HA applied
5

2

at 10 CFU/cm , HA alone treatment, and the untreated control (Table 2.05).

61

Table 2.06 Means and LSD comparisons for treatment effects on dollar spot disease incidence on different dates in 2010.
Treatments

Rate

Tx-1
2 x 10
Tx-1
2 x 10
Tx-1 + HA
2 x 10
Tx-1 + HA
2 x 10
10TC30
2 x 10
10TC30
2 x 10
10TC30 + HA
2 x 10
10TC30 + HA
2 x 10

Rate Unit
5
7
5
7
5
7
5
7

7/13

7/21

8/11

8/20

8/29

9/13

9/24

10/3

2

3.4 a

12.8 a

10.3 a-d

14.3 ab

20.8 a

30 a

30 ab

25 a

2

4.8 a

5.3 bc

7 b-e

5.8 c-e

9.3 bc

8.3 b

16.3 c-e

13.3 cd

2

4.8 a

7.3 a-c

11.5 a-c

17.5 a

23.8 a

28.8 a

33.8 a

23.8 a

2

5a

4.3 bc

2.9 e

2 e

5.5 bc

8.5 b

13.8 de

11 d

2

5a

10.8 ab

13.5 ab

14.3 ab

21.8 a

31.3 a

30 ab

21.3 ab

2

3.5 a

2.5 c

2 e

2.4 de

4.3 bc

8 bc

8.8 ef

11 d

2

4.8 a

9 a-c

8 b-e

9.3 b-d

23.8 a

27.5 a

22.5 b-d

19.3 a-c

2

6a

8.3 a-c

4.5 c-e

7 c-e

11 b

9.8 b

13 de

15 b-d

2

5.3 a

10.8 ab

12 ab

11.5 a-c

20 a

28.8 a

26.3 a-c

21.3 ab

2

3.5 a

2.8 c

3.5 de

5.8 c-e

1.6 c

0.3 c

0.1 f

1.3 e

5.8 a

11.3 ab

16 a

16.3 ab

27.5 a

35 a

32.5 ab

22.5 ab

CFU/cm
CFU/cm
CFU/cm
CFU/cm
CFU/cm
CFU/cm
CFU/cm
CFU/cm

HA
7.09

g/92.9m

0.03

L/92.9m

Banner
Untreated

Means followed by the same letter in a column are not significantly different according to Fisher’s LSD (P>0.05).

62

Table 2.07 Means and LSD comparisons for treatment effects on anthracnose disease incidence on different dates in 2010.
Treatments

Rate

Rate Unit

Tx-1
2 x 10
Tx-1
2 x 10

7

Tx-1 + HA
2 x 10
Tx-1 + HA
2 x 10

2 x 10
2 x 10

2 x 10
2 x 10

5

7

10TC30 + HA
10TC30 + HA

5

7

10TC30
10TC30

5

5

7

7/21

8/2

8/11

8/20

8/29

2

21.3 a-c

16.3 a

9.3 bc

15 ab

8.8 a

2

26.3 a-c

10.8 ab

6.3 c

6.8 c-e

5.5 a-c

2

36.3 a

15 a

10.5 a-c

11.3 a-d

6.8 a-c

2

14.5 bc

17.5 a

7.3 c

8.8 b-d

3.5 cd

2

18.8 a-c

19.3 a

11 a-c

13 a-c

4.3 b-d

2

27 ab

10.5 ab

7c

6.3 de

4.3 b-d

2

17.5 a-c

16.8 a

10.5 a-c

10.5 a-d

7.5 ab

2

18.8 a-c

8.8 ab

5.5 cd

9.3 b-d

4 b-d

2

23.8 a-c

17.5 a

16.3 a

13.8 ab

5.8 a-c

2

7.5 c

0b

0.3 d

2.3 e

1.4 d

23.8 a-c

19.5 a

14.3 ab

16.3 a

5.5 a-c

CFU/cm
CFU/cm
CFU/cm
CFU/cm
CFU/cm
CFU/cm
CFU/cm
CFU/cm

HA
7.09

g/92.9m

0.03

L/92.9m

Banner
Untreated

Means followed by the same letter in a column are not significantly different according to Fisher’s LSD (P>0.05).

63

Table 2.08 Means and LSD comparisons for treatment effects on turf quality on different dates in 2010.

Rate
Unit

Treatments

Rate

Tx-1

2 x 10

Tx-1

2 x 10

Tx-1 + HA

2 x 10

Tx-1 + HA

2 x 10

10TC30

2 x 10

10TC30
10TC30 +
HA
10TC30 +
HA

2 x 10

HA

7.09

g/92.9m

Banner
Untreated

0.03

L/92.9m

7
5

7
5

7

2 x 10
2 x 10

5

5

7

7/13

CFU/cm
CFU/cm
CFU/cm
CFU/cm
CFU/cm
CFU/cm
CFU/cm
CFU/cm

2
2
2
2
2
2
2
2

2
2

7/21

8/2

8/11

8/20

8/29

9/13

9/24

10/3

5a

4.5 b

4.8 d

4f

4c

3.8 de

3.8 c

3.5 ef

4d

4.8 a

4.8 b

5.5 b-d

5 c-e

5.5 b

4.8 cd

5b

4.3 c-e

4.8 cd

4.8 a

5b

5.8 b-d

4.3 ef

3.8 c

3.5 e

3.5 c

3.3 f

4d

5a

4.8 b

5.8 b-d

5.5 bc

5.5 b

5.3 bc

5b

5 bc

5.3 c

4.5 a

4.5 b

5 cd

4.5 d-f

4.3 c

3.8 de

3.5 c

3.8 d-f

4.3 d

5a

4.8 b

6.5 b

6.3 ab

5.5 b

6 ab

4.8 b

5.3 b

6.5 b

5a

4.5 b

5.3 cd

4.5 d-f

4c

3.8 de

4.3 bc

3.8 d-f

4.3 d

4.5 a

4.8 b

6 bc

5.3 cd

4.8 bc

4.8 cd

5b

4.5 b-d

4.8 cd

4.8 a

4.5 b

5.5 b-d

4.8 c-f

3.8 c

4 de

3.8 c

3.8 d-f

4d

5a
4.5 a

5.8 a
4.8 b

8.5 a
5.5 b-d

6.5 a
4.8 c-f

7a
4c

6.5 a
3.5 e

7a
3.8 c

8.5 a
3.3 f

7.5 a
4d

Means followed by the same letter in a column are not significantly different according to Fisher’s LSD (P>0.05).

64

Figure 2.01. Population size of P. aureofaciens Tx-1 in foliage and thatch.

8
7
a

a

a

a

ab

ab

b*

a

a

a

a

a
Tx-1

Log (CFU cm-3)

6
Tx-1 + HA
5
10TC30
4
10TC30 + HA
3
2
1
0
7/14

8/27

10/10

Sampling Dates

*, Means followed by the same letter are not significantly different according to Fisher’s LSD (P>0.05).

65

2010
In 2010, dollar spot disease incidence data that were collected from eight rating dates are
presented in Table 2.06. 10TC30 applied at 10

7

CFU/cm

2

significantly suppressed the

development of the disease on seven rating dates, which is the same duration of disease control
7

2

provided by Banner Maxx (Table 2.06). The other three treatments applied at 10 CFU/cm (Tx1, Tx-1 + HA, and 10TC30 + HA) provided significant dollar spot control on six dates (11-Aug
5

2

to 3-Oct). No differences were found among 10 CFU/cm treatments with the only exception of
10TC30 + HA, which showed significant control of the disease on 11-Aug (Table 2.06).
The severity of anthracnose in 2010 was recorded at five rating dates is presented in
Table 2.07. Significant disease control was achieved on two rating dates (11-Aug and 20-Aug)
by biological treatments applied at 10

7

2

CFU/cm , which is two rating dates shorter than

suppression provided by chemical fungicide (Table 2.06). All biological treatments applied at
5

2

10 CFU/cm and HA alone treatment showed no effect on disease incidence (Table 2.07).

Turf quality ratings taken in 2010 at nine rating dates are reported in Table 2.08. Among
7

2

all treatments applied at 10 CFU/cm , 10TC30 provided significantly improved turf quality on
six rating dates, and the longest disease control period among all biological treatments (Table
2.08). Tx-1 and HA combination, Tx-1 alone, and 10TC30 + HA treatments applied at 10
2

7

CFU/cm offered significant improvement on five, four, and three rating dates, respectively

66

5

2

(Table 2.08). 10 CFU/cm treatments and HA alone treatment had no effect on turf quality
(Table 2.08).
7

2

Populations of the top 1 cm soil (foliage and thatch) of the 10 CFU/cm treatments were
monitored in 2010 and presented in Figure 2.01. In 8/27, the 10TC30 + HA treatment had
significantly higher log population size than Tx-1 alone treatment (Figure 2.01). No difference in
the population was found among the treatments on any other rating dates (Figure 2.01).
Discussion
Humic acid has been shown as a promising UV sunscreen for Tx-1 in vitro.

To

investigate whether the improvement of UV tolerance provided by HA might affect disease
5

control efficacy under field condition, Tx-1 with or without HA treatments were applied at 10
2

7

2

CFU/cm and 10 CFU/cm and tested over two years (2009-2010). When applied at 10

7

2

CFU/cm , Tx-1 and Tx-1 + HA treatments resulted in similar dollar spot suppression (Table 2.03,
7

2

2.06). Compares to Tx-1+HA applied at 10 CFU/cm , Tx-1 alone on one rating date provided
significant control of dollar spot in 2009 (Table 2.03). Both treatments also provided significant
control in 2010 (Table 2.06). This is the first study demonstrating that Tx-1 inhibits anthracnose
7

2

in vivo. Results show Tx-1 and Tx-1 + HA applied at 10 CFU/cm treatments provided similar
levels of suppression of anthracnose (Table 2.04, 2.07). The similarity between Tx-1 and Tx-1 +
HA treatments was observed in turf quality data as well, which show the same level of
improvement on turf quality was achieved on similar rating dates in both years (Table 2.05, 2.08).
Moreover, no significant difference of the population size of Tx-1 and Tx-1 + HA was observed
67

5

2

in 2010 (Figure 2.01). When applied at 10 CFU/cm , Tx-1 alone treatment provided significant
dollar spot control at six rating dates in 2009, while Tx-1 + HA showed no effect on dollar spot
incident (Table 2.03). No significant difference in disease incident and turf quality between Tx-1
and Tx-1 + HA was observed in 2010 (Table 2.06-2.08). HA alone treatment showed no effect
on disease severity and turf quality for over two years (Table 2.03-2.08). All together, these
results indicate that the addition of HA has limited effect on disease control efficacy of Tx-1 and
HA itself has no impact on disease development.
One possible explanation of this is degradation of HA in the field. It has been shown that
microbial population in aerobic environments are able to degrade humic materials (Trump, 2006).
Exposure of HA to UV radiation from sunlight significantly promotes the bio-degradation
process (Trump, 2006). Since it has been proposed that the UV inactivation efficacy of HA could
be concentration dependent (Cantwell, 2008), it is likely that the bio-degradation of HA caused
by microbes in the field might decrease the concentration to a level that it is unable to provide
efficient UV protection for Tx-1. To understand more about the degradation process of HA,
concentrations of HA in the field need to be traced and investigated in future field studies.
The competition with the other micro-flora is crucial for an introduced biocontrol agent
to survive and colonize (Thomashow, 1988). The survival and colonization of bacteria or
microbial biocontrol agents in UV intensive habitats, such as the leaf surface, largely depend on
tolerance to radiation (Sundin, 2004). HA has been shown to be able to protect numerous species
of bacteria from UV radiation (Corin, 1998; Templeton, 2006; Cantwell, 2008; Lee, 2009). It is
reasonable to hypothesize that the application of HA might not only protect Tx-1, but other
microbes as well. This improved UV tolerance provided by HA to the other microbes on the leaf

68

would benefit their survival hence increasing the competition in the area. This might be a
possible explanation for the observation that in 2009, when all the treatments were applied after
5

2

4:30 pm., Tx-1 + HA at 10 CFU/cm failed to inhibit dollar spot (Table 2.04), and at 10

7

2

CFU/cm rate, Tx-1 alone provided on one rating date more control of dollar spot. Moreover,
after the application schedule was changed to noon in 2010, the overall solar UVR was more
intensive than 2009, during which HA could be degraded faster, this differences in disease
control efficacy among Tx-1 alone treatments and Tx-1 + HA treatments at both application rates
were not been observed (Table 2.06, 2.07). To fully understand the impact of HA on other
microbes on the leaf, population size of these microbes needed to be traced in further studies.
The disease suppression efficacy of 10TC30 with and without HA was evaluated in the
field in 2010. 10TC30 applied at 10

7

CFU/cm

2

was the first biological treatment that

significantly controlled dollar spot on 21-Jul (Table 2.06). It also provided longer period of
improved turf quality than Tx-1 treatments (Table 2.08).

10TC30 has shown improved

UVC/UVB tolerance in vitro (Figure 1.06). This increased UV-B tolerance might provide
10TC30 an advantage in colonizing the area faster than Tx-1, which could be a likely
explanation for this longer period of dollar spot control. 10TC30 + HA provided similar dollar
spot and anthracnose control as Tx-1 treatments (Table 2.06, 2.07). The addition of HA to
7

2

10TC30 at 10 CFU/cm rate failed to provide control for dollar spot on 21-Jul (Table 2.06). It
might be a result of HA protecting other microbes and increasing the competition in that area.
7

2

Population sizes for treatments applied at 10 CFU/cm were monitored in 2010. The
7

2

population sizes of all the 10 CFU/cm treatments were not different from each other after 24 h
69

7

of first application (Figure 2.01), and the value were all one log factor lower than 10 which
been applied. This observation is in the agreement with previous field study with Tx-1, which
reported the threshold population of disease control was one log factor lower than the daily
application rate (Dwyer, 1999).The population size of 10TC30 + HA at 27-Aug was significantly
larger than Tx-1 alone treatment (Figure 2.01). The increased UV-B tolerance of 10TC30 and
UV protection provided by HA might be the reasons which lead to the increase population size.
5

2

It could also be a possible explanation that when applied at 10 CFU/cm , 10TC30 + HA was the
only treatment that inhibited dollar spot on one rating date (8/11) (Table 2.06). The increased
population size for 10TC30 + HA treatment was not observed at the last sampling date (Figure
2.01), it is possible this was due to the shorter days which decreased the intensity of UV
radiation and the temperature. UV-A wavelengths attribute 95% of total energy within solar
UVR (Sundin, 2004), 10TC30 has shown no change in UV-A tolerance in vitro (Table 1.06), this
could be a possible reason for the fact that population of 10TC30 alone treatment was no
different to Tx-1.
It has been suggested that the mechanism for Tx-1 to colonize and compete in the canopy
is antibiosis-asisted competition (Sigler, 2001). Based on data reported by Powell (1993), the
antibiotic of Tx-1 was secreted primarily during the stationary phase, during which the
population size of Tx-1 was around 9 to 9.5 log of CFU. Hence, it has been proposed that the
antibiotics produced by Tx-1 during the incubation period may be the major factor in fungal
inhibition (Dwyer, 1999; Sigler, 2001). Data reported by Powell (1993) and Dwyer (1999)
support this hypothesis by showing that the heat-killed Tx-1, and purified antibiotic of Tx-1
(PCA) were sufficient for disease control. As a result, it is reasonable to hypothesize that the

70

increased population size of 10TC30 + HA might not be large enough to promote the production
of the antibiotics. It is a possible explanation for the observation that the advantage in population
size of 10TC30 +HA treatment did not result in an improvement in disease suppression efficacy
(Table 2.06, 2.07). To further understand the disease control efficacy of Tx-1 in the field, the
relationship between the population of Tx-1 and its antibiotic production need to be investigated
in vivo.
In most cases, biological control agents are delivered to an ecological unsuitable
environment (Deacon, 1991). Besides UV inhibition, the survival and biological antagonistic
activity of biocontrol agents are highly influenced by other environmental conditions such as
humidity, pH, nutritional availability, temperature (Lahlali et al., 2011). Hence, the improvement
in only UV tolerance might not be conclusive for increasing the overall persistent and disease
control efficacy of Tx-1. In order to fully optimize the disease control efficacy of Tx-1, the
impacts from other environmental factors need to be studied.

71

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72

Baker, K.F. (1987). Evoloving concepts of biological control of plant pathogens. Annual
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Eisenstark, A. (1987). Mutagenic and lethal effects of near-ultraviolet radiation (290-400 nm)
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patch on fairway height turf. HortScience 39(7), 1750-1753.
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Jacobs, J.L., Sundin, G.W. (2001). Effect of Solar UV-B Radiation on a Phyllosphere Bacterial
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