UNCOVERING BIOACTIVE METABOLITES FROM THE AGRICULTURALLY
IMPORTANT STRAIN QST 2808, BACILLUS PUMILUS, THROUGH BIOASSAYGUIDED FRACTIONATION
A Thesis
Presented to the faculty of the Department of Chemistry
California State University, Sacramento
Submitted in partial satisfaction of
the requirements for the degree of
MASTER OF SCIENCE
in
Chemistry
(Biochemistry)
by
Sarah Hovinga
SUMMER
2014
© 2014
Sarah Hovinga
ALL RIGHTS RESERVED
ii
UNCOVERING BIOACTIVE METABOLITES FROM THE AGRICULTURALLY
IMPORTANT STRAIN QST 2808, BACILLUS PUMILUS, THROUGH BIOASSAYGUIDED FRACTIONATION
A Thesis
by
Sarah Hovinga
Approved by
___________________________________, Committee Chair
Mary McCarthy-Hintz
___________________________________, Second Reader
Roy Dixon
___________________________________, Third Reader
Tamara Meragelman De Pedrosa
_________________
Date
iii
Student: Sarah Hovinga
I certify that this student has met the requirements for format contained in the University
format manual, and that this thesis is suitable for shelving in the Library and credit is to
be awarded for the thesis.
__________________________, Department Chair
Linda Roberts
Department of Chemistry
iv
___________________
Date
Abstract
of
UNCOVERING BIOACTIVE METABOLITES FROM THE AGRICULTURALLY
IMPORTANT STRAIN QST 2808, BACILLUS PUMILUS, THROUGH BIOASSAYGUIDED FRACTIONATION
by
Sarah Hovinga
Modern agriculture faces many challenges in the twenty-first century.
Sustainability and productivity must move together in stride if humans expect to
minimize the impact to the environment with pesticides and fertilizers while still
producing the volume and quality of food needed to feed a growing world population.
Biological control is an important tool to reduce both synthetic pesticide and fertilizer
application amounts, by offering disease control and plant growth promotion, while
lowering synthetic chemical load on the environment. Bacteria of the genus Bacillus are
great biological control options considering their natural origins, vast production of
v
bioactive metabolites, ability to promote the growth of plants, ease of producing endproducts at a manufacturing scale, and potential for stability as a product due to the
resilient spores they form. In this work, fermentation whole broth (WB) of a specific
strain of Bacillus pumilus, QST 2808, was investigated for bioactive metabolites of
agricultural relevance. An amino sugar, active against two crop disease targets, downy
mildew and bacterial spot, was isolated and characterized from a B. pumilus QST 2808
fermentation product. Two additional fractions were identified, distinct from the amino
sugar, one with activity against bacterial spot and one with activity against cucumber
anthracnose. With the identification of these three bioactive fractions, this work adds to
the knowledge of Bacillus biological control possibilities for developing safe and
effective crop protection products.
_______________________, Committee Chair
Mary McCarthy-Hintz
_______________________
Date
vi
ACKNOWLEDGEMENTS
I want to thank Dr. Mary McCarthy Hintz, Dr. Roy Dixon, and Dr. Tamara
Meragelman De Pedrosa for their time and energy in participating on my committee and
for their thoughtful comments and edits on my thesis. I would also like to thank Dr.
David Sesin for guiding and supporting me throughout the thesis research and Dr. Jorge
Jimenez, Dorte Lindhard, and Arnaldo Rivera for their support in the lab with
methodology, instrumentation, and trouble-shooting. I also thank Laura Lampa and
Cecilia Wilson for their expertise in microbiology and plant pathology, respectively,
which contributed to this project.
vii
TABLE OF CONTENTS
Page
Acknowledgements…………………………………………………………………………vii
List of Tables…………………………………………………………………………..xi
List of Figures………………………………………………………………….…….xiii
Chapter
1. INTRODUCTION………………………………………………………………….1
1.1 Disease in Agriculture……………………………………………....……...1
1.2 The Importance of Natural Products……………………………………….4
1.3 Managing Plant Disease – Integrated Pest Management…………………..5
1.4 Brassica Downy Mildew………………………………………………….10
1.5 Cucumber Anthracnose…………………………………………………...13
1.6 Bacterial Spot……………………………………………………………..14
1.7 The Biology of Bacillus…………………………………………………..16
1.8 The Chemistry of Bacillus………………………………………….……..18
1.9 Objective, Scope, and Significance of Study……………………………..23
2. EXPERIMENTAL METHODS…………………………………………………..25
2.1 Chemicals and Reagents Used……………………………………………25
2.2 Bacterial whole broth (WB) Production…………………………………..25
2.3 Ultra-filtration of WB……………………………………………………..26
2.4 Liquid-liquid Extractions and Cell Extractions…………………………...27
viii
2.5 Ion-Exchange Chromatography (Small-Scale)…………………………...28
2.6 Ion-Exchange Chromatography (Large-Scale)…………………………...29
2.7 Size-exclusion Chromatography………………………………………….30
2.8 Solid-phase Extraction……………………………………………………31
2.9 Ninhydrin Spray…………………………………………………………..32
2.10 Reverse-Phase High-Performance Liquid Chromatography (HPLC)…...32
2.11 Derivatization Assay for Amino Sugar Quantification………………….33
2.12 Colorimetric Assay for Amino Sugar Quantification……………………35
2.13 Thin-layer Chromatography……………………………………………..37
2.14 Solubility Testing of Colletotrichum orbiculare (CA) Active……….....38
2.15 Mixed-mode Chromatography…………………………………………..38
2.16 Lyophilization for NMR ………………………………………………...39
2.17 Minimum Inhibitory Concentration in vitro Assay……………………...39
2.18 Agar Diffusion Assay……………………………………………………42
2.19 Germ Tube Assay………………………………………………………..43
2.20 Brassica Downy Mildew in planta Assay………………………….……44
3. RESULTS AND DISCUSSION……………………………………………….…46
3.1 Cell vs. Permeate Activity………………………………………………..48
3.1.1. BDM Activity………………………………………………………….48
3.1.2. CA and Xcv Activity…………………………………………………..50
3.2 Liquid-liquid Extractions and Cell Extractions…………………………..52
ix
3.2.1 Xcv and CA activity……………………………………………………52
3.2.2 BDM activity……………………………………………………………53
3.3 Ion-Exchange Chromatography…………………………………………..54
3.3.1 Xcv, CA, and BDM activity…………………………………………….57
3.4 Scaled-up Ion-Exchange Chromatography……………………………….58
3.4.1 Xcv, CA, and BDM Activity……………………………………………62
3.5 Size-exclusion Chromatography………………………………………….65
3.5.1 Xcv and BDM Activity…………………………………………………69
3.5.2 Mixed-Mode HPLC of Size Exclusions Fractions……………………...72
3.5.3 Derivatization of Size Exclusions Fractions……………………………76
3.6 Isolation of Amino Sugar by Solid-phase Extraction……………………..86
3.7 Colorimetric Assay………………………………………………………..87
3.8 Probing Solubility of Flow-through………………………………………88
3.9 Thin-layer Chromatography on CA-Active Fraction……………………..89
4. CONCLUSIONS……………………………………………………………….....99
References…………………………………………………………………………...102
x
LIST OF TABLES
Tables
1.
Page
The parameters for the small-scale ion-exchange flash column
chromatography ……………………………… .……………………………. 29
2.
The parameters for the large-scale ion-exchange flash column
chromatography ……………………………….… .. …………………………30
3.
The gradient used with samples tested in the NP-screen method .. ………….. 33
4.
The gradient conditions for the amino sugar HPLC assay. Results are
expressed as mg of amino sugar per ml of starting material……..………….. 35
5.
The efficacy of liquid-liquid extraction samples on Xcv and CA compared
to antibacterial and antifungal positive controls……..………………………. 53
6.
Efficacy of ion-exchange fractions against Xcv and CA and corresponding
amino sugar concentrations………………………………...………………....57
7.
Bioactivity of large-scale ion-exchange fractions in MIC against Xcv and
CA and corresponding amino sugar concentration from the derivatization
HPLC assay…………………………………………………………………...63
8.
Efficacy against Xcv of the nine combined fractions from the size-exclusion
separation………………………………………………………………...…....70
9.
Concentration of the amino sugar as determined by the colorimetric assay
xi
of three replicates of fractions from the small-scale ion-exchange column..…88
xii
LIST OF FIGURES
Figures
Page
1.
The disease triangle(3)…………………………………………………………3
2.
A head of broccoli affected by Brassica downy mildew disease(18)...............11
3.
Cucumber anthracnose symptoms extending all the way to the
deliverable commodity (24)…………………………………...……………...13
4.
Bacterial spot of tomato (33)…………………………………………...……..15
5.
The life cycle of Bacillus (35)…………………………...……………………18
6.
Surfactin, the most well-known member of the surfactin family of secondary
metabolites produced by Bacillus (39)…………………………………….….20
7.
Iturin A, a secondary metabolite produced by Bacillus (40)………………….21
8.
Fengycin, a secondary metabolite produced by Bacillus (37)………………...22
9.
Other known secondary metabolites produced by Bacillus: bacillaene,
difficidin, macrolactin, and bacilysin (38)……………… …...………………22
10.
The set-up of the high-pressure filtration system to separate bacterial cells
from permeate (synonym: supernatant). The bottle on the right shows the
xiii
clear, dark-brown permeate exiting the system and to the left of this bottle
is the opaque, viscous whole broth (WB) that contains both permeate and
cells……………………………………………………………………………27
11.
The effect of the cell pellet extract and permeate samples compared to WB
against BDM. Samples were applied as a spray on Brassica before inoculation
with the pathogen and rated ~ 1 week later for disease……………………….49
12.
The efficacy of the cell pellet extract, permeate, and WB samples against
BDM..................................................................................................................50
13.
The efficacy of the cell pellet extract and permeate samples against CA and
Xcv……………………………………………………………………......…..51
14.
The efficacy of liquid-liquid extraction samples on BDM……………………54
15.
HPLC chromatograms of fractions from the small-scale ion-exchange flash
column Detection at 210 nm. A blank run before sample injection showed no
detectable peaks…………………………………………………………….…55
16.
Amino sugar calibration curve using the derivatization assay…………….….56
17.
Efficacy of ion-exchange fractions against BDM……………...……………..58
18.
The chromatograms of the scaled-up ion-exchange fractions compared to the
amino sugar standard using mixed-mode HPLC using the ELSD detector.
xiv
Signals over 1200 mV exceed the maximum input in the Agilent analog to
digital convertor……………………………………………………………....61
19.
Efficacy of the large-scale ion-exchange fractions against BDM…….......…..63
20.
The agar diffusion activity of the large-scale ion-exchange fractions against
Xcv. The two top plates show the zones of inhibitions as they could be seen by
holding the plate up to light and the bottom two plates is this same exact image
illustrating where the zones are located by using red circles to outline
them……………………………………………………………………….…..65
21.
Fractions 13 and 14 from the size-exclusion column that stained positive for
amino sugars with ninhydrin staining…………………...…………………....66
22.
Example of fractions from the size-exclusion column separation that were
combined because they gave similar UV traces in the NP-screen at
210nm………………………………………………………………………....67
23.
The chromatogram of fraction 13 from the size-exclusion column with the NPscreen HPLC method by UV at 210 nm………………..………………….….68
24.
The chromatograms from fraction 13 and the amino sugar standard using the
NP-screen HPLC method with ELSD detection……….……………………..69
25.
Efficacy of the nine combined fractions from the size-exclusion separation
against BDM………………………………...…………………………….......71
xv
26.
The chromatograms of fractions 14 and 15 by UV at 210 nm………………..72
27.
The chromatograms of combined fractions 3A and 4A and the amino sugar
standard at differing concentrations using the mixed-mode chromatography
method using ELSD detection……….………………………………….…….74
28.
Standard curve of the amino sugar standard injected at different concentrations
using the mixed mode chromatography method with ELSD detection…….....75
29.
Figures 29 A through E: The chromatograms of combined fractions 3A
and 4A alongside the amino sugar standard using the derivatization
assay at 260 nm............................................................................................77-81
30.
The chromatograms of combined fractions 3A and 4A and the new amino
sugar standard, all tested at sample concentrations of 100 μl of 1.0 mg/mL
solutions using the derivatization assay read at
260nm………………………………………………………………………....83
31.
Calibration curves for each of the samples when 100 μL, 50 μL, and 25 μL of
1.0 mg/mL concentrations were derivatized and injected……………..……...84
32.
Amount of amino sugar found in the different steps of the SPE cartridge steps
compared to the starting permeate material using the derivatization HPLC
assay to quantify amino sugar concentration in each………………..………..87
xvi
33.
The TLC plate of the amino sugar standard, and the flow-through and water
wash from the small-scale ion-exchange column………………..……………91
34.
Development of different concentrations of the water wash fraction after
ninhydrin processing on a preparatory cellulose TLC plate…………………..92
35.
Chromatograms generated from the NP screen HPLC method to determine the
best solvent with which to extract the separated fraction from the preparatory
TLC plate…………………………………………………………...…………93
36.
A and B: A visual plate map of the ten different fractions that were excised
from the TLC preparatory plate……………………………………...………..95
37.
Zones 1 and 10 compared to the negative (water) and positive (WB) controls
for activity against CA………………………………………………….…….96
xvii
1
Chapter 1
INTRODUCTION
1.1 Disease in Agriculture
Even in these modern times, agriculture still faces a problem that has been around
for centuries: disease. Diseases of agricultural crops can arise in many forms all around
the world, can affect many crops, and can affect all parts of a plant. From soil fungi that
attack the root systems, to bacterial blights that infect flowers and lead to infected fruit,
plants everywhere are susceptible to a constant battery of pests. Not only is this battery a
problem for plants, but it is a problem for the humans that rely on the foods they produce
to feed populations. With new causative agents for disease being discovered every
month, and with a world population expected to exceed 9 billion in 2050, this is not a
problem that will go away (1).
Plant diseases can be very destructive and cause severe losses in yield and
marketability of agricultural commodities. From this, not only arises the problem of
supplying enough food to feed the human population, but socio-economic issues also
surface. More loss of yield in one type of crop vs. another, for example, will lead farmers
to plant more of the less disease-affected crop, which can reduce the diversity of plant
species that are commercially produced. In a worst-case scenario, cascade effects like
this could potentially lead to malnutrition of humans due to lack of essential nutrients
from a diverse diet. A similar situation of favoring one crop over another for economic
reasons could lead farmers to plant cultivars that cannot be used for food, but rather
2
activities like drug trafficking. Such is the case in Afghanistan where, instead of
traditional farming practices of the past that relied mainly on wheat, farmers may instead
choose to plant opium since it is more profitable (2). The issue of disease control for
plants and which crops we plant in the future spans farther than just feeding people; it
will shape the way our world populations and societies develop. With increasing
population, food will increasingly be in demand. Disease can block the production of
safe, healthy, nutritious food to satisfy that demand.
Disease in plants is caused by two main factors: abiotic physiological factors and
biotic ones that cause infection due to parasitic organisms. Physiological factors that
cause disease in plants can be narrowed down to a few main categories: nutrition,
temperature, water, and toxicity from chemicals. Abiotic factors can be controlled
somewhat easier than infectious ones, given that the farmer knows what the physiological
disorder is. Blossom-end rot in tomato and pepper, for example, is caused by calcium
deficiency and can be remedied with fertilizers that contain this essential nutrient. Biotic
diseases stem from viruses, bacteria, fungi, phytoplasmas, oomycetes, and nematodes.
Insects contribute greatly to crop damage as well, and are a main target for many
pesticides, but where infection is concerned, they mostly vector viruses and bacteria to
susceptible host plants. In order for biotic disease to occur, all three points of the
“disease triangle” — host, pathogen, and environment — must be present (Figure 1). For
example, botrytis bunch rot of grapes (caused by Botrytis cinerea) favors humid, mild
conditions, so even if the fungus and plant are present and touching one another on a very
dry cold day, disease will not develop (3).
3
Figure 1: The disease triangle (3).
Biotic factors require not only identification of the disease and appropriate
treatment, but this treatment needs to be applied in a smart and effective way to
contribute to safe farming practices so that future generations of crops can benefit from
the same treatment (3). Resistance of pathogens to chemical treatment is quite common
and results from the improper use of pesticides. This renders a specific treatment less
effective for future crops, necessitating an industry devoted to finding new solutions.
In agriculture, the classes of compounds in many of the commonly used pesticides
have been around for at least 30 years and have been or are in the process of being phased
out due to toxicity, environmental concerns, and/or resistance problems (4,5). This
highlights the need for continued discovery and development of intelligently designed,
effective, and safe control measures. These novel chemical treatments play an important
part in the regulatory field, where closer scrutiny is being placed on emerging products.
Novel compounds are also crucial for successful pesticides in a legal capacity, since they
allow companies to stake claims on the invention of the compounds. If a pesticide
company is to be profitable, their work in discovering and developing a product needs to
4
be encouraged and facilitated into the marketplace, and the compounds and/or
manufacturing processes need to be protected by law (patents, trade secrets, etc.) so that
profits are preserved.
Considering the problems facing current pesticide use, one can see the need for
novel active ingredients and the complex legal and regulatory networks necessary to
make them successful.
1.2 The Importance of Natural Products
Technically speaking, if a compound has never been used in a commercial
application, chances are that affected pathogens will not develop resistance to it if it is
used properly. These new molecules open up a wide range of possibilities for disease
control and can mean high efficacy for the users and big profits for the responsible
company. Thus, there is a drive to discover novel compounds for use in disease
management. Many companies seek new compounds through natural product screening.
Natural products are typically gathered from plant material or are made from bacteria and
fungi that were gathered from plant and soil samples.
The goal of a natural products screening platform is to find novel secondary
metabolites with new modes of action (in this case, for plant disease management).
Currently, only about 50 modes of action are known, but considering the complex
physiology of cells, there may be many more possibilities for control with natural
products.
5
The resistance of agricultural pathogens to established chemical treatments has
generated the need for high-throughput screening to find new chemical treatments against
specific disease targets (6). In the pharmaceutical industry, screening efforts have steered
away from natural products and have been replaced with robotic combinatorial chemistry
screening, where chemicals with similar structures are synthesized automatically rather
than derived from natural sources such as plants or bacteria (7). Screening in this way
cuts down on much of the labor previously associated with a traditional natural products
screening platform. Many of the high-throughput strategies associated with
combinatorial chemistry have been adapted by the agricultural industry for natural
products screening as the rapid search continues for new modes of action.
1.3 Managing Plant Disease – Integrated Pest Management
In order to make a diseased crop, or one that is vulnerable to disease,
commercially successful, usually a combination of practices is used to mitigate the risk.
These practices are necessary to preserve the crop (thus avoiding famine due to a
catastrophic crop die-out) and to maintain the value of the crop once brought to market
(through yield and quality). One of the most unfortunate examples of famine caused by
disease is the incredible devastation known as the Irish Potato Famine in the 1840’s,
which was caused by the fungal-like oomycete pathogen Phytophthora infestans (8). The
loss of about 1 billion dollars to American corn growers due to the southern corn leaf
blight fungus (caused by Cochliobolus maydis, anamorph Bipolaris maydis) is an
example of the types of economic losses that can be experienced (8).
6
Growers must be aware of environmental conditions to properly anticipate which
disease(s) their plants may be vulnerable to. Key to the management of the disease is
breaking the disease cycle once it is present. The “disease cycle” usually refers to the life
cycle of the particular organism attacking the crop. Also very important is the correct
identification of the pest, since this relates to what part of the disease cycle one should
aim to interrupt and what practices one wants to use to combat it. Commonly, there are
four categories thought to be important to disease control: exclusion, eradication,
resistance, and protection (8). It is important to keep all of these factors in mind, as none
provides a silver bullet. Fortunately, integrated pest management, incorporating more
than one of the above strategies, is becoming more common. This is especially important
when taking into consideration issues, such as resistance, that arise because of over-use
of one particular method.
Exclusion practices are usually enforced when plant material is being shipped
from one location to another and is focused on preventing the spread of disease from a
known infected area to a pathogen-free area. Disease can be spread by many components
related to the shipping of plants, such as packing material, but the most common and
effective way of practicing exclusion is by providing clean seed, transplants, and
rootstocks to farmers so they do not unknowingly proliferate invading pathogens. Safe
practices like using clean or unshared farming equipment within a farm can also be
implemented to use exclusion principles so that disease is not spread between fields (8).
Eradication measures involve fighting a pest once it has already entered into an
area and is not fully established yet. These practices can involve things such as soil
7
removal, heat-sterilization of soil, leaving fields fallow, crop rotation, pruning away
diseased tissues in infected fields, weeding (since pests may find refuge in these plant
materials), burning fields, and soil fumigation. The latter two are the most controversial,
as their use poses direct risks to environmental safety and human health. Methyl
bromide, for example, is a soil fumigant used for sterilization of fields, and is toxic both
to the environment (by depleting stratospheric ozone levels) and to humans that come
into contact with it (9). The case of methyl bromide also exemplifies the dire need for the
continual research into alternatives. Since the signing of the Montreal Protocol in 1987,
the use of methyl bromide has been phased out by 196 countries, except for its use in
crops that have no viable alternative that would protect the yield and quality of the crop
(10). One obvious exception to the Montreal Protocol can be seen when driving through
the coastal strawberry fields of California.
The third category of an overall integrated pest management system is
resistance—not resistance of the pathogen to a chemical treatment, but rather selecting
for pathogen-resistant crops. Many scenarios are possible where a plant can become or is
resistant to a disease. Traditionally, plant breeding has been the primary funnel for
resistant crops, where after generations of different crosses made by humans, a resistant
plant may arise that still retains nutritional and marketable qualities. Recently, plant
mutations that confer resistance have been induced by chemical or physical means.
Induced systemic resistance and systemic acquired resistance of plants is also an active
field of study wherein a plant activator (either a chemical or an organism) may trigger a
plant to increase its immune response to an invading pest on its own, but these
8
mechanisms are not well-understood. And last, but certainly not least, genetically
modified crops are available, in which genes that confer the desired disease-control or
growth traits have been inserted directly into the plant’s genome. Unfortunately,
resistance of pathogens can still develop to the modified crop, meaning control strategies
by other means are still necessary.
In the fourth category, protection practices involve establishing a barrier between
the vulnerable plant and the pathogen. Usually, this barricade is a chemical pesticide, but
one can also separate plant from pathogen by physical, temporal, or spatial means. In
order to properly use the protection strategy, one must not wait until infection has already
occurred to implement the practice. If the disease pressure has risen to a substantial level
in the field, products can still be applied in a curative way, but control becomes more
difficult. Synthetic pesticides are the most commonly used strategy in the protection
category and have been used for more than one hundred years (8). Early pesticides
included inorganic elements like copper, sulfur, and mercury, some of which are still
used today. For example, copper is currently widely used as a bacteriocide, where the
product is sprayed on the leaf surface to provide a chemical barrier to infections from
pathogens such as Erwinia.
Many of the first pesticides were broad-spectrum, while many pesticides
nowadays focus on a single mode of action of the target organism. This can be both
beneficial and detrimental. Broad-spectrum pesticides, while efficacious against a wide
range of organisms (even ones that would not otherwise cause infection), may be toxic to
beneficial organisms, such as honeybees and ladybugs. Narrow-range products usually
9
stop the invading pest by only affecting one or a couple of specific metabolic processes in
the target pathogen cell, but inherent to this strategy, resistance can develop quickly.
Therefore, programs are developed for growers that use a combination of different
chemistries implementing multiple modes of action to slow the development of
resistance. In a way, this practice can make narrow-spectrum pesticides collectively
“broad-spectrum”, while still protecting beneficial insects.
To prevent resistance, it is also important to consider the dosage on the label and
the disease pressure that is present in a given field. Resistance is a risk when growers
apply lower than the dose recommended on the label of a pesticide as a cost-cutting
measure. Just like antibiotics in the medical field, applying a dose that is not strong
enough to kill the pest completely but strong enough to weed out weaker individuals in
the pest community, can lead to the production of new generations of agricultural
“superbugs”. Overall, adding to the diversity of control measures and applying pesticides
in smart ways reduces the risk of resistance.
Biological control is another example of the diversity of treatment options for
growers. The use of biological pesticides is an emerging market, which uses living
organisms (the product) to control other living organisms (the pests). There is
tremendous opportunity for crop improvement using “biologicals”, such as beneficial
bacteria and fungi, due to the wealth of natural products produced by these microbes and
their history of symbiotic relationships with plants. However, this treatment option also
has its share of problems. For example, biological pesticides are usually not as
predictable as synthetic pesticides, due to the increased variability introduced by using
10
living organisms in the control strategy. The lessened environmental impact, however, of
biopesticides has gained this control method attention due to increasing concern of the
public and the industry about health and sustainability issues in agriculture.
Synthetic pesticides are often seen as harsher products on the environment and
more toxic when compared to biopesticides, since the latter come from natural sources
and compounds made by microorganisms often decompose quicker than synthetic ones,
meaning less worries about chemical residues and retention in the environment (11). The
different modes of action of biologicals, both secondary metabolites and plant
interaction-driven, offer diversity in contributing to overall crop health. When used in
combination with synthetic pesticides in an integrated pest management program,
biologicals have the potential to reduce the overall dosage of each that is needed to
achieve similar control, which lessens the total environmental impact that purely
chemical treatment would otherwise have.
Overall, there are many different methods one can implement to control a crop
disease. Knowledge of the crop, geography, weather, disease susceptibility, disease life
cycle(s), and treatment strategies all add into the integrated control of plant disease.
However, the disease management strategies of many crops have room for improvement.
Disease management in broccoli, cucumber, and tomato are discussed below.
1.4 Brassica Downy Mildew
Among broccoli (Brassica oleraceae) diseases, downy mildew can be one of the
worst. It is found worldwide and is capable of destroying up to 100% of crop yields (12-
11
17). Young plants (seedlings) are the most susceptible, but infections in older Brassica
plants can contribute to yield losses due to poor produce quality and can leave the plant
susceptible to secondary infections by other microorganisms (15,17). An infected head
of broccoli can be seen in Figure 2 where the interior of the inflorescence is collapsed
due to abnormal development.
Figure 2: A head of broccoli affected by Brassica downy mildew disease (18).
Originating from the soil, this pathogen (Hyaloperonospora parasitica) is
capable of infecting plants by infiltration of the roots, growing systemically throughout
the plant, all the way to the leaves. Once on the leaves and after reproduction, spores of
the pathogen can spread from plant to plant by wind, where infection can begin again on
a new host by direct penetration of the leaf cuticle (13,14).
12
Symptoms include spotty, yellow, green or black lesions on upper surfaces of
leaves, with the underside of leaves appearing white and fluffy from the production of
spores (12-14). Defoliation often occurs and, while not fatal, strongly stunts plant growth
(13,14,17). If broccoli heads are infected, this can cause brown or grey discoloration,
while infection within the stems can appear as dark brown or black streaks (12).
Practices for lowering the incidence of this disease include spray treatments, crop
rotation, proper disposal of infected plant material, and growing plants in enclosed
structures to avoid exposure to soil and wind sources of inoculum (13,14). It is also
advisable to limit watering as much as possible to discourage spore germination, increase
ventilation in enclosures to reduce humidity (humidity encourages spore germination),
and to maintain proper nutrient requirements for the growing plants, since deficiencies in
minerals such as potassium have been shown to increase crop susceptibility to downy
mildew (13,14). Research into host and non-host resistance is an on-going topic in
downy mildew control, and there are certain varieties available, such as “Marathon”, that
are resistant to downy mildew, but these may not meet all of the quality characteristics
that farmers look for (15,17,19). Chemical treatment for this pathogen is available, such
as Mancozeb, Metiram and Zineb (13,14), yet, the issue of pathogen resistance still
arises. Organic treatments, like copper, are approved for Brassica downy mildew control
(20). Considering the challenges that this industry faces, there exists a large opportunity
for biological control strategy development.
13
1.5 Cucumber Anthracnose
One of the most important diseases of cucurbits is anthracnose caused by
Colletotrichum orbiculare, which is identifiable by the circular brown to red lesions on
leaves, and which commonly affects watermelon, muskmelon, and cucumber (21-23). It
can infect stems, fruits, and leaves, and can greatly hinder plant quality and yield (21-23),
as can be seen in Figure 3.
Figure 3: Cucumber anthracnose symptoms extending all the way to the deliverable
commodity (24).
The fungus thrives in warm, wet weather, and can spread easily from plant to plant under
these conditions in spore form, since water splashes facilitate the transfer (23).
Chemical treatments are available for this disease, such as azoxystrobin, Captan,
and Mancozeb, but, as for all synthetic treatments, there are issues of pathogen resistance,
environmental strain, and residue concerns (21,23). Although some varieties of
watermelon are resistant to certain isolates of anthracnose, other cucurbit species and
14
varieties are not inherently resistant, hence treatments strategies are still required (23).
There are microbial products on the market for the control of this disease, such as certain
Streptomyces strains of bacteria, with some isolates capable of controlling the disease up
to 93% (22), but there is certainly room for growth in the area of biological control of
anthracnose.
1.6 Bacterial Spot
Xanthomonas campestris pv. vesicatoria (Xcv) causes bacterial spot of pepper and
tomato, which can devastate entire fields all over the world if the humid, warm conditions
favorable to this pathogen are met (25-29). Even in drier climates, as long as heavy
overhead irrigation that maintains humid conditions is used, plants are at risk for
infection (26). This is of concern to growers who produce transplants for farmers, since
mainly overhead irrigation is used in the transplant industry (26,29,30). The main points
of bacterial entry are through stomata or wounds on leaves, but infection can also arise
from contaminated seed (26,29,31). Xcv is also capable of overwintering in soil, on
previously infected plant debris, and on or inside of non-host plants, making it a concern
even before a new crop is planted (26,31).
Once inside the plant, the bacteria release proteins called type III effectors that
allow the bacteria to grow inside the plant, where they can multiply into millions of cells
within a 24-hour period (26,32). Every above-ground part of tomato and pepper has the
potential to be infected: stems, petioles, leaves, flowers, and fruit (26,27). Infection leads
to many symptoms, the most serious being leaf drop and direct damage to the fruit itself,
15
as can be seen in Figure 4. Leaf drop exposes fruit to sun-stress, which can directly scald
the fruit, while direct infection of the fruit itself can lead to secondary infections from
other organisms; both symptoms can lower final fruit quality and marketable weight up to
52% (26,27,29).
Figure 4: Bacterial spot of tomato (33).
A variety of management tools can be practiced to reduce the incidence of
bacterial spot, however none offers a “cure-all”, and, for the most effective control, all
practices should be performed together in an integrated disease management system.
Excess nitrogen should be avoided to limit leaf size of peppers and tomato, reducing the
surface area of plant tissue that airborne bacterial cells can land on. Over-head watering
can be changed out to drip irrigation watering near the base of the plant, reducing the
chances of creating airborne cells within water droplets that may eventually land on
16
leaves and infect tissue (26,30). Treated or sterilized seed should be used whenever
possible (from resistant varieties if available), and growers should begin with field soil
and equipment that are pathogen-free as determined by testing, or sterilization (26,27,29).
Crop rotation should be practiced and all weeds should be removed to prevent persistence
and survival in the field (26).
Chemical treatments can be used, but none currently exist for treating established,
multiplying populations inside the plant, and, as with all chemical treatments, the
possibility of resistance is a concern (26). While biological control agents have shown
promise, none have demonstrated a significant enough decrease in the level of disease to
warrant their sole use as the bacterial spot control method and would still need to be part
of an integrated chemical program.
1.7 The Biology of Bacillus
One control strategy that offers itself for providing multiple crop solutions to
pathogen resistance, toxicity, and yield issues among others is the use of Bacillus for
agricultural applications.
Bacteria in the genus Bacillus have been intensively studied for their abilities to
produce a wide array of chemical compounds beneficial to humans in a wide-variety of
industries (discussed below). Combined with their ability to survive at high temperatures
and for long periods of time in a “hibernation-type” form called an endospore, bacteria in
this genus lend themselves to be good potential microbes for the development of
biopesticides.
17
The genus Bacillus resides in the Bacteria domain, Firmicutes phylum, Bacilli
class, Bacillales order, and Bacillaceae family (34). It is thought to originate from the
soil and is a gram positive, rod-shaped bacterium.
During normal growth without lack of nutrients, the bacterium multiplies and
divides through the formation of vegetative cells, which elongate, divide at the center,
and continue in this fashion during the logarithmic growth stage. Upon starvation, a prespore begins to form at a pole of the vegetative cell. This pre-spore becomes engulfed by
the vegetative or mother cell so that there is a cell within a cell (the soon-to-be
endospore), and the mother cell begins to add layers to the pre-spore exterior to form a
stable spore with a spore coat. After the layer addition has ended, the spore matures and
can withstand high temperatures (at least up to 100°C), chemical solvents, ionization,
hydrolytic enzymes, detergents, extreme pH, and long periods of time (it is proposed
maybe even millions of years) in a dry state (35). This sporulation process can be seen in
Figure 5.
18
Figure 5: The life cycle of Bacillus (35).
Due to this extremely stable endospore form, it is very amenable to industrial
formulation processes which often uses harsh conditions (like high-temperature drying)
to form functional products for the end-user.
1.8 The Chemistry of Bacillus
Bacteria of the genus Bacillus are well-known producers of a variety of
compounds that are used in many industries from food consumption to antibiotics, which
make them excellent candidates for biopesticide products (36). The most industrially
important species of Bacillus is B. thuringiensis, which is known for its insecticidal
19
properties due to the production of toxins known as δ-endotoxins (36,37). B. subtilis, a
member of this genus capable of producing a wide array of different antibiotic and
antifungal compounds, on average has 4-5% of its genome devoted to the production of
these secondary metabolites, so in a way, these bacteria can be viewed as tiny factories
for production of bioactive molecules (37).
One of the most studied types of Bacillus secondary metabolites are the
lipopeptides, which are known not only for their antimicrobial properties, but also for
effects that allow Bacillus to spread and grow on plant roots. This colonization
stimulates plants’ host defense mechanisms, allowing the beneficial bacterial population
to persist and allow the plant to thrive, respectively (37). Lipopeptide families known to
be produced by Bacillus include surfactins, iturins, and fengycins (pliplastatins) (37,38).
While each lipopeptide family is bio-active in itself, when combined together, they have
synergistic biological activity (37).
Surfactins are cyclic heptapeptides with a cyclic lactone ring that is formed by a
β-hydroxy-interlinkage (Figure 6). Because of their amphiphillic properties, surfactins
perturb membrane stability (such as in the membrane of an invading pathogenic cell) at
low concentrations, start to form pores in membranes at higher concentrations, and at
high enough concentrations, will completely solubilize the membrane and cause cell
death (37).
20
Figure 6: Surfactin, the most well-known member of the surfactin family of
secondary metabolites produced by Bacillus (39).
Iturins are also cyclic heptapeptides and are linked to a β-amino fatty acid chain
with 14-17 carbons, as can be seen in Figure 7. They are thought to form ion-conducting
pores in membranes, leading to osmotic destabilization. While surfactins have antiviral
and antibacterial activity, iturins have antifungal activity, limited antibacterial activity,
and no antiviral activity (37).
21
Figure 7: Iturin A, a secondary metabolite produced by Bacillus (40).
Fengycins (pliplastatins) comprise the third family of lipopeptides found in
Bacillus and are lipodecapeptides with a β-hydroxy fatty acid chain composed of 14-18
carbons (that can be saturated or not) and they have a lactone ring in the peptide moiety,
as can be seen in Figure 8. While the mode of action that fengycins have against
microbes is less understood than the other lipopeptides, they do interact with membranes
and are known to have antifungal activity.
22
Figure 8: Fengycin, a secondary metabolite produced by Bacillus (37).
In addition to the lipopeptides, other antimicrobial compounds are well known to
be produced by Bacillus, such bacilysin and the polyketides macrolactin, difficidin, and
bacillaene, which are shown in Figure 9 (38,41).
Figure 9: Other known secondary metabolites produced by Bacillus: bacillaene,
difficidin, macrolactin, and bacilysin (38).
23
Other bioactive compounds known to be produced by Bacillus includes but is not
limited to bacillibactin, bacteriocins, subtilin, zwittermicin A, siderophores, auxin,
gibberellins, cytokinin, abscisic acid, sporulenes, pumilin, 2,3-butanediol, acetoin,
cereulide, and homocereulide.
With the potential to produce so many types of compounds with a myriad of
functions, one can easily see how species of the genus Bacillus could be a good fit for
any industry with the need for new bioactive compounds with unique modes of action
and low possibilities of target resistance.
1.9 Objective, Scope, and Significance of Study
In this work, fermentation whole broth (WB) of a specific strain of Bacillus
pumilus, QST 2808, is investigated for potential bioactive metabolites. It is hypothesized
that this strain possesses at least one and possibly many metabolites with activity against
three important crop diseases: downy mildew, bacterial spot, and cucumber anthracnose.
Previous work had identified an amino sugar (under trade secret) in the bacterial whole
broth as being bioactive against other diseases, but secondary metabolites from B.
pumilus have not been investigated for potential activity against these three specific
diseases.
Specific Aim 1: The primary objective of this study was to identify and
characterize active constituent(s) for each of the three diseases indicated above. If more
than one active constituent was present, it was desired to at least show evidence as to why
it was hypothesized as such to pave the way for subsequent research. Specific Aim 2:
24
Isolation schemes were desired for at least one compound to give an indication as to how
the collection of this/these compound(s) could be scaled-up. Specific Aim 3: It was also
desired to investigate other methods for quantification of active metabolite(s) in order to
approach higher throughput, less labor-intensive procedures.
In this study, a metabolite active against two crop disease targets, downy mildew
and bacterial spot, was isolated and characterized from the WB from B. pumilus QST
2808 fermentation. This proved to be the previously identified amino sugar whose exact
chemical name must remain confidential to Bayer CropScience LP. An isolation scheme
was developed for this metabolite for future preparation purposes and for use as a
standard to monitor amino sugar levels for quality control of fermentation runs. Two
other fractions were identified, one with activity against bacterial spot and one with
activity against cucumber anthracnose. These will be further purified and characterized
in subsequent work. With the identification of these three bioactive fractions, this study
adds to the knowledge about bioactive compounds in Bacillus, with the eventual goal of
developing safe and effective crop protection products.
As a final note, Bacillus pumilus strain QST 2808 is protected by various patents
in the US and throughout the world (e.g., U.S Patent No. 6,245,551). Anyone wishing to
perform studies with this microorganism should contact Bayer CropScience LP to
determine whether a license is necessary and available.
25
Chapter 2
EXPERIMENTAL METHODS
2.1 Chemicals and Reagents Used
Acetylacetone, sodium carbonate, N,N-dimethyl p-aminobenzaldehyde,
ninhydrin, acetic acid, fluorenylmethyloxycarbonyl chloride (FMOC-Cl), Quadris,
chlorotetracycline, and cycloheximide were purchased from Sigma, trifluoroacetic acid
(TFA) was purchased from Pierce Chemical Company, D-glucosamine hydrochloride
was purchased from Acros Organics, and all other chemicals, reagents and solvents were
purchased from Fisher Scientific. Milli-Q-filtered, de-ionized water was used for all
experiments. Colletotrichum orbiculare (CA) and Hyaloperonospora parasitica (BDM),
both isolated from infected plant tissue, and Xanthomonas campestris pathovar:
vesicatoria (Xcv), strain AD17, were used for bioassay-guided fractionation.
2.2 Bacterial whole broth (WB) Production
Bacteria were grown in 12 L bioreactors (Applicon) in soy-based medium that
provided the nutrient requirements for amino sugar production using a standard seed train
inoculum process that is confidential to the company. After fermentation, the WB was
harvested and stored at 4°C.
26
2.3 Ultra-filtration of WB
Separation of bacterial cells and permeate (everything liquid outside of the cells)
was performed using an ultra-filtration, hollow-fiber system (Asahi Kasei Corporation).
The hollow-fiber system was cleaned before use by flushing large amounts of 0.1 M
sodium hydroxide through the tubing. Hot water was then used to flush out the sodium
hydroxide until the pH (Thermo Scientific, Orion 3 star) of the liquid coming out of the
filtration machine matched the pH of the water. Remaining liquid was flushed out until
the system went dry, and then WB was pumped through the system and permeate
(synonym: supernatant) was collected as soon as the liquid exiting the machine turned
dark brown as can be seen in Figure 10. The permeate and the retained cells (retentate)
were both saved.
27
Figure 10: The set-up of the high-pressure filtration system to separate bacterial
cells from permeate (synonym: supernatant). The bottle on the right shows the
clear, dark-brown permeate exiting the system and to the left of this bottle is the
opaque, viscous WB that contains both permeate and cells.
2.4 Liquid-liquid Extractions and Cell Extractions
The permeate was extracted sequentially with solvents using conical, large
volume centrifuge tubes (Corning, 431123). First, two tubes containing 100 mL of
permeate each were extracted three times with 75 mL of ethyl acetate each. Each mixture
was vigorously shaken and then centrifuged (Sorvall RC6+) at 7,000 rpm for 10 minutes
at 4°C. The ethyl acetate layer was removed, and extraction and centrifugation was
repeated twice more on the raffinate. All of the ethyl acetate layers were combined
28
together. The extraction and centrifugation process was repeated on the raffinate with nbutanol, and all of the n-butanol layers were combined together. The remaining aqueous
layers from the two conical tubes were combined together.
The raffinate was extracted with 100 mL of n-butanol to 50 mL of raffinate for 48
hours, after which the raffinate was filtered through a 0.2 μm filter (Millipore) to remove
bacterial cells and other large debris.
Small (1 mL – 3 mL) aliquots of all layers were dispensed onto metal drying pans
and placed in an oven (Fisher) overnight for weighing the next day on an analytical scale
(Sartorius, Extend ED124S). All layers were evaporated to dryness using a rotary
evaporator (Büchi, RE121) and re-suspended to 200 mL in solvent (15% aqueous
ethanol) for the n-butanol and ethyl acetate layers and water for the aqueous layer) so that
the samples were reconstituted in the volume they were originally in. The retentate was
re-suspended in 50 mL of 15% ethanol.
2.5 Ion-Exchange Chromatography (Small-Scale)
For separating the permeate based on charge, a styrene-divinylbenzene gel with a
sulfonic acid functional group (Dowex-50 hydrogen form with 4% cross-linkage and
100-200 mesh) stored in 5% acetic acid was used. One hundred mL of the permeate were
loaded through a glass column (2.5x20.5 cm) filled with 20 mL of resin at a rate of ~ 0.4
mL/min. The excess 5% acetic acid (the remaining solvent above the resin bed) was
eluted and discarded before loading and collecting the permeate material that was not
retained on the column (the flow-through). Three eluents (water, 0.2 N ammonium
29
hydroxide, and 0.1 M sodium chloride, in that order) were used in a 2.5 volumetric ratio
to the volume of resin used (see Table 1) to collect fractions of different ionic
characteristics. Each new solvent was added after the liquid above the resin just
approached the level of the resin bed. After the addition of the next eluent, 5 additional
mL of eluate was collected before switching the collection bottle. The pH of each
fraction was measured and re-adjusted to ~4.9 (as close as reasonably possible) with 1 M
hydrochloric acid and 1 M sodium hydroxide.
Resin
Feed (Permeate)
Flow-through
Volume
(mL)
20
100
n/a
Water
50
2.5
50
2.5
50
2.5
0.2 N Ammonium
Hydroxide
0.1 M Sodium Chloride
Ratio
1
5
n/a
Original
pH
n/a
4.9
1.24
4.61
Adjusted
pH
n/a
n/a
3.98
not
adjusted
8.48
8.2
5.1
4.84
Table 1: The parameters for the small-scale ion-exchange flash column
chromatography.
2.6 Ion-Exchange Chromatography (Large-Scale)
To collect more amino sugar in larger quantities for future use, a scaled-up
version of the small-scale Dowex-50 separation was performed. Using the same type of
resin as in the small-scale separation, 400 mL were packed into a glass column (30x4.8
cm), using acetic acid as the packing solvent. The excess acetic acid (the remaining
liquid amount above the resin bed) was drained to just above the resin bed and discarded
before adding 1.5 L of the permeate. A ~15 mL/min flow rate was maintained with a
30
hand pump connected to the top opening of the column. When feed and eluent additions
were made to the column, the pump was removed momentarily. To collect fractions of
different polarities, three eluents (water, 0.2 N ammonium hydroxide, and 0.1 M sodium
chloride, in that order) were added in a 1.875 volumetric ratio to the amount of resin used
(see Table 2). Each eluent was added after the liquid above the resin just approached the
level of the resin bed. After the addition of subsequent eluents, but before switching the
collection bottle, 100 additional mL was collected. The pH of each fraction was
measured and re-adjusted as close as reasonably possible to ~4.9 with 1 M hydrochloric
acid and 1 M sodium hydroxide (see Table 2).
Resin
Feed (Permeate)
Flow-through
Water
0.2 N Ammonium
Hydroxide
0.1 M Sodium Chloride
Volume
(mL)
400
1500
n/a
750
750
750
Ratio
1
3.75
n/a
1.875
Original
pH
n/a
4.9
0.98
2.01
Adjusted
pH
n/a
n/a
4.94
4.99
1.875
1.88
3.34
1.24
4.91
4.96
Table 2: The parameters for the large-scale ion-exchange flash column
chromatography.
2.7 Size-exclusion Chromatography
To further separate material by size exclusion, a 16x90 cm column was used with
a polyacrylamide resin was used (Bio-gel P-2, Bio-Rad, 100-1,800 molecular weight, <45
µm wet bead size). The gel was first washed in 90% methanol, then 100% methanol, and
then 100% deionized water, and was then changed to the running eluent, 10% methanol.
31
The 0.2 N ammonium hydroxide fraction from the ion-exchange column (in 22.5 mL)
was dried using a rotary evaporator and was re-suspended in 2 mL of deionized water.
Two hundred μL were retained and diluted in 22.5 mL to make a ~0.1X concentration for
bioassays.
As soon as the solvent front reached the bed, the rest of the fraction material was
gently pipetted onto the top of the bed, ensuring that the top surface area of the bed was
covered evenly and was not disturbed. After pipetting the material onto the column, the
flow was quickly resumed with a hand-pump, at a flow rate of ~0.35 mL/min. Fifty-eight
fractions (200 drops each) were collected and the flow was stopped.
2.8 Solid-phase Extraction
To attempt another method for amino sugar purification, a “catch and release”
style solid-phase extraction (SPE) technique was used. A 50 mg cartridge with the
cation-exchanger tosic acid resin (Silicycle) was pre-conditioned with 1 mL of methanol.
One mL of permeate was loaded onto the column and pumped through at ~1 mL/min, and
the flow-through was collected. The cartridge was then washed with 1 mL of methanol,
and collected as the wash. Finally, 1 mL of 2 M methanolic ammonium hydroxide (made
by adding 267 μl of 15 M ammonium hydroxide to 1.733 ml of methanol) was added to
the column, and this last fraction was collected as the “release”.
32
2.9 Ninhydrin Spray
Since ninhydrin spray stains primary amines purple and secondary amines
orange/yellow due to the reaction with the amine group and the formation of Ruhemann's
purple and an iminium salt (orange/yellow color), respectively, this was a useful, quick
way to determine the presence or absence of amino sugars from column fractions and in
zones on thin-layer chromatography (TLC) plates.
A 0.2% solution of ninhydrin spray was prepared by dissolving 0.2 g of ninhydrin
in 100 mL of ethanol. A thin layer was sprayed on the material of interest to be
developed; this was allowed to dry for 30 minutes underneath a fume hood. Using a
hairdryer, heat was applied to the material of interest until the purple, orange, or yellow
color appeared, but not so much heat so that browning of the ninhydrin occurred (about 1
minute of on-and-off heat).
2.10 Reverse-Phase High-Performance Liquid Chromatography (HPLC)
Reverse-phase HPLC was used to analyze ion-exchange chromatography and
size-exclusion column fractions (later referred to as the Natural Products screen, or NP
screen). This direct-injection, NP screen method was meant as a general separation
method rather than targeting specific compounds in order to be able to judge which
fractions could be pooled together and further analyzed. An Agilent Technologies 1200
Series HPLC and detector were used.
Fifty μL of sample was injected onto a C-18 reverse-phase Varian Microsorb
column (3 µm particle size, 4.6 x100 mm) at 45°C at a flow rate of 1 mL/min with 0.05%
33
TFA in water (solvent A) and 0.05% TFA in acetonitrile (ACN) (solvent B) using the
gradient program shown in Table 3. Absorbance was recorded at 210, 254, 280, and 340
nm. A blank methanol injection was made before and after all samples in the sequence.
Time
0
30
40
43
47
50
% ACN with 0.05%
TFA
14
55
95
95
14
14
Table 3: The gradient used with samples tested in the NP-screen method.
2.11 Derivatization Assay for Amino Sugar Quantification
To quantify the amino sugar present in samples and fractions, a derivatizationHPLC assay for amino sugars was used. Twenty-five mg of each sample was added to
duplicate microcentrifuge tubes (Eppendorf) using an analytical scale and 975.0 µL of
water was added to each. The samples were vortexed (Scientific Industries, Vortex
Genie) for 30 seconds and then centrifuged for 10 minutes at 12,000 rpm. Five mg of
potassium bicarbonate was added to two labeled microcentrifuge tubes, and 75 μL of the
supernatant was added; these were vortexed for 10 seconds and then left to sit at room
temperature until dissolved.
FMOC-Cl (5 mg per sample in assay) was weighed into scintillation vials
(Fisherbrand, 0333925) under a fume hood, 1,4-dioxane was added (425 μL per sample
in assay), and the mixture was lightly shaken until dissolved. Four hundred and twenty-
34
five μL of the FMOC-Cl-1,4-dioxane solution was added to the sample supernatant tubes.
This was vortexed for 20 seconds, sonicated at 50°C for 30 minutes, centrifuged at
12,000 rpm, and filtered through a 0.45 μm filter (Whatman) into HPLC vials (National
Scientific, C4000-1W).
A three-point calibration curve was performed with each sample set, wherein
different amounts of the derivatized amino sugar standard were injected (whose exact
identity must remain confidential to Bayer CropScience LP). Twenty five μL, 50 μL, and
100 μL of the 1.0 mg/mL stock were added to microcentrifuge tubes with 5 mg of
potassium bicarbonate, in duplicate; this was vortexed for 10 seconds and left to sit at
room temperature until dissolved. Four hundred and twenty-five μL of the FMOC-Cl1,4-dioxane solution from above was added to the 25 μL and 50 μL standards. The 100
μL standard received slightly less volume of a more concentrated FMOC solution to
maintain 5 mg FMOC per tube. These were vortexed for 20 seconds, sonicated at 50°C
for 30 minutes, centrifuged at 12,000 rpm, and filtered through a 0.45 μm filter into
HPLC vials (National Scientific, C4000-1W). Four hundred μL of a 12.5 mg/mL,
FMOC-Cl-1,4-dioxane solution was added to the 100 μL standard sample tube and was
treated like the rest.
Thirty μL of each sample was injected onto an XTerra Phenyl HPLC column (15
cm in length, 4.6 mm inner diameter and 5μm pore size) at 25°C, with a flow rate of 1.0
mL/min (this column was different from the column in section 2.10). Absorbances were
measured at 210 and 260 nm. The derivatized peak that elutes at ~6.6 minutes is the
amino sugar peak and can be further identified by comparing the ultraviolet trace of this
35
peak in the samples tested to the standards. The total run time was 20 minutes. The
solvent gradient with 0.05% TFA in water, 0.05% TFA in ACN, and methanol was as
shown in Table 4.
Time
(min)
0.0
6.0
12.0
14.1
18.0
Water+0.05%TFA ACN+0.05%TFA Methanol
80.0
10.0
10.0
40.0
50.0
10.0
10.0
80.0
10.0
80.0
10.0
10.0
80.0
10.0
10.0
Table 4: The gradient conditions for the amino sugar HPLC assay. Results are
expressed as mg of amino sugar per ml of starting material.
2.12 Colorimetric Assay for Amino Sugar Quantification
To investigate an easier method for quantifying amino sugar concentration than
the derivatization assay, a colorimetric method was performed (42). A solution of 4%
(v/v) acetylacetonein and 1.25 N sodium carbonate was made by dissolving 6.625 g
sodium carbonate in 100 mL of deionized water. This was stored at 4°C in a light-proof
bottle. Ehrlich’s reagent was made by dissolving 1.6 g N,N-dimethyl paminobenzaldehyde in a 30:30 mixture of ethanol and concentrated hydrochloric acid.
This was stored at 4°C in a light-proof bottle.
Samples to be tested were centrifuged at 8,000 rpm for 10 minutes at 4°C, and the
top portion of the supernatant was used for the next step. Samples (in duplicate) were
diluted 100 fold by adding 1.0 mL of sample to 9.0 mL water twice. A standard curve
from WB samples prepared in the manner above was performed alongside with duplicate
36
samples diluted 10, 50, 100, and 200 fold in water. A deionized water control was used
as a blank. Alkaline acetylacetone solution (0.25 mL) was added to each sample, and
samples were capped and placed in a 90°C water bath for 60 minutes. After letting cool
to room temperature, 2.0 mL of ethanol was added to each tube, and these were vortexed
for 10 seconds. Ehrlich’s reagent was added to each tube (0.25 mL), these tubes were
vortexed for 10 seconds, and the absorbance at 530 nm was read between 30 and 60
minutes, using deionized water to blank the spectrophotometer (Thermo Scientific, Evo
60).
To calculate the amount of amino sugar in each sample, first the reading from the
deionized water control sample was subtracted from all samples’ readings to yield the
“corrected absorbance of standard” or the “corrected absorbance of sample”. The WB
from the standard curve set that was diluted 100 fold was set to 100% amino sugar
concentration so that all values were relative to this and this was used as the standard.
The corrected absorbance of the sample was divided by the corrected absorbance of the
standard in order to obtain a ratio of the reading of the sample vs. the standard. This ratio
was then multiplied by the dilution of the sample divided by the dilution of the standard
in order to normalize any effects of dilution or concentration that the sample had.
Finally, this was multiplied by the concentration of amino sugar in the standard
determined by the derivatization assay (see equation below). In this way, concentration
units of amino sugar samples could be translated from the colorimetric assay to the
derivatization assay in case comparison of samples sets from the two different methods
was necessary.
37
[Amino sugar in Sample] = [Standard] x (Corrected Absorbance of
Sample/Corrected Absorbance of Standard) x (Dilution of Sample/Dilution of Standard)
2.13 Thin-layer Chromatography
To further separate purified fractions, thin-layer chromatography (TLC) was used.
Acetic acid, n-Butanol, and de-ionized water (10 mL:40 mL:40 mL) were mixed and
incubated overnight at room temperature to allow complete separation of the layers. The
bottom layer was drained off and the top layer was retained for use as the solvent in TLC
experiments.
A TLC chamber with a lid was used with a paper towel pressed on the side wall
with ~100 mL of solvent in the bottom in all experiments. Plates were allowed to
develop until the solvent line was ~1-2 cm away from the top and were left to dry for ~1
hour in a fume hood before further use.
For preparative quantities, glass-backed 500 μm or 1000 μm thick, 20x20 cm,
cellulose or micro crystalline cellulose plates (300 μm or 50 μm particle size) with an
ultra-violet indicator (254 nm) were used (Selecto Scientific and Avicel, respectively).
For analytical tests, flexible, 100 μm-thick, 20x20 cm, cellulose plates (300 μm particle
size) with an ultra-violet indicator (254 nm) were used (Selecto Scientific).
For extracting the material from the preparatory TLC plate, sections of the
cellulose plate were scraped off with a razor blade and extracted with water overnight in
20-80 mL (depending on how much material was in a certain zone) in an Erlenmeyer
flask with a stir bar on a stir plate. The next day, this material was transferred to a 50 mL
38
conical tube, centrifuged at 4,200 rpm, the supernatant was filtered through a 0.45 μm
filter, and the fraction was rotary evaporated to dryness.
2.14 Solubility Testing of Colletotrichum orbiculare (CA) Active
Bioassays of the initial separation indicated that a separate fraction was
responsible for CA activity. Solubility analysis was carried out on the purified CA active
fraction. One mL of the flow-through from the small-scale Dowex-50 isolation in section
2.5 was dried using a rotary evaporator and re-dissolved in 1 mL of various solvents
sequentially, drying each with a Reacti-vap (III, Thermo Scientific) between new
solvents. Chloroform, ethyl acetate, methanol, acetonitrile, and water were tested and
solubility was monitored visually by looking for particulates and opaqueness.
2.15 Mixed-mode Chromatography
To investigate another HPLC method for amino sugar separation/comparison
/quantification, a mixed-mode column scheme was used with the same HPLC system as
was used for the NP-screen (Agilent Technologies 1200 Series), but using an evaporative
light-scattering detector (ELSD) set to 50°C and 3.4 bar (Sedex 75, Sedere), in addition
to the UV detector. This additional method was attempted in an effort to reduce sample
preparation time required by the derivatization assay. A purified porous silica, 150x4.6
mm mixed-mode (octadecylsilane with anion and cation-exchange ligands) column was
used (3 μm particle size and 13 nm pore size, Scherzo SM-C18). The solvent system
39
used was 3.0 mM ammonium acetate and 80:20 80 mM ammonium acetate: acetonitrile.
The elution was isocratic, with a flow of 0.5 mL/min.
2.16 Lyophilization for NMR
For nuclear magnetic resonance (NMR) analysis, fraction 4 from size exclusion
chromatography (section 2.7) was lyophilized overnight in glass scintillation vials
(Fisherbrand, 0333925) by freezing the fraction at a slant at -80°C for at least 30 minutes,
then placing in the lyophilizer to sublime overnight. Before adding the sample, the vial
was first heated for ~ 10 seconds using a hair-dryer and placed in the freeze-dryer for 30
minutes, after which the vial was weighed and the sample added. The vial containing the
fraction was weighed after lyophilization to obtain the total weight of the purified
fraction. The sample was sent to an off-site NMR facility for structure elucidation
(Acorn, Inc.).
2.17 Minimum Inhibitory Concentration in vitro Assay
The minimum inhibitory concentration (MIC) method is a high throughput assay
to test dilutions of samples against fungi and bacteria in vitro. The smallest concentration
that inhibits the growth of the organism is called the MIC. This number represents the
dilution factor of the sample before the activity disappeared, so a higher number means
greater activity. This value can be used to compare the efficacy of different samples.
40
All procedures were performed aseptically. For bacterial inoculum, Xanthomonas
campestris pv. vesicatoria (Xcv) was streaked out from a -80°C glycerol stock onto a
nutrient agar Petri plate (Hardy Diagnostics) 2 days before the experiment. The next day
(1 day before the experiment), 1 colony was inoculated into 10 mL of nutrient broth
(Hardy Diagnostics) in a baffled 250 mL flask with a metal lid and shaken overnight at
30°C and 200 rpm.
Xcv was grown in the manner described above to an optical density of 2 at 600 nm
(OD600). The OD600 was determined for 2-fold dilutions of the bacterial culture. Colonyforming units (CFU) were determined for all dilutions and the original culture by diluting
each sample 1:10 in phosphate buffered saline, six consecutive times, vortexing between
dilutions. Each of these dilutions were plated (100 μL onto nutrient agar plates) and
incubated for 2 days at 30°C. Colonies were enumerated after the incubation period, and
the CFU/mL was calculated for each dilution. The OD600 was plotted against the
CFU/mL. An equation was obtained to determine the concentration of cells given OD
readings between 0.1 and 2: y = 0.407ln(x) + 8.9244, where y = log(CFU/mL) and x =
OD.
When the inoculum was desired on the day of testing, it was grown as before, the
CFU/mL was determined from its OD, and an inoculum of 107 colony-forming units
(CFU)/mL was made by diluting the overnight culture into the appropriate amount of
nutrient broth.
For fungal CA inoculum, spores were harvested from a 2-3 week-old plate that
was made by streaking spores from a storage plate (stored at 20°C) onto potato dextrose
41
agar (Hardy Diagnostics) and allowed it to grow at 20°C. Spores were harvested by
flooding the plate with sterile deionized water, rubbing the agar with an L-rod to dislodge
the spores, filtering the spore suspension through cheesecloth and quantifying with a
hemocytometer (Bright Line, Sigma Z359629) under a phase-contrast microscope
(Olympus, BX41). An inoculum of 105 spores/mL was made by diluting the spore
suspension into deionized water.
In preparation for the MIC assay, samples were centrifuged (Heraeus,
Biofugepico) at 8,000 rpm and 4°C for 15 minutes, and the supernatant was filtersterilized using a 0.2 μm filter (Millipore). All samples were diluted with deionized water
in a 1 mL, 96-well dilution block (Thermo Scientific), 1:1, 11 times, for a total of 12
concentrations per sample (including the sample at the original concentration). Two
replicate dilution rows were performed for each sample. Water was used as a negative
control. Positive controls were also used: chlorotetracycline at 7 ppm for Xcv and
cycloheximide at 25 ppm for CA. Control compounds were weighed from a stock
powder using an analytical scale (Sigma) and diluted in deionized water.
Each sample dilution series was tested in quadruplicate with an organism in clear,
flat-bottomed, 96-well polystyrene microplates (two dilution series replicates located on
one plate, and two on another plate; Thermo Scientific). An additional plate with the
same sample dilution series was left un-inoculated so that at the end of the test, the OD
effect of the samples and reagents alone could be subtracted from the final reading of the
inoculated plates, in order to differentiate OD effects due to sample from OD effects due
to growth. In other words, the non-inoculated plate was a blank used for the plate reader
42
(spectrophotometer) so that all that was being measured at the end was the growth of the
organism.
For bacterial assays, wells were first filled with 100 μL of nutrient broth, then 25
μL of the sample (from the dilution series) was added, and then 50 μL of inoculum or
blank nutrient broth was added. All plates were placed at 30°C for 2 days to incubate.
For fungal assays, wells were filled with 100 μL of potato dextrose broth (BD)
with 100 ppm of chloramphenicol (Sigma), 25 μL of sample, and 50 μL of inoculum or
blank water. All plates were placed at 20°C for 2 days.
Results were obtained spectrophotometrically using a microtiter plate reader
(Perkin Elmer, Victor 3). The OD600 value was inserted into an equation that compares
the growth in the treated wells with the growth in the water control:
Percent growth =
(OD of Treatment With Organism) - (OD of Treatment Without Organism) X 100%
(OD of Water With Organism) - (OD of Water Without Organism)
The percent growth was graphed versus the dilution series of the sample. The
dilution before the percent growth increases dramatically is reported as the MIC value,
and the higher the value, the more potent a sample is (since higher values mean more
dilutions were made to the original sample). As long as the percent growth remains
under 20%, the sample is still seen as controlling the growth of the organism.
2.18 Agar Diffusion Assay
For testing small volumes of sample for bioactivity against bacteria or fungi, a
classic agar diffusion method was used on Petri plates. Bacterial and fungal inocula were
43
prepared as for the MIC assay above, 150 μL were spread evenly with an L-rod onto a
nutrient agar Petri plate, and the inocula on these plates were allowed to dry for 10
minutes while the plate lids remained on. Eight mm holes were aseptically punched into
the agar, and each of these wells was filled with 100 μL of the sample to be tested.
Samples were tested in duplicate, and water was included as a sample for a negative
control. These plates were incubated 1 day at 30°C for bacteria and 3 days at room
temperature for fungi. After incubation, the diameter of the zone of inhibition was
measured.
2.19 Germ Tube Assay
For testing small volumes of sample for bioactivity against CA, a germ-tube assay
was used. The efficacy of a sample against CA is related to how many CA spores
germinate when exposed to the sample overnight. Fungal inoculum was prepared as for
the MIC assay, and 100 μL was pipetted into a divot slide, along with 100 μL of the
sample to be tested. Samples were tested in duplicate, and water was used as a negative
control. Slides were incubated overnight at room temperature in a moist box (an inverted
Tupperware container with a moist paper towel at the bottom) to prevent sample
evaporation. The following day, slides were evaluated for the amount of germination by
phase microscopy (Olympus, BX41).
44
2.20 Brassica Downy Mildew in planta Assay
In order to evaluate the effectiveness of samples against the obligate pathogen
Hyaloperonospora parasitica (BDM), an in planta assay with broccoli seedlings was
used. Whole-broth samples were tested, undiluted or as 20%, 10%, or 5% (v/v) dilutions
in water.
Broccoli seeds (¼ teaspoon; Johnny’s Selected Seeds) were sprinkled evenly
across the surface of a 6.35 cm plastic pot filled with potting mix (Sunshine #3) for each
replicate to be tested and were grown to the cotyledon stage (6-8 days from planting).
Treatments were sprayed on both the upper and lower leaves with an artist’s airbrush
until run-off. An untreated control was used to monitor disease severity (UTC) and a
fungicide check (Quadris [azoxystrobin] 10 ppm, diluted in water from a stock powder,
Syngenta) was used as a positive control. All samples from the dilution series were
tested in quadruplicate. All pots were randomized in growing trays and the sprayed
leaves were allowed to dry for one day at room temperature.
The pathogen was maintained on live Brassica plants in a dew chamber (Percival,
I-36D/DL) at 14-16oC, and the culture was transferred to new Brassica seedlings every 7
days. A handful of heavily sporulated leaves (6 days after infection of new seedlings)
were used for the inoculum. These were placed in a water bottle with ~500 mL of water,
the bottle was shaken, and the liquid was passed through two layers of cheesecloth to
filter out mycelium. The spores were quantified using a hemocytometer (Bright-line) and
a microscope (Olympus, BX41) and diluted to 5.0 x 103 – 2.0 x 104 sporangia/mL using
deionized water.
45
Treated plants were misted evenly with the spore suspension using a spray bottle,
about 25 mL per tray, or ~1 mL per plant. The trays were covered with plastic domes,
and these were set inside a Percival growth chamber at 14-16oC and then rated for
pathogen growth 6-7 days later.
Bioactivity was rated on a percent scale for visual determination of how well the
disease was controlled compared to the infected control (0-50% control, or no activity)
and the uninfected control (100% control, or perfect activity): 100% = perfect control,
99%-98% = excellent control, 97%-90% = very good control, 89%-80% = satisfactory
control, 79%-50% = poor control, and 50%-0% = no activity.
46
Chapter 3
RESULTS AND DISCUSSION
This study focused on analyzing whole broth (WB) of a Bacillus pumilus strain
QST 2808 for bioactivity against three organisms responsible for causing plant disease:
Hyaloperonospora parasitica, which causes Brassica downy mildew (BDM);
Xanthomonas campestris pv. vesicatoria (Xcv), which causes bacterial spot of tomato;
and Colletotrichum orbiculare, which causes cucumber anthracnose (CA). The objective
was to isolate and characterize at least one compound that is active against one or more of
these three disease agents. If more than one active constituent was present, it was desired
to at least show evidence as to why it was hypothesized as such to pave the way for
subsequent research.
A summary of work is as follows, with details on this overall process in the
following subsections. The strategy was to first identify whether the activity was
associated with the bacterial cells (i.e. compounds bound to the exterior of the cells)
and/or if activity resided in the supernatant of the whole broth (i.e., compounds secreted
from the cells). The solubility of active compound(s) in organic solvents was then
characterized in order to understand what could be used as diluents in future assays.
Liquid-liquid extractions were performed on cell and supernatant samples with different
solvent types in order to address these initial questions. Bacillus cells are difficult to lyse
and many compounds can bind to the exterior, hence, extractions of cells mostly involved
47
solubilizing cell-bound chemical components rather than compounds trapped within cells.
The bioactive fractions were found to reside in the supernatant and to be water-soluble.
Further chromatographic separations based on size and charge to purify and
characterize the active fractions in the WB were performed. In the ion-exchange
chromatography studies, two distinct fractions, one active against CA, and another against
BDM and Xcv, were discovered, hence, at least two active compounds were present in the
WB. Size separation chromatography was performed on the BDM and Xcv active
fraction, and a fraction was identified to be responsible for this activity. This fraction
was of suitable purity for nuclear magnetic resonance (NMR), and it was identified as an
amino sugar.
As a side-project not crucial to the overall goal of discovering active natural
product chemistry, ion-exchange chromatography was performed with the permeate
(synonym in this thesis: supernatant) from the fermentation WB on a large scale in order
to judge the feasibility of duplicating the separation and to isolate larger quantities of
material. This scaled-up separation did not replicate the fractionation pattern seen in the
small-scale ion-exchange method. With the large-scale method, however, two distinct
Xcv-active fractions were found compared to only one Xcv-active fraction in the smallscale method. This indicated that a third active compound was present in the whole
broth. Various side methodologies were also attempted to improve the quantification and
separation of the amino sugar, but none were optimal to adopt as permanent quality
control methods.
48
Through these experiments, the knowledge of QST 2808 was increased, which
made further postulation into the mode of action of this product against crop pests
possible.
3.1 Cell vs. Permeate Activity
Active compounds bound to bacterial cells will not necessarily be in the liquid
portion of the culture when separating cell from permeate/supernatant samples.
Therefore, the activity in both the cell pellet extract and the permeate/supernatant
(everything that is in the liquid portion of the WB and not associated with the cells)
against BDM, CA, and Xcv was compared to that of the WB. The cell pellet extract was
prepared by extracting the cell pellet with n-butanol, drying the n-butanol layer, and resuspending it in water. Due to the need to filter-sterilize samples for assays involving CA
and Xcv, and due to particulate matter (cell debris, insoluble fermentation solids, cells,
etc.) interfering with the spectrophotometer, only the cell pellet extract and the permeate
were tested against CA and Xcv.
3.1.1. BDM Activity
The pellet extract, the permeate, and the WB were tested in the BDM assay
diluted to 20, 10, and 5% (v/v) WB equivalent (with undiluted WB or undiluted sample at
the concentration it would be found at in WB being 100%). Bioactivity was rated on a
percent scale for visual determination of how well the disease was controlled compared
49
to the infected control (0-50% control equating to no activity) and the uninfected control
(100% control, or perfect activity). No activity resulted in the Brassica leaves
completely shriveling due to the infection destroying the tissue, whereas perfect activity
appeared as healthy leaves that looked like a control where no disease was present in the
first place. If the activity was in between these two measurements, a qualitative
assessment was made depending on how relatively damaged the plants were.
The effect of all samples tested on BDM is shown in Figure 11 and quantified in
Figure 12. It appeared that the majority of the activity was in the permeate, since the
activity of 20% and 10% permeate against BDM was comparable to the activity of these
same concentrations of WB. As the percentage of the permeate and WB dropped to 5%,
the activity decreased. Low activity was seen at all dilutions tested of the cell extract.
From these results, it appeared that most of the active compounds towards BDM are
found primarily in the permeate.
Figure 11: The effect of the cell pellet extract and permeate samples compared to
WB against BDM. Samples were applied as a spray on Brassica before inoculation
with the pathogen and rated ~ 1 week later for disease.
50
Figure 12: The efficacy of the cell pellet extract, permeate, and WB samples against
BDM.
3.1.2. CA and Xcv Activity
For testing in the MIC assay against Xcv, samples were diluted into deionized
water, 1:2, 11 times, for a total of 12 concentrations per sample (including the undiluted
sample). An additional plate with the same sample dilution series was left un-inoculated
so that the OD effects of the samples and reagents alone could be subtracted from the
final reading of the inoculated plates. In this way, it was possible to differentiate OD
effects due to sample from OD effects due to growth. The OD was inserted into an
equation (described in section 2.17) that compares the growth in the treated wells with
the growth in the water control. The percent growth was graphed versus the dilution
51
series of the sample. The dilution before the percent growth increases dramatically is
reported as the MIC value.
The activity of the permeate and cell pellet extract towards CA and Xcv can be
seen in Figure 13. Similar to the results with BDM, the permeate had higher activity
against CA and Xcv than did the cell pellet extract, although this was not as pronounced
in the CA efficacy. There was slight activity in the cell extract for both pathogens,
indicating that it is possible that other active metabolites against these pathogens could be
cell-associated and/or n-butanol-soluble (since the cell extract was performed with nbutanol).
12
Average MIC
10
8
6
4
MIC (Xcv)
MIC (CA)
2
0
Figure 13: The efficacy of the cell pellet extract and permeate samples against CA
and Xcv.
In all three pathogen bioassays, most of the activity was located in the permeate
and not the cell extract; therefore, the permeate was used in most of the subsequent
separation experiments.
52
3.2 Liquid-liquid Extractions and Cell Extractions
Next, the relative polarity of the active compound(s) in the WB was determined
using liquid-liquid extractions.
Sequential WB extractions resulted in volumes of 390 mL, 540 mL, and 100 mL
of the ethyl acetate extract, n-butanol extract, and aqueous raffinate, respectively. The
extracts were dried and weighed. The original concentrations of all extracts were
determined to be WB, 98.4 mg/mL; ethyl acetate, 0.2 mg/mL; n-butanol, 1.4 mg/mL;
aqueous, 76.8 mg/mL. Based on masses, most of the material partitioned into the
aqueous layer.
3.2.1 Xcv and CA activity
For bioassays, extracts were re-suspended in 15% aqueous ethanol. MICs
(against Xcv and CA) and a BDM in planta bioassay were performed on all of the
fractions. The activity that the various liquid-liquid extracts had against Xcv and CA can
be seen in Table 5.
Against Xcv, the majority of the activity, besides that in the permeate, was
observed in the aqueous fraction since higher numbers were observed in these two
samples (meaning that these samples could be diluted farther out on the microplate before
their activity stopped). There was slight activity in the n-butanol fraction, indicating that
53
other active metabolites against this pathogen could be cell-associated and/or n-butanolsoluble.
It appeared for CA, that once extracted and separated from the WB, the limit of
detection in the MIC assay was too low so that no activity was detected in the ethyl
acetate, n-butanol, or aqueous samples.
Permeate
Ethyl acetate
n-butanol
Aqueous
Chlorotetracycline
Cycloheximide
MIC (Xcv)
7.5 ± 0.7
no activity
1±0
8±0
5±0
not tested
MIC (CA)
1±0
no activity
no activity
no activity
not tested
2±0
Table 5: The efficacy of liquid-liquid extraction samples on Xcv and CA compared
to antibacterial and antifungal positive controls.
3.2.2 BDM activity
Samples were tested against BDM at 20%, 10%, and 5% (v/v). The results can be
seen summarized in Figure 14. Similar to Xcv activity, most of the activity was in the
aqueous layer; this was similar to the activity seen in the different concentrations of
permeate and WB tested. This suggested that most of the highly active components
against BDM could be found in the aqueous layer since negligible activities were seen in
the ethyl acetate and n-butanol layers.
54
Figure 14: The efficacy of liquid-liquid extraction samples on BDM.
Thus far, similar activities were seen in similar fractions for both Xcv and BDM
since the activities were tracking with one another for both bacterial and oomycete
control.
3.3 Ion-Exchange Chromatography
To separate compounds in the permeate by charge, flash cation-exchange
chromatography was performed using water, ammonium hydroxide, and sodium chloride
as eluents. Ion-exchange fractions of 81.8 mL, 19 mL, 22.5 mL, and 15.5 mL were
collected of the flow-through, water wash, ammonium hydroxide wash, and sodium
chloride wash, respectively. After drying and weighing aliquots of the liquid fractions to
55
measure the distribution of material, 2.077 g, 209 mg, 531 mg, and 195 mg, were found
to be in the flow-through, water wash, ammonium hydroxide wash, and sodium chloride
wash, respectively. Judging the fractions by weight, material was present in all fractions
collected, but the majority did not adsorb to the resin and flowed through.
The NP-screen HPLC method was performed on the ion-exchange fractions.
These results can be seen in Figure 15. The fractions with the largest detectable peak
areas were the flow-through and the ammonium hydroxide fractions.
Figure 15: HPLC chromatograms of fractions from the small-scale ion-exchange
flash column Detection at 210 nm. A blank run before sample injection showed no
detectable peaks.
56
Up until this point, unknown compound(s) were responsible for the various
bioactivities demonstrated. Previous work on whole broth from this bacterium had
identified an amino sugar as a possible candidate; hence, column fractions were analyzed
using the amino sugar derivatization assay.
In the derivatization HPLC assay, the limit of amino sugar detection was found to
be low (~80 mAu*s peak area, or 0.8 μg injected before derivatization) since samples
injected with volumes corresponding to this peak area were still detectable and fit a
standard curve with a high correlation coefficient (Figure 16). A blank injection yielded
no detectable peaks corresponding to the retention time of the amino sugar. The amino
sugar was present in all fractions, which indicated either that the column was overloaded,
or that the solid phase did not retain the compound well as can be seen from the
derivatization results in Table 6.
8000
Peak Area mAu*s
7000
6000
5000
4000
3000
2000
1000
0
0
20
40
60
80
100
120
Amount of Amino Sugar Standard Injected (μL of a 1.0 mg/mL
y = 70.672x + 27.385
solution)
R² = 0.9911
Figure 16: Amino sugar calibration curve using the derivatization assay.
57
3.3.1 Xcv, CA, and BDM activity
In order to compare bioactivity with amino sugar concentrations, column fractions
were also tested against Xcv and CA (in MIC), and BDM in planta. No eluent seemed to
be particularly good at eluting the component that is active against Xcv and BDM, as the
activity against these pathogens was spread across all column fractions (Table 6 for Xcv
and Figure 17 for BDM). The fraction with the majority of the activity for both, however,
was the ammonium hydroxide wash.
Against CA, the activity profile of the fractions was completely different. No
activity was found in the ammonium hydroxide wash, and the activity appeared only in
the flow-through and the water wash. It can be concluded that the compound responsible
for CA activity did not have high affinity for the ion-exchange resin, flowed through upon
column loading, and was eluted with water (Table 6).
Permeate (feed)
Flow-through
Water wash
Ammonium Hydroxide
Wash
Sodium Chloride Wash
MIC
(Xcv)
MIC
(CA)
Amino Sugar
Concentration
(mg/g of WB)
7.5 ± 0.7
4±0
5±0
4±0
1±0
4±0
59.71
10.8
16
6 ± 0.7
5±0
0±0
0±0
45
16
Table 6: Efficacy of ion-exchange fractions against Xcv and CA and corresponding
amino sugar concentrations.
58
Figure 16: Efficacy of ion-exchange fractions against BDM.
Looking at the amino sugar results from the HPLC assay (Table 6), it was
interesting to note that this compound was spread across many fractions, with the
majority of the material located in the ammonium hydroxide wash. This was the same
pattern as seen with Xcv and BDM activity and to this point, the amino sugar was tracking
with these activities. Conversely, less amino sugar was in the flow-through and water
washes, where the CA activity was the highest, so it seemed that a different active could
be responsible for this antifungal activity.
3.4 Scaled-up Ion-Exchange Chromatography
For isolation of larger amounts of bioactive ion-exchange fractions to facilitate
subsequent characterization work, it was investigated if the ion-exchange technique on a
59
small-scale would mirror a scaled-up one. Volume of resin was multiplied by a factor of
20 and eluents and amount of eluent collected were multiplied by a factor of 15 from the
small-scale column scheme, so that 1.5 L of flow-through, 0.75 L of water wash, 0.75 L
of ammonium hydroxide wash, and 0.75 L of sodium chloride wash were collected, to
yield fractions of 87.3 mg, 16.3 mg, 6.3 mg, and 8.4 mg, respectively, once dried. Since
the solid material obtained from the large-scale cation-exchange technique was much
smaller (less than a 20-fold increase) than expected based on the small-scale solid
masses, this indicated that further optimization would be needed on a large scale for at
least mimicking the small-scale procedure, such as smaller volumes of feed.
In order to visualize the purity of these fractions and to see if any compounds had
retention times that corresponded to that of the amino sugar standard, the mixed-mode
chromatography method was first used to analyze these fractions, followed by the
derivatization method.
Even though it was still in the process of development, the mixed-mode amino
sugar method was the first technique attempted because of its decreased sample
preparation time and the ability to re-use the sample remaining in the HPLC vial after
injection. Products of the derivatization assay could not be reused. It was also preferable
to use the mixed-mode method instead of the NP-screen since the underivatized amino
sugar is not detectable with UV but rather with ELSD, as can be seen in the amino sugar
standard chromatogram of Figure 18.
Since the column fractions were still crude, high purity was not expected, and
most fractions over-loaded the column so that individual peak resolution was not
60
possible. The amino sugar standard yielded two peaks with the mixed-mode HPLC
method, one at 6.010 minutes and another at 9.536 minutes (Figure 18). The 6.010minute peak was most likely due to an impurity, since later work on a pure amino sugar
standard showed that with this method, the amino sugar corresponded to a peak around
9.5 minutes (not included in this work). In Figure 18, no fraction contained material that
eluted at ~6 minutes, but all contained a compound(s) that eluted between 9.5 and 10.1
minutes. The ammonium hydroxide wash appeared interesting in particular because it
contained a compound eluting at 9.567 minutes, which closely matches the elution time
of the peak from the standard. However, most of these peaks were off-scale and showed
peak shapes indicative of column overloading so retention times of those peaks are
uncertain. Additionally, it could not be determined if the off-scale peaks represented one
compound or more. Re-injections could have been made, but the chromatograms of the
mixed-mode were not the main focus since this method was not optimized. The
quantification using the derivatization procedure was used in the small-scale ionexchange separation, hence, it was also desired to use this method in the large scale ionexchange separation amino sugar quantification to compare values easily.
Samples were analyzed using the amino sugar derivatization assay to assess in
which fraction the amino sugar was the most concentrated. As shown in Table 7 in the
bioassay result section, the amino sugar was retained more on this flash column run than
in the small-scale run.
61
Figure 17: The chromatograms of the scaled-up ion-exchange fractions compared to
the amino sugar standard using mixed-mode HPLC using the ELSD detector.
Signals over 1200 mV exceed the maximum input in the Agilent analog to digital
convertor.
62
3.4.1 Xcv, CA, and BDM Activity
In order to monitor the bioactivity that these column fractions contained, samples
were tested for bioactivity against Xcv and CA in MIC (Table 7), and against BDM in
planta (Figure 19). The sodium chloride wash was the fraction with the most activity
against Xcv, and the flow-through also had some activity. The flow-through was the only
fraction with activity against CA. The sodium chloride wash was the only fraction that
showed activity against BDM (albeit slight). It is possible that the compound(s)
responsible for activity against Xcv and BDM was/were still partly retained on the resin
inside the column and would need more quantities of eluent to remove it/them.
The results of the scaled-up column did not mirror those of the small-scale ionexchange column in that the ammonium hydroxide wash did not contain the highest
amino sugar content and activity against BDM and Xcv. Instead, the sodium chloride
wash contained the most amino sugar concentration and activity against BDM and Xcv.
The CA activity somewhat mirrored the results seen with the small-scale ion-exchange
slightly more, in that the flow-through showed activity, and this activity was distinct and
separate from the sodium chloride wash, where the majority of the amino sugar eluted.
However, where there had been CA activity in the water wash from the small-scale
column, there was none in the large-scale process.
63
Permeate (feed)
Flow-through
Water wash
Ammonium Hydroxide Wash
Sodium Chloride Wash
Quadris
UTC
MIC (Xcv)
MIC (CA)
Amino Sugar
Concentration
(mg/mL)
9±0
2±0
no activity
no activity
6±0
not tested
not tested
1±0
2±0
no activity
no activity
no activity
not tested
not tested
67.3
2.7
2.7
2.4
18.5
n/a
n/a
Table 7: Bioactivity of large-scale ion-exchange fractions in MIC against Xcv and
CA and corresponding amino sugar concentration from the derivatization HPLC
assay.
Average % Disease Control
100
90
80
70
60
50
40
30
20
10
0
20%
10%
Permeate
5%
20%
10%
Flow-through
5%
20%
10%
Water Wash
5%
20%
10%
5%
Ammonium Hydroxide
Wash
20%
10%
5%
Sodium Chloride Wash
Quadris
UTC
Figure 18: Efficacy of the large-scale ion-exchange fractions against BDM.
In addition to the MIC bioassay, these samples were assayed against Xcv using an
agar diffusion test. Activities more or less mirrored what was seen in the MIC assay,
with the feed, flow-through and sodium chloride wash all having activities, as can be seen
in Figure 20. An interesting observation was noted with the agar diffusion test, however,
in that the types of activities seen on the plates differed among the fractions. In the feed,
64
two distinct growth inhibition zones could be seen: a large hazy zone of growth, where
the organism was inhibited but not killed, and a smaller clear zone within the hazy zone,
where the organism had been killed. In the flow-through fraction, only the small clear
zone was present, and in the sodium chloride wash, only the large hazy zone was present.
Illustrations of all zones can be seen in Figure 20. This seemed to suggest that two
different activities, and possibly compounds, were responsible for the activity seen
against Xcv, since both types of activity were present in the source material, and, in the
separated fractions, the two different activity types were separated by their apparent
interaction with the ion-exchange resin: one did not adhere and flowed through, and the
other eluted with the sodium chloride wash.
65
Figure 19: The agar diffusion activity of the large-scale ion-exchange fractions
against Xcv. The two top plates show the zones of inhibitions as they could be seen
by holding the plate up to light and the bottom two plates is this same exact image
illustrating where the zones are located by using red circles to outline them.
3.5 Size-exclusion Chromatography
In order to further purify the active fraction against BDM and Xcv, the ammonium
hydroxide wash from the small-scale ion-exchange column was separated using a size-
66
exclusion resin. Fifty-eight total fractions were collected, each consisting of 8.5 mL. To
the naked eye, two bands of yellow to brown fractions appeared — in fractions 7-15 and
18-24. Fractions from the size-exclusion column were monitored using ninhydrin spray
in order to determine which fractions might contain compound(s) with amino groups.
Fractions 9-18 showed color after ninhydrin spraying, with 13 (purple) and 14 (brown)
being the most intense, as can be seen in Figure 21, which indicated that fraction 13
contained compound(s) with primary amine groups and fraction 14 contained
compound(s) with primary and secondary amine groups (due to the brown color mostly
likely cause by the combination of purple and orange/yellow staining).
Figure 20: Fractions 13 and 14 from the size-exclusion column that stained positive
for amino sugars with ninhydrin staining.
Based on general chemical characteristics, these fractions were combined into
smaller, more manageable groups for bioassay and further chemical elucidation. The NP-
67
screen HPLC method was used to evaluate similarities of the UV traces, and fractions
were pooled together based on similarity among neighboring fractions. For example,
fractions 6 and 7 were included in the same group, because compounds with similar
retention times appeared in both, as can be seen in Figure 22.
Figure 21: Example of fractions from the size-exclusion column separation that
were combined because they gave similar UV traces in the NP-screen at 210nm.
Fraction 13 appeared to have a lot of material at the very beginning of the
separation indicating little retention on the column, as can be seen in Figure 23. Since it
also stained purple by ninhydrin spraying, and the amino sugar was not detected by UV, a
side analysis was performed comparing the ELSD traces of fraction 13 and the amino
sugar standard in the NP-screen HPLC method. After drying and re-suspending in 1 mL
water, fraction 13 was diluted 100 times in water and run through the NP screen and
compared to the 1.0 mg/mL amino sugar standard (Figure 24). The amino sugar standard
yielded four major peaks by ELSD: two prominent peaks at 1.524 minutes (with a
shoulder on the forward edge of the peak) and 1.653 minutes, and two smaller peaks at
68
1.825 and 2.106 minutes. Fraction 13 yielded one major peak at 1.631 minutes with a
shoulder on the forward edge of the peak. Since the chromatogram of fraction 13 had a
peak that corresponded to a peak in the chromatogram of the standard, it was possible
that the two samples contained the same compound, and it was possible that this
compound could be the amino sugar. However, to make certain of this, a purer standard
should be used. Judging by the number of peaks present in the amino sugar standard, the
need for a more pure standard was clear, especially since the sample injected before this
sample showed no peaks eluting at the same retention time as the hypothesized
contaminants (peaks other than 1.6 minutes). Interestingly, fraction 13 appeared more
pure than the standard by this method of analysis (Figure 24).
Figure 22: The chromatogram of fraction 13 from the size-exclusion column with
the NP-screen HPLC method by UV at 210 nm.
69
Figure 23: The chromatograms from fraction 13 and the amino sugar standard
using the NP-screen HPLC method with ELSD detection.
3.5.1 Xcv and BDM Activity
Size-exclusion chromatography fractions 1-7, 8-12, 13, 14-15, 16-19, 20-21, 2230, 31-39, and 40-58 were pooled together, based on similarity by HPLC, to make 9
different fractions of 2.2, 20.4, 34.9, 167.9, 3.8, 3.3, 3.2, 1.1, and 2.6mg, once dried,
respectively. The nine new pooled fractions (1A through 9A) were tested in MIC for Xcv
activity and against BDM. For both Xcv and BDM, the majority of the activity resided in
combined fractions 3A and 4A (Table 8 and Figure 25, respectively). This was
70
interesting because these combined fractions contained the original size-exclusion
fractions, 13 and 14, respectively, where the ninhydrin staining was the most intense and
the mass highest. As stated above, based on in the NP-screen ELSD results, fraction 13
contained compounds that corresponded to a similar retention time than the amino sugar
standard.
Fraction
1A
2A
3A
4A
5A
6A
7A
8A
9A
Feed
MIC (Xcv)
no activity
0.5± 0.7
3.5 ± 0.7
8.5 ± 0.7
no activity
no activity
0±0
0±0
0±0
6±0
Table 8: Efficacy against Xcv of the nine combined fractions from the size-exclusion
separation.
71
100
Average % Disease Control
90
80
70
60
50
40
30
20
10
1A
2A
3A
4A
5A
6A
7A
8A
9A
20%
100%
20%
100%
20%
100%
20%
100%
20%
100%
20%
100%
20%
100%
20%
100%
20%
100%
20%
100%
0
Retain
Figure 24: Efficacy of the nine combined fractions from the size-exclusion
separation against BDM.
Fractions 14 and 15 were combined to make combined fraction 4A, and combined
fraction 4A showed the highest activity against BDM. Neither fraction 14 nor fraction 15
showed any compounds by UV at 210 nm in the NP screen (Figure 26). It is noted that
the amino sugar cannot be detected by UV, therefore this would be expected with the NP
screen.
72
Figure 25: The chromatograms of fractions 14 and 15 by UV at 210 nm.
Since combined size-exclusion fractions 3A and 4A demonstrated the highest
activity against Xcv and BDM, and these bioactivities so far had correlated to amino sugar
concentration in the cation-exchange separation, further chemical characterization was
performed to determine if the amino sugar was present.
3.5.2 Mixed-Mode HPLC of Size Exclusions Fractions
To avoid a derivatization step and preserve material, a mixed-mode HPLC
method was attempted for fraction analysis first. Samples (15 μL) were injected onto a
Scherzo column. This method was not optimized and, therefore, was only used as an
indicator of amino sugar presence and to estimate the purity of each fraction. As detected
by ELSD, two peaks appeared in the chromatogram of the amino sugar standard using
this method: one between 6.831 and 7.789 minutes and another between 8.427 and 8.985
minutes, as can be seen in Figure 27.
73
It seemed that with this method, retention time was not always consistent, since
three different injections of the same amino sugar standard eluted at different times (two
at 8.9 minutes and one at 8.4 minutes), so development would need to be done on this
method to improve its consistency and precision. It is possible that the column needed to
equilibrate for a longer period of time in the mobile phase, since the first injection (the
amino sugar standard at 1.0 mg/mL) resulted in a retention time that was different from
that of the other two amino sugar samples.
In the chromatogram for combined fraction 3A (Figure 27), three peaks appeared,
at 5.757, 6.957, and 8.937 minutes. Since this fraction contained a compound that eluted
at a similar retention time to the second and third injections of the amino sugar standards
(at 8.9 minutes), it seemed to indicate that this fraction could possibly contain the amino
sugar.
In the chromatogram for fraction 4A (Figure 27), one large, off-scale, peak
appeared at 9.076 minutes, which is very close to the retention time of the amino sugar.
A dilution of combined fraction 4A would most likely make the peak appear within the
limits of detection and may have helped to resolve it better.
74
Figure 26: The chromatograms of combined fractions 3A and 4A and the amino
sugar standard at differing concentrations using the mixed-mode chromatography
method using ELSD detection.
A dilution series of the amino sugar standard was run using this method and the
linearity of the peak areas by ELSD detection in the region between 8.427 and 9.076
75
minutes were plotted vs. the amino sugar concentration. The response of amino sugar
Peak Area (mAU*s)
concentration to corresponding peak area appeared to be mostly linear (Figure 28).
y = 3607x - 13.68
R² = 0.9832
4000
3500
3000
2500
2000
1500
1000
500
0
0
0.2
0.4
0.6
0.8
1
1.2
Amino Sugar Standard Concentration (mg/mL)
Figure 27: Standard curve of the amino sugar standard injected at different
concentrations using the mixed mode chromatography method with ELSD
detection.
Since the common area for peaks to appear for the amino sugar standard and
combined fraction 3A resided in the region between 8.427 and 9.076 minutes, the
concentration of combined fraction 3A was estimated based on the amino sugar standard
curve. Peak area for combined fraction 3A at 8.937 minutes was 10872 mAu*s, which
corresponded to a relative concentration of 3.02 mg/mL. The concentration could not be
estimated for combined fraction 4A due to the peak being off-scale. Since this method is
neither optimized nor quantitative, and ELSD is not an appropriate method to use for
actual concentration (ELSD should only be used for comparison between standards) the
concentration for combined fraction 3A was taken only as a rough estimate based on the
76
amino sugar standard run alongside it. Judging by the lack of other peaks present, these
fractions appeared to be fairly pure.
3.5.3 Derivatization of Size Exclusions Fractions
The derivatization HPLC assay was also run on combined fractions 3A and 4A to
see if the amino sugar was present and, if so, to estimate the concentration by comparing
to the standard. For this assay, both fractions were freeze-dried and weighed out as
normal for the HPLC assay. As can be seen in Figures 29 A through E, for combined
fraction 3A, the peak area corresponding to the derivatized amino sugar (at 6.6-6.7
minutes) was 5325.3 mAu*s, while, for combined fraction 4A, the peak area
corresponding to the derivatized amino sugar was 9136.5 mAu*s. This corresponds to
amino sugar concentrations of 688.6 mg/g and 2266.5 mg/g for those fractions,
respectively. Since the upper limit of the linear calibration range for the amino sugar
standard was 7278.6 mAu*s, the amount of amino sugar present in combined fraction 4A
could only be estimated as a minimum. The concentrations of samples are calculated
based on known standard concentrations, where the standard purity is set at 100%, and
combined fraction 4A by this calculation was 227% pure by mass compared to the
standard. This was an indication that the standard used for comparison was less pure than
combined fraction 4A, and the separation technique used to get combined fraction 4 was
superior to the separation technique used to produce the standard.
77
Figure 28A
78
Figure 29B
79
Figure 30C
80
Figure 31D
81
Figure 32E
Figures 33 A through E: The chromatograms of combined fractions 3A and 4A
alongside the amino sugar standard using the derivatization assay at 260 nm.
At this point in the project, it was discovered that the amino sugar could be
purchased with a purity of 98%, so a follow-up derivatization HPLC assay was
performed on the column fractions along with the purer “new amino sugar standard” for
comparison. All fractions were adjusted to 1.0 mg/mL with water, as was the new amino
82
sugar standard, and calibration curves were performed on all samples by derivatizing 100
μL, 50 μL, and 25 μL of sample. Chromatograms of the 100 μL amounts of each sample
can be seen in Figure 30, where the amino sugar peak appears at 6.3 minutes, and
calibration curves comparing the peak areas of the three derivatized amounts of each
sample can be seen in Figure 31.
83
Figure 34: The chromatograms of combined fractions 3A and 4A and the new
amino sugar standard, all tested at sample concentrations of 100 μl of 1.0 mg/mL
solutions using the derivatization assay read at 260nm.
84
Figure 35: Calibration curves for each of the samples when 100 μL, 50 μL, and 25
μL of 1.0 mg/mL concentrations were derivatized and injected.
85
When comparing the 6.3 minute peak areas of the 100 μL injection amounts of
each fraction to the 98% pure new amino sugar standard across the different
concentrations of samples that were derivatized (Figure 30), it was evident that combined
fraction 4A contained the majority of the amino sugar whereas combined fraction 3A
contained much less. In terms of purity, when these peak areas were compared to the
new amino sugar standard, it was found that combined fraction 4A was ~87% pure and
that combined fraction 3A was ~7% pure.
At this point, combined fraction 4A and the purchased amino sugar were sent for
NMR analysis to an offsite facility (Acorn NMR). There was no NMR facility in the
laboratory where this work was performed, and proprietary issues precluded transfer of
the samples to California State University, Sacramento, for NMR analysis. Proprietary
issues also prevent the publication of NMR spectra of the amino sugar or fraction likely
to contain the amino sugar in this thesis. The NMR spectrum of combined fraction 4A
looked clean and had little background noise, which suggested that the fraction was
relatively pure, as the HPLC data had demonstrated. Most of the proton signals appeared
in the region between 3 ppm and 4 ppm, which suggested that the molecule was a
glycoside. The NMR spectrum of combined fraction 4A was compared to the NMR
spectrum of the purchased amino sugar, and the two appeared very similar to one another,
so it was determined that the isolated compound in combined fraction 4A was likely to be
the same compound as the purchased amino sugar. NMR analysis was also done at the
same time with an analogue of the amino sugar, and it was noted that the position of the
86
amino group in the compound in combined fraction 4A and the purchased amino sugar
differed from that in the analogue.
3.6 Isolation of Amino Sugar by Solid-phase Extraction
Another method of isolating the amino sugar was attempted using a catch-and
release-style elution technique with solid-phase extraction (SPE) cartridges with tosic
acid as the solid phase (“catch”), methanol as the wash, and 2 M ammonium hydroxide in
methanol as the elution solvent (“release”). Four samples - permeate (feed), flowthrough, wash, and release - were compared to one another using the amino sugar
derivatization quantification assay to judge how well the solid phase adsorbed the amino
sugar and how well the release eluted it. Most of the material did not adhere to the solid
phase and eluted in the flow-through. The wash step continued to elute material that did
not adhere to the resin, and so little remained to elute in the release step. It is possible
that by trying another type of SPE cartridge with a different solid phase that this
technique could possibly work, but more experimentation would need to be performed.
The results of this assay can be seen in Figure 32 as a comparison of peak area.
87
16000
14000
12000
mAu*s
10000
8000
6000
4000
2000
0
Feed
Flow-through
Wash
Release
Figure 36: Amount of amino sugar found in the different steps of the SPE cartridge
steps compared to the starting permeate material using the derivatization HPLC
assay to quantify amino sugar concentration in each.
3.7 Colorimetric Assay
In order to try another method for amino sugar quantification, a colorimetric assay
was employed based on the Elson-Morgan reaction. The experiment was repeated three
separate times with the same sample set, all within a week of one another. The samples
chosen were from the small-scale ion-exchange experiment.
Similar trends were seen amongst the different sample sets (run on different days)
in terms of relative amino sugar content being higher or lower among the different
fractions, but absolute values seemed to fluctuate greatly between the tests of the same
fractions on different days (Table 9). Upon investigation of the standard curve of WB
samples, the response of absorbance relative to WB dilution was not linear which
88
explained the inconsistent results. This method was not pursued further as a means of
amino sugar quantification.
Amino Sugar Concentration (mg/mL)
Day 1
Day 2
Day 3
Average Standard
Deviation
14.5
9.4
15.1
13
3.1
Flow-through
17.3
13.5
20.7
17.2
3.6
52.8
22.3
51.8
42.3
17.3
20.2
14.1
21.3
18.5
3.9
59.7
47.1
56.2
54.3
6.5
60.9
60.9
60.9
60.9
0
0
0
0
0
0
Water Wash
Ammonium Hydroxide Wash
Sodium Chloride Wash
Permeate
WB Standard
H2O
Table 9: Concentration of the amino sugar as determined by the colorimetric assay
of three replicates of fractions from the small-scale ion-exchange column.
3.8 Probing Solubility of Flow-through
Since characterization remained to be done on the CA-active fraction, the
solubility of the antifungal fraction was tested in order to see its compatibility with
various solvents. In water, full dissolution was seen, but in chloroform, slight
particulates were seen, and in ethyl acetate, methanol and acetonitrile, particles,
opaqueness and material adhering to the glass were noticed, so it seemed the fraction was
only soluble in water.
89
3.9 Thin-layer Chromatography on CA-Active Fraction
In order to further separate and elucidate what type of compound could be
responsible for the antifungal CA activity, a series of thin-layer chromatography (TLC)
experiments were performed on the water wash sample from the small-scale ionexchange experiment.
It was first determined what type of separation would be seen with a cellulose
plate using the solvent system of the top layer of 4:1:4 n-BuOH:HOAc:H2O mixture. For
this test, both the flow-through and the water wash from the small-scale ion-exchange
separation were tested on an analytical cellulose plate, since both had activity against CA.
The old amino sugar standard (1.0 mg/mL) was tested alongside these for comparison.
After developing the plate, allowing it to dry, spraying with ninhydrin spray, and
applying heat, as described in sections 2.9 and 2.13, distinct zones could be seen in all
fractions tested (Figure 33). Even though ninhydrin spray was meant for staining amino
sugars, and it was known that the CA activity was not due to the amino sugar that had
BDM and Xcv activity, it was still used since amino sugars were known to still be present
in some quantity in this sample (albeit low). It appeared that the flow-through and water
wash had similar separation patterns, except that the flow-through had a longer, more
spread out orange zone at the bottom of the plate. This was most likely due to the large
amount of secondary amino groups present in the flow-through with different retention
times than those in the water wash fraction and/or could be due to over-loading of the
TLC plate. When the flow-through was tested in the derivatization assay previously, the
90
amino sugar that had BDM and Xcv activity was still present in low amounts, therefore it
was also possible that other compounds with amine groups were present. There were two
distinct areas that stained purple in each fraction, one more concentrated in the center of
the plate, and one more spread out towards the top of the plate, so primary amines were
present. Since individual bands were not present in the purple and large orange zones
that were smeared, further work needed to be done with thicker plates so as to not
overload the TLC plate material. The amino sugar standard also stained orange and
eluted in a similar area as the other orange-stained regions in the other fractions did. It
was interesting to note that with the standard, no purple stains were seen as would be
expected for a primary amino sugar, as it was known to be, so a higher concentration
would need to be loaded of the amino sugar in order for it to appear after staining. The
orange/yellow color in the standard indicated a contaminant with a secondary amine was
present. Since both fractions showed ability to be retained on the TLC plate and to be
carried with the solvent system and therefore developed, it was decided to move forward
with this system for separation of the water wash fraction, with further work focusing on
optimizing the plate material and loading volume.
91
Figure 37: The TLC plate of the amino sugar standard, and the flow-through and
water wash from the small-scale ion-exchange column.
Based on the results of this test, it was decided to separate these fractions on a
preparatory TLC plate in order to obtain enough material for bioassay testing and to
further purify the fraction responsible for CA activity. In order to first ensure that good
separation would be seen with the water wash fraction on a cellulose TLC preparatory
plate, different amounts of the fraction were applied to the plate. Following separation,
the plate was allowed to dry, it was sprayed with ninhydrin spray, and heat was applied.
Distinct zones were seen on the preparatory plate, but they were different than those seen
on the analytical plate in all amounts tested, as can be seen in Figure 34. Separation on
the preparatory plate appeared better than on the analytical plate. This seemed like a step
forward for testing different zones with bioassays.
92
Figure 38: Development of different concentrations of the water wash fraction after
ninhydrin processing on a preparatory cellulose TLC plate.
Further testing was done to ensure that the sample could be easily recovered from
the plate with water as the solvent. Two hundred and fifty μL of the water wash fraction
was applied to the TLC plate, it was developed, allowed to dry, and the entire plate was
scraped with a razor blade into a round-bottom flask with 100 mL of water and agitated.
The liquid was separated from the solid by filtering through an empty SPE cartridge, and
the filtrate was dried in a rotary evaporator and re-suspended in 1000 μL of water. To
determine if there were any compounds of differing polarity, washes of the solid phase
with 100 mL of acetonitrile and methanol were performed in the same manner as with the
water. These washes were also dried using a rotary evaporator and re-suspended in 1000
μL of water. As a control, a corresponding sample for the feed for the plate (the water
wash) was prepared by adding 250 μL of the sample to 750 μL of water. All four
fractions were analyzed using the NP screen, as can be seen in Figure 35.
93
Figure 39: Chromatograms generated from the NP screen HPLC method to
determine the best solvent with which to extract the separated fraction from the
preparatory TLC plate.
From the NP-screen HPLC results (Figure 35), it was evident that this method
was not appropriate for separation of the material, as most of the material appeared to be
eluting in the void volume and not retaining on the column. Regardless, it allowed a
qualitative analysis of which solvents allowed for better solubility of the compounds from
the TLC pate. As can be seen from the above figure, most of the material eluted from the
plate in the water wash, and very little material came off with the subsequent washes, so
94
more confidence was gained knowing that water could be used to extract the material
from the cellulose of the TLC plate once developed with the water wash fraction.
However, a small amount of material was recovered via the methanol wash; these could
have been impurities in the TLC plate itself, since none of this material was seen in the
original fraction.
Since the preparatory TLC plate worked well for the separation of compounds in
the water wash fraction from the small-scale ion-exchange column, it was used to move
forward with purification of the CA active fraction. A 1.4 mL aliquot of the fraction was
applied to a plate, the plate was developed, allowed to dry, and a one-inch section on the
side of the plate was sprayed with ninhydrin and heated. This allowed the visualization
of zones to excise, extract with water, and test for bioassay without ninhydrin
contamination (Figure 36). Ten zones were excised, extracted with 20 mL of water each
(except for zone ten, which was extracted with 40 mL since it had more material),
filtered, dried, and then re-suspended in 1.4 mL, so that each fraction could be tested at
the concentration it would be found in the original water wash fraction. Fractions 1
through 10 consisted of 0.3, 0, 0.3, 0, 1.9, 1.5, 0.8, 0, 0.5, and 2 mg, respectively, once
dried, although with the method for determining mass (± 0.1 mg), these values were
uncertain for fractions less than 0.5 mg. Fractions 1-5 showed the most evidence for
presence of amino sugars due to the purple ninhydrin staining of those regions.
95
A
B
Figure 40 A and B: A visual plate map of the ten different fractions that were
excised from the TLC preparatory plate.
These fractions were tested for activity against CA using the germ tube assay, in
which CA spores were directly challenged with fractions in liquid and the germination (or
lack thereof) was monitored and compared to untreated controls (Figure 37). Out of the
ten zones, Zone 1 and Zone 10 appeared to have the most bioactivity on an absolute
basis, inhibiting the germination of the fungal spores the most whereas the other bands
showed similar activity to the water (negative) control. The water control spores had
germinated fully; indicating that good growth should be seen in the absence of inhibition.
In the WB (positive) control, complete inhibition of spore germination was seen,
indicating that some part of the WB was active. Zones 1 and 10 looked the most similar
to the WB control, out of all of the zones tested.
96
Figure 41: Zones 1 and 10 compared to the negative (water) and positive (WB)
controls for activity against CA
These fractions were injected into the mixed-mode column in order to see if they
contained the amino sugar standard. Since these fractions and the source feed fraction
had activity against CA, and this activity did not correlate with amino sugar
concentration/presence, it was expected the fractions would not contain the amino sugar.
Five μL of Zone 1, Zone 10, and the amino sugar standard were injected separately, then
each zone was mixed one to one with the amino sugar standard and 5 μL were injected.
These chromatograms can be seen in Figure 38. In the amino sugar standard, two peaks
appeared: one at 6.169 minutes and the second peak at 9.603 minutes. With Zone 1, one
peak appeared at 9.965 minutes and in Zone 10, one peak appeared at 9.630 minutes.
When Zone 1 and the amino sugar standard were mixed together, a broad peak with a
97
shoulder on the right appeared at 9.897 minutes, and when Zone 10 and the amino sugar
standard were mixed, two distinct peaks could be seen: one at 9.597 minutes and another
at 9.912 minutes. Since, when each zone was mixed together with the amino sugar, the
peak morphology(ies) changed from those of the amino sugar alone, or the zones alone, it
was clear that these compounds were different from the amino sugar. It can also be
concluded that the compound in Zone 1 is not the same compound as the one in Zone 10.
98
Figure 38: HPLC chromatograms using the mixed-mode method with ELSD
comparing the old amino sugar standard, Zone 1 and Zone 10 separately and Zones
1 and 10 mixed 1:1 with the amino sugar standard.
99
Chapter 4
CONCLUSIONS
In this study, a non-cell associated, water-soluble amino sugar compound that had
activity against BDM and Xcv was isolated from the permeate of bacterial whole broth
(WB) from strain QST 2808, a Bacillus pumilus. This separation was achieved with
high-pressure separation of the permeate from cells and ion-exchange and size-exclusion
chromatographies, and was confirmed by NMR. In this permeate, distinct and separate
bioactivities, other than this amino sugar, were also observed in different fractions,
indicative of other bioactive compounds towards CA and Xcv. This indication of other
bioactive compounds could be a good starting point to expand the value and discovery of
other active secondary metabolites produced by this bacterium for use in agriculture.
A scaled-up version of the ion-exchange chromatography was attempted for the
isolation of the amino sugar to determine the reproducibility and possibility of isolating
the amino sugar on a larger scale. It was determined that the large-scale isolation differed
significantly from the small-scale and, thus, would need further work.
In order to investigate other methods of isolating the amino sugar, an SPE
cartridge technique was attempted using a “catch and release” style set-up with tosic acid
as the solid phase. Most of the amino sugar did not adsorb to the resin, so other solid
phases would need to be investigated to determine if a method such as this would be
feasible.
100
Two methods, mixed-mode chromatography and a colorimetric assay, were
investigated for quantification of the amino sugar to try to steer away from the timeconsuming nature of the current derivatization assay. The colorimetric assay was not
reproducible enough to rely on. The mixed-mode chromatography method using ELSD
looked more promising, since a linear response could be seen, but the accuracy of peak
elution time was a concern, highlighting the need for method improvement through
mobile phase equilibration and possibly solvent system experimentation.
Overall, more was learned about the active components of the WB of this
bacterium, in terms of the specific bioactivity that different compounds had against
important crop diseases like Brassica downy mildew, bacterial spot of tomato, and
cucumber anthracnose. Other chemical methods for analysis and isolation were
attempted and improved upon in order to be able to study it more comprehensively and
easily in the future.
Natural product chemistry produced by microbes can prove to be just as effective
as synthetic compounds, especially considering many synthetic compounds were
originally discovered from biological sources. Assuming that an average leaf in an
agricultural field is 50 cm2, and the average volume of liquid applied to a leaf is 1
μL/cm2, a leaf would hypothetically receive 50 μL of a treatment. WB is essentially the
concentrated product before dilution for agricultural field application. Considering that
the amino sugar is present in WB at levels of ~60 mg/mL, and assuming an average
concentration of WB used in the field is 5%, that leaf would be receiving 150 μg of active
metabolite. Comparing that to a synthetic compound such as Quadris, whose active
101
compound azoxystrobin can be found in a concentrated liquid product at ~250 mg/mL,
one can see how the effective quantities of biologically produced secondary metabolites
are within the same range as that of a synthetic product. The activity of each compound
will vary depending on pest target and concentration needed for efficacy against that
target, however one can see how on a concentration/leaf basis, if a strong enough
biologically-produced compound is discovered and produced at levels in the WB
equivalent to that of a synthetic compound found in its concentrated product, the
practicality of using WB in a real-world scenario is very likely and competitive.
This study adds to many other examples in the literature of the importance
Bacillus organisms can play in the production of industrially important secondary
metabolites that can be immediately applied to disciplines such as agriculture for the
establishment of sustainable farming practices for the future.
102
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