!!   NOVEL PHYTOANTICIPINS FROM 
PRUNUS MAACKII
: POSSIBLE FACTORS IN 
DISEASE RESISTANCE
  By  Linzi Jo Kaniszewski
                           A DISSERTATION
  Submitted to
 Michigan State University
 in partial fulfillment of the requirements
 for the degree of
  Plant Pathology 
Ð Doctor of Philosophy
  2015!ABSTRACT
  NOVEL PHYTOANTICIPINS FROM 
PRUNUS MAACKII
: POSSIBLE FACTORS IN 
DISEASE RESISTANCE
  By  Linzi Jo 
Kaniszewski
  Armillaria ostoyae
, the cause of Armillaria root rot, is a major problem in cherry 
orchards in the northwest region of Michigan. One mechanism of resistance to 
pathogen invasion is the production of antifungal compounds. 
Prunus maackii
 was the 
only Prunus
 species
 tested
 that contained significan
t antifungal activity based on Thin 
Layer C
hromatography bioassays.
 The purpose of this research was
 aimed at understanding the nature of resistance/defense found in 
P. maackii
 to Armillaria root rot. 
A series of additional bioassays were performed to examine the range of antimicrobial 
activity and the potency of the secondary metabolites contained in the periderm of 
P. 
maackii.
  Periderm
-incorporated media was used to screen fo
r growth inhibition of 
Armillaria
 species along with a group of non
-host pathogens. 
Prunus maackii
 periderm 
amended-media was fungistatic at very low concentrations and also displayed fungitoxic 
properties. X
-ray crystallography was used to determine the s
tructures of two antifungal 
components of 
P. maackii
 periderm as 3,5,7
-trihydroxy
-6 methoxyflavone and 3,5,7-trihydroxy
-6-methoxyflavanone.  This is the first report of crystallization and 
antimicrobial activity for both compounds. 
!"""!           To my 
parents Joseph P. Kaniszewski and Kathi J. Kaniszewski;
 There are no words that I could ever write that would hold a light to your
 brilliance, wisdom and values.
             !"#!ACKNOWLEDGEMENTS
   I could effortlessly fill countless pages expressing my 
appreciation
 to all of the wonderful people of the Plant, Soil and Microbial Sciences Department at Michigan 
State University. I am grateful beyond words to my mentor and dear friend Dr. Raymond 
Hammerschmidt for each and every opportunity and for simply b
elieving in me.  Thank 
you for investing your time and efforts into shaping my skillset and for allowing me to 
learn from your expertise, which is truly an understatement when referring to the 
physiology and biochemistry of resistance
 and disease
. I will continue to think of you 
each time I draw out an aromatic compound, forever motivating me to do all that I can 
with your investment. 
 Thank you to my committee mem
bers for their time and direction
.  To Dr. Amy 
Iezzoni, I am 
thankful
 for the opportunity to l
earn from your plant breeding research and 
for the time that youÕve taken throughout the years to help me gain experience in your 
field. A special thank you to Dr. Frank Telewski for all of your insightful discussions
, for 
the extra time that youÕve taken 
to help me achieve my goals and
 for your 
encouragement to pursue my dreams
. Thank you to Dr. Richard Staples for your 
diligent efforts towards the identification of unknown samples
, for teaching me 
new crystallization techniques and for the extra time that you have spent helping me to better 
understand critical concepts in x
-ray crystallography. 
 I would like to express my most sincere gratitude and appreciation to my parents 
and sisters. To my mom, 
Kathi Jo (Russell), for your remarkable editing skills, for your 
constant push to achieve more and for your help overcoming obstacles of any kind and 
to my dad, Joseph Peter Kaniszewski, for always brainstorming with me 
- sharing your 
!#!creative and unique i
deas, for imparting your self
-taught organic gardening knowledge, 
and for your help collecting new samples for future 
P. maackii
 research. You have both 
made me who I am today 
Ð thank you for everything. 
 I would like to sincerely thank my sister Brynne fo
r providing a place to live when 
first starting at MSU 
Ð I will remain
 forever 
grateful 
for your constant support, love and 
patience. 
To my
 sister Brooke, for 
always 
lifting my spir
its and for the 
numerous 
lunches 
and care packages, making each day
 brighte
r and
 so much 
lovelier. Thank you to my 
brother
-in-law Joel Buckley, my nephews and niece, Braden, Collin and Quinn for the 
immense joy that you have all brought to my life. A very special thanks to my 
grandparents, Phyllis and Howard Russell for infusing 
agriculture into my background 
with your farm in Fairgrove, MI and to Francis and Walter Kaniszewski for inspiring my 
curiosity in biology with every camping trip made to Grand Marais, MI. Thank you to my 
Aunt Lydia Maurer, for all of your 
uplifting words 
that helped me
 push through the 
subjects that I struggled 
with most
 and for helping me find my purpose in life as a 
teacher. To Leonard Russell, for taking such great care of the Russell farm and for his 
discussions on plant disease.
 I am extremely gratefu
l to Dr. Chris Vandervoort for your
 generous donation of 
time towards characterizing unknown samples using HPLC
-MS and to Dr. Daniel Jones 
for additonal HPLC
-MS analyses, Dr. Daniel Holmes and Kermit Johnson for NMR 
analyses, Audrey Sebolt for teaching me 
new skills and to Dr. Tyre Proffer for your help 
fostering new experimental ideas. To Shelley Redman, Linda Colon, Lee Duynslager, 
and Jeff Nobis who
 have all helped make East Lansing feel like a second home. I am 
appreciative 
to all of the lab members tha
t have made this research project possible. I 
!#"!owe tremendous thanks to cur
rent lab members Chris Behrens, 
Celeste Dmytryszyn
 and Michael Staples
 for their friendship and support.
  To former lab members for the
ir contributions on this project including 
Cory
 Outwater, Emmie Bishop, Janette Jacobs
, Jordan Crandall
, Nayara Marangoni, and 
Ryan Dickinson
. Thank you to my fellow graduate students that have helped me 
throughout my academic journey 
Ð Helen Jiang, Nancy Dykema, Jane Jiang, Carolina 
Escobar, Saltanat 
Kurmanbekovna, Sandesh Dangi, and many others who will remain 
lifelong friends.
 Finally, thank you to my husband Ryan Taylor 
Ð You truly know how to make 
each day count, which is evident by your sincere, thoughtful and honest character. 
Thank you for the w
eekends spent on campus, for helping me review test information 
and for always thinking of ways to make my life easier. 
 !                     !#""!TABLE
 OF CONTENTS
   LIST OF TABLES
 ............................................................................................................ ix  LIST OF FIGURES
 ........................................................................................................... x  LITERATURE REVIEW
 .................................................................................................... 1!ARMILLARIA ROOT ROT
 ............................................................................................. 1 Causal Agent.
 ............................................................................................................... 1 Signs and Symptoms.
 ................................................................................................... 2 Dissemination & Spread of Pathogen.
 .......................................................................... 4 Virulence & Pathogenicity.
 ............................................................................................ 6 Management.
 ................................................................................................................ 8 Resistance.
 ................................................................................................................. 10 LITERATURE CITED
 .................................................................................................. 11 LITERATURE REVIEW
 .................................................................................................. 16!ROLE OF SECONDARY METABOLITES IN PLANT DEFENSE
 ............................... 16 Primary Metabolism.
 ................................................................................................... 16 Secondary Metabolism.
 .............................................................................................. 16 Flavonoids.
 ................................................................................................................. 18 Phenolic Compounds in the Genus 
Prunus
. ............................................................... 22 Plant Defense.
 ............................................................................................................ 23 Techniques for Phenolic Detection.
 ............................................................................ 25 LITERATURE CITED
 .................................................................................................. 27 CHAPTER ONE
 .............................................................................................................. 34!ANTIFUNGAL BIOCHEMICAL COMPARISON OF 
PRUNUS
 SPECIES
 ....................... 34!ABSTRACT
 ................................................................................................................. 34 Introduction
 ................................................................................................................. 35 Materials and Methods
 ............................................................................................... 38 Results
 ........................................................................................................................ 43 Discussion
 ................................................................................................................... 59 LITERATURE CITED
 .................................................................................................. 61 CHAPTER TWO
 ............................................................................................................. 66!IDENTIFICATION OF FLAVONOIDS FROM 
PRUNUS MAACKII
 TISSUES
 ................. 66!ABSTRACT
 ................................................................................................................. 66 Introduction
 ................................................................................................................. 67 Materials and Methods
 ............................................................................................... 70 Results
 ........................................................................................................................ 73 Discussion
 ................................................................................................................... 96 LITERATURE CITED
 ................................................................................................ 103  !#"""!   CHAPTER THREE
 ....................................................................................................... 107!THE ANTIMICROBIAL SPECTRUM OF PERIDERM TISSUE FROM 
PRUNUS 
MAACKII
 ....................................................................................................................... 107!ABSTRACT
 ............................................................................................................... 107 Introduction
 ............................................................................................................... 108 Materials and Methods
 ............................................................................................. 110 Results
 ...................................................................................................................... 115 Discussion
 ................................................................................................................. 137 APPENDIX
 ................................................................................................................ 140 LITERATURE CITED
 ................................................................................................ 156 CHAPTER FOUR
 ......................................................................................................... 159!PRUNUS MAACKII
 FUTURE RESEARCH
 .................................................................. 159!LITERATURE CITED
 ................................................................................................ 163                              !"$!LIST OF TABLES
   Table 1.1. Samples used to screen for 
Armillaria
 growth inhibition
 ................................ 39  Table 3.1. Armillaria
 species used to evaluate the antimicrobial activity of 
P. maackii
 periderm
 ....................................................................................................................... 112 !Table 3.2. Pathogens of non
-Prunus
 species used to evaluate antimicrobial activity of 
P. 
maackii
 periderm
 .......................................................................................................... 114 !Table 3.3. Dose
-response of 
P. maackii
 periderm
-amended media on the growth and 
viability of 
A. ostoyae
 (warren isolate).
 ......................................................................... 116                          !! !!$!LIST OF FIGURES
   Figure 1.1. Separation of 
P. maackii
, P. serotina
 and P. mahaleb
 samples visualized 
with UV (365 nm). Samples were developed on silica TLC plates using chloroform 
- methanol 9:1 (v/v) (top) and on cellulose TLC plates using water as the solvent 
(bottom).
 ......................................................................................................................... 44 !Figure 1.2. The periderm extracts from 
P. maackii, P. serotina 
and P. mahaleb
 were 
developed on cellulose TLC plates, in a water solvent and sprayed with 
C. cucumerinum
. Four microliters of acetone extract (0.20g/mL) was 
used and eight 
microliters of hot water extract (0.10g/mL) and autoclave extract (0.10g/mL) were 
applied to the TLC plate. ................................................................................................ 48 !Figure 1.3. The extracts from 
P. maa
ckii, P. serotina 
and P. mahaleb
 were applied to 
silica TLC plates, developed in chloroform 
Ð methanol (9:1 v/v) and sprayed with 
C. cucumerinum
. Four microliters of acetone extract (0.20g/mL), eight microliters of hot 
water extract (0.10g/mL) and autoclav
e extract (0.10g/mL) were applied to the TLC 
plate. ............................................................................................................................... 48 !Figure 1.4. UV detection (365 nm) and TLC bioassay results for the crude periderm 
extract of 
P. maackii 
(PM) and 
P. maackii
 x Clare hybrid seedling (PM x).  All samples 
were 0.20g/mL, 10 µL were applied to each lane. Silica TLC plates were used and 
developed in chloroform 
Ð methanol (9:1 v/v).
 ............................................................... 50 !Figure 1.5. UV detection (365 nm) and TLC bioassay of 
P. maackii
 periderm tissue (P), 
cambial tissue (C), periderm from roots (R), and wood (W). All samples were 0.20g/mL, 
10 µL were applied to each lane. Silica TLC plates w
ere used and developed in 
chloroform 
Ð methanol (9:1 v/v).
 ..................................................................................... 52 !Figure 1.6. Extracts from 
P. serotina, P. mahaleb
 and P. maackii 
were compared. 
Extracts were applied to a silica TLC plate (0.20 g/mL), developed in chloroform 
Ð methanol (9:1), sprayed with the DPN reagent (left), vanillin reagent (middle) and 
C. cucumerinum 
(right).
 ...................................................................................................... 54 !Figure 1.7. UV detection (365 nm) and vanillin reagent comparison of periderm tissue 
from mature and young 
P. maackii
 trees. Crude extracts were applied to silica TLC 
plates (0.20 g/mL) and were developed in chloroform
-methanol (9:1 v/v).
 .................... 55 !Figure 1.8. The growth of 
A. ostoyae
 on periderm
-amended media (10 g/L YMPG) of 
P. 
maackii 
(1), 
P. serotina
 (2) and 
P. mahaleb
 (3). The control growth is shown on YMPG 
nutrient
-medium (4). C
ultures were grown for 3 wk.
 ...................................................... 57 !Figure 1.9. Survey of 
Prunus 
species and rootstocks (10 g periderm tissue/L YMPG) for 
their ability to inhibit the growth of 
A. 
ostoyae
. Samples were grown for 3 wk.
 .............. 57 !!$"!Figure 1.10. Survey of 
Prunus
 species and rootstocks (17 g/L YMPG) for their ability to 
inhibit the growth of 
A. ostoyae
. Samples were grown for 2 wk.
 .................................... 58  Figure 2.1. HPLC
-MS analysis of periderm extract from older tissues collected from a 
mature 
P. maackii
 tree. Potential names were assigned based on retention times and 
masses.
 .......................................................................................................................... 74 !Figure 2.2. HPLC
-MS comparison of crude periderm extract; samples were collected 
from a young 
P. maackii 
tree (top) and older tissues were collected from a mature tree 
(bottom).
 ......................................................................................................................... 75 !Figure 2.3. MS/MS of unknown compound with 301 m/z from periderm tissue of 
P. 
maackii
. .......................................................................................................................... 76 !Figure 2.4. The UV spectrum of the unknown compound with 301 m/z from the periderm 
tissue of 
P. maackii
. ....................................................................................................... 77 !Figure 2.5. Modified bioassay of isolated fractions fr
om the periderm of 
P. maackii
. .... 79 !Figure 2.6. UV detection (365 nm) used to determine the purity of three biologically 
active fractions 2, 3 and 4 collected from crude 
P. maackii
 periderm extract.
 ............... 80 !Figure 2.7. Single crystal used for x
-ray crystallography analysis and the corresponding 
structure of 3,5,7
-trihydroxy
-6-methoxyflavone.
 ............................................................. 81 !Figure 2.8. Single
 crystal used for x
-ray crystallography analysis and the corresponding 
structure of 3,5,7
-trihydroxy
-6-methoxyflavanone.
 ......................................................... 82 !Figure 2.9. Potential hydrogen bonding
 of 3,5,7-trihydroxy
-6-methoxyflavanone 
identified using x
-ray crystallography.
 ............................................................................ 83 !Figure 2.10. Single crystal used for x
-ray crystallography analysis and the 
corresponding structure of the stereoisomer, 3,5,7
-trihydroxy
-6-methoxyflavanone.
 .... 84 !Figure 2.11. Potential hydrogen bonding of 3,5,7
-trihydroxy
-6-methoxyflavanone 
(stereoisomer) identified using x
-ray crystallography.
 .................................................... 85 !Figure 2.12. Single crystal used for x
-ray crystallography analysis and the 
corresponding structure of (
S)-5,7-dihydroxy
-8-methoxy
-2-phenyl chroman
-4-one. ...... 86 !Figure 2.13. Potential hydrogen bonding of (
S)-5,7-dihydroxy
-8-methoxy
-2-phenyl chroman
-4-one identified using x
-ray crystallography.
 ................................................... 87 !Figure 2.14. UV detection (365 nm) and bioassay of semi
-purified fractions both 
containing alnusin (195mg/mL 80% MeOH), approximately 80% pure on left lane; right 
lane contains approximately
 40% alnustinol (140mg/mL 80% MeOH).
 ......................... 89 !$""
!!Figure 2.15. Most of the compounds, including antimicrobial metabolites, present in the 
crude periderm extract of 
P. maackii 
were able to form crystals.
 ................................... 91 !Figure 2.16. Antimicrobial compounds present in the leaves and buds of 
P. maackii
. .. 93 !Figure 2.17. 5, 10 and 15 µL of crystal extract containing dihydrowogonin (left) 
compared under UV light (365 nm) to the crude extract (right) of 
P. maackii 
leaves 
(40mg/mL 80% acetone). Estimated
 Rf value of dihydrowogonin is in the red box.
 ...... 95 !Figure 2.18. Diagram of the possible phenylpropanoid pathway found in 
P. maackii
. ... 99 !Figure 2.19. Diagram of possible biosynthetic pathways of flavonoids containing B
-ring 
substituents.
 ................................................................................................................. 100 !Figure 2.20. Diagram of the possible biosynthesis of alnustinol and alnusin.
 .............. 101  Figure 3.1. Dose
-response of 
P. maackii
 periderm on the growth of 
A. ostoyae
 (warren 
isolate).
 ......................................................................................................................... 116 !Figure 3.2. Dose
-response (12 g 
Ð 0.4 g/L) of 
P. maackii
 periderm on the growth of 
A. 
ostoyae
 (warren isolate). TukeyÕs HSD, 
p = 0.05. * = Significantly different from the 
control.
 .......................................................................................................................... 119 !Figure 3.3. The growth of 
A. ostoyae 
(warren), 
A. gemina 
(35
-5), 
A. mellea
 (49
-5), and 
A. gallica 
(ISRA
-5) on periderm
-amended media (12 g/L YMPG). * = Significantly 
different from the control. TukeyÕs HSD, 
p = 0.05.
 ....................................................... 121 !Figure 3.4. The growth of 
A. ostoyae 
isolates on 
P. maackii
 periderm
-amended medium (12 g/L YMPG). * = Significantly different from the control. TukeyÕs HSD, 
p = 0.05.
 .... 122 !Figure 3.5. The growth of 
one isolate of 
A. gemina
 on P. maackii 
periderm
-amended medium (12 g/L YMPG). * = Significantly different from the control. TukeyÕs HSD, 
p = 0.05. ............................................................................................................................. 123 !Figure 3.6. The growth of 
A. mellea 
isolates on 
P. maackii
 periderm
-amended medium (12 g/L YMPG). * = Significantly different from the control. TukeyÕs HSD, 
p = 0.05.
 .... 123 !Figure 3.7. The growth of 
A. gallica
 isolates on 
P. maackii 
periderm
-amended medium (12 g/L YMPG). * = Significantly different from the control. TukeyÕs HSD, 
p = 0.05.
 .... 124 !Figure 3.8. Crystal formation on the surface of 
P. maackii
 periderm
-amended medium. ...................................................................................................................................... 125 !!$"""
!Figure 3.9. Growth of 
A. ostoyae
 isolates when transferred from 
P. maackii 
periderm
-amended medium to plain YMPG nutrient
-medium. * = Significantly different from the 
control. TukeyÕs HSD, 
p = 0.05.
 .................................................................................... 128 !Figure 3.10. Growth of 
A. gemina
, isolate 35
-5, when transferred from 
P. maackii 
periderm
-amended medium to plain YMPG nutrient
-medium. * = Significantly different 
from the control. TukeyÕs 
HSD, 
p = 0.05.
 ..................................................................... 129 !Figure 3.11. Growth of 
A. mellea 
isolates when transferred from 
P. maackii
 periderm
-amended medium to plain YMPG nutrient
-medium. * = Significantly different from the 
control. TukeyÕs HSD, 
p = 0.05.
 .................................................................................... 130 !Figure 3.12. Growth of 
A. gallica
 isolates when transferred from 
P. maackii
 periderm
-amended medium to plain YMPG nutrient
-medium. * = Significantly different from the 
control. TukeyÕs HSD, 
p = 0.05.
 .................................................................................... 131 !Figure 3.13. Periderm tissues fr
om five young P. maackii
 trees were screened for their 
ability to inhibit the growth of 
A. ostoyae
.  All trees were able to significantly inhibit 
pathogen growth. TukeyÕs HSD, 
p = 0.05.
 .................................................................... 133 !Figure 3.14. Screening for antifungal activity in older tissues collected from mature 
P. 
maackii
 trees. All trees were able to significantly inhibit pathogen growth. TukeyÕs HSD, 
p = 0.05.
 ....................................................................................................................... 134 !Figure 3.15. Periderm
-amended media from both young and mature 
P. maackii 
trees 
were able to significantly inhibit 
A. ostoyae 
growth. Samples were grown for three 
weeks. TukeyÕs HSD, 
p = 0.0
5. .................................................................................... 134 !Figure 3.16. The periderm
-amended medium from 
P. maackii
 was inoculated with 
several non
-host pathogens. Isolates were grown for two weeks. TukeyÕs HSD test, 
p = 0.05. ............................................................................................................................. 136   Figure A.1. Growth comparison of 
A. solani, C. orbiculare, C. carbonum,
 and C. cucumerinum 
on P. maackii 
mulch
-amended medium and YMPG nutrient
-medium. .. 141 !Figure A.2. Growth of non
-host pathogens on 
P. maackii
 mulch
-amended medium. .. 142 !Figure A.3. Growth of non
-host pathogens on 
P. maackii
 leaf-amended medium. ...... 143 !Figure A.4. HPLC chromatogram of the semi
-purified fraction collected from 
P. maackii 
periderm tissue using dry
-column chromatography. Alnustinol showed a retention time 
of approximately 6.77 (301 ion) and alnusin with a retention time of approximately 8.4
4 (299 ion) in negative ion mode.
 .................................................................................... 144 !Figure A.5. HPLC chromatogram of the semi
-purified fraction collected from 
P. maackii 
periderm tissue containing alnustinol 
with a retention time of approximately 6.83 and 
!$"#
!alnusin (nearly 80% pure) with a retention time of approximately 8.46 (299 ion) in 
negative ion mode. ....................................................................................................... 145 !Figure A.6. Crude extract of 
P. maackii
 wood tissue containing alnustinol with a 
retention time of approximately 6.79 and alnusin with a retention time of approximately 
8.45 in negative ion mode. ........................................................................................... 146 !Figure A.7. Crude extract of 
P. maackii
 bud tissue containing alnustinol with a retention 
time of approximately 6.74 and alnusin with a retention time of approximately 8.43 in 
negative ion mode. ....................................................................................................... 147 !Figure A.8. Crude extract of 
P. maackii 
cambial tissue containing alnustinol with a 
retention time of approximately 6.78 and alnusin with a retention time of approximately 
8.44 in negative ion mode. ........................................................................................... 148 !Figure A.9. Crude extract of 
P. maackii
 periderm tissue containing alnustinol with a 
retention time of approximately 6.76 and alnusin with
 a retention time of approximately 
8.43 in negative ion mode. ........................................................................................... 149 !Figure A.10. Crude extract of 
P. maackii 
exposed root tissue containing alnustinol with a 
retention time of approximately 6.77 and alnusin with a retention time of approximately 
8.43 in negative ion mode. ........................................................................................... 150 !Figure A.11. Crude extract of 
P. maackii 
non-exposed root tissue containing alnustinol 
with a retention time of approximately 6.77 and alnusin with a retention time of 
approximately 8.43 in negative ion mode.
 .................................................................... 151 !Figure A.12. Crude extract of 
P. maackii
 leaves containing alnustinol with a retention 
time of approximately 6.81 and alnusin with a retention time of approximately 8.63 in 
negative ion mode. ....................................................................................................... 152 !        !%!LITERATURE REVIEW
 !ARMILLARIA ROOT ROT
  Causal Agent. 
 Armillaria root 
rot
 is one of the most destructive diseases of trees, including many 
fruit tree species, in the temperate regions of the world (Worrall, 2004). This may be 
attributed to the broad host range of the pathogen, the need for resistant hosts, the lack 
of chemical 
control, and 
the longevity of the mycelium in the soil. Management of this 
disease in Michigan is critical because of the economic importance of fruit production. 
 Michigan was the leading state in cherry production at 11.6 million pounds in 
2012 and 217.9 million pounds in 2013 (NASS, 2014). Pathogenic species in the Genus 
Armillaria
 have an extensive host range
, including sour cherry, sweet cherry, peach, 
apple, and oak trees in Michigan
. Since 
Prunus
 cerasus, tart cherry, is one the most 
common hosts for
 Armillaria 
and cherry production is i
n the northern part of Michigan 
where 
Armillaria 
is prevalent, there is a need for research on resistant root stock 
development. Armillaria ostoyae
 attacks both sour and sweet cherry and has been 
found to be widely dis
tributed in Michigan, including Leelanau, Grand Traverse, Benzie, 
Manistee, Mason, and Oceana. 
Armillaria 
mellea
 has been reported on sou
r cherry and 
oak trees in Manistee and Oceana counties
 (Proffer et al, 1987).
 In addition to cherry 
and oak trees, peac
h and almond trees are also susceptible to root rot caused by 
A. 
mellea 
(Guillaumin et al., 1991).  
 Peach trees have been found to be more susceptible 
to Armillaria root rot than apple trees
 because they
 succumb faster and more uniformly 
to infection caus
ed by Armillaria
 compared to apple trees (Kable, 1974). 
 !&!Hyphal walls of fungi are complex in chemical composition. For example, the 
walls of 
A. mellea
 consist of polysaccharides, proteins and lipids (
S⁄nchez et al., 1990
). Identification based on morphology is limited, due to the inconsistencies in the 
production of basidiocarps, which are transient and not produced on a regular basis 
(Baumgartner et al., 2011).
 Due to the limited morphological features, a molecular 
diagnostic approach has been employed for 
the identification of fungal pathogens, 
particularly 
those 
species that spread through the soil (Guillaumin,
 1996).
 Some 
Armillaria
 species are closely related, including 
A. ostoyae
 and A. gemina
. Polymorphism in the IGS region of 
A. gemina
 may provide evidence for
 common 
ancestry (Kim et al., 2006). Other species may coexist; 
A. cepistipes
 often coexists in 
the same geographic area as 
A. ostoyae
, including Europe and North America 
(Prospero et al
, 2004).
  Signs and Symptoms. 
 The earliest symptoms of root rot infection by 
A. mellea
 include yellowing and 
browning of the foliage, reduction in growth and defoliation (Singh, 1980). Once the 
infection of the host has been established, this pathogen cau
ses rot within the wood 
tissue, which leads to slow degeneration and eventual death of the host. Most 
Armillaria
 species are facultative necrotrophs. The parasitic phase involves the colonization and 
death of cambial tissue in roots resulting in necrosis b
eneath root bark. The fungus 
survives on dead tissue in the saprophytic phase making management difficult 
(Baumgartner et al., 2011). 
   !'! The slow growth of 
A. ostoyae
 and the lack of symptoms is also problematic for 
management of the disease since the host species appear healthy for several years, 
post infection, and prior to a rapid decline. Although the hosts do not exhibit symptoms 
until the later stages of infectio
n, the infected wood 
exhibits bioluminescence in the dark 
(Webster & Weber, 2010). Bioluminescence has been reported from mycelia of 
Armillaria gallica, A. mellea, A. ostoyae, A. tabescens, A. calvescens, A. cepistipes, A. 
gemina, A. nabsnona
, and A. sinap
ina (Mihail, 2015). 
 Periderm formation, resinosis, compartmentalization, and callus formation occur 
in conifer roots in response to infection by 
Armillaria
 species (Robinson & Morrison, 
2001). Conifer roots form traumatic resin canals
 (TRCs)
 in response t
o A. ostoyae
 colonizing the 
cambial tissues
 and can be used to determine the timing of infection.
 However, t
his is a non
-specific response that can also be a result of a wide range of 
biotic and abiotic stimuli (Cruickshank et al., 2006). 
 In the field, rh
izomorphs of the parasitic 
Armillaria
 species, 
A. mellea, A. ostoyae
 and A. tabescens
, appear as mycelial fan
-like ribbons. The more saprotrophic species, 
A. calvescens, A. gallica
 and A. sinapine
 have rhizomorphs that were more cylindrical, 
melanized cord
s (Mihail & Bruhn, 2005). It has been reported that 
A. mellea
 rhizomorph 
production is limited in the soil (Guillaumin et al., 1993). Rhizomorph development may 
be stimulated by micro
-fungi that are commonly found in the forest soil and oak 
rhizosphere including 
Penicillium lanosum, Penicillium notatum, Cylindrocarpon 
destructans, Penicillium spinulosum
, and Nectria grammicospora
 (Kwa
!na
 et al., 2004). 
Growth of rhizomorphs in soil has 
been found to be optimal at 20¡C. At temperatures 
!(!above 26¡C, the rhizomorph apices are susceptible to drying out. Rhizomorph growth is 
also inhibited in heavy
-textured and very moist soil (Rishbeth, 1978).
 Hyphae from both 
A. mellea
 and A. ostoyae
 were ab
le to directly penetrate the 
root bark of 
Picea sitchensis
 without prior wounding. Neither species produced 
rhizomorphs when root bark was not wounded.  However, both species produced 
rhizomorphs
 when exposed to
 wounded tissues (Solla et al., 2002). Both 
A. gallica
 and A. cepistipes
 developed more rhizomorphs than 
A. ostoyae
. Mushroom production 
however was greater in 
A. ostoyae
 compared to 
A. gallica
 and A. cepistipes
 (Legrand et 
al., 1996).
  Dissemination & Spread of Pathogen.
 Armillaria
 species produce basidia on the lining of the gills
, which
 give rise to 
haploid spores
 with single nuclei
 (Ullrich & Anderson, 1988). 
Although it is thought that 
sexual basidiospores spores result in new infection, it has not been clearly 
demonstrated in a
 natural environment. 
However, it is 
clear that 
Armillaria
 species 
produce an abundance of mushrooms, which give rise to 
a significant
 amount of basidiospores
 costing
 the organism an immense amount of energy. The benefit of this 
energy cost of spore production needs to be further investigated (Worrall, 2004).
  Recent 
research 
suggest
s that spore dispersal may play an important role in the 
colonization of new habitats. 
Spore dispersal in 
A. mellea
 was found to be spatially 
restricted. Homogeneity between separate sub
-populations may indicate that long
-distance spore dispersal occurs rarely 
(Travadon et al., 2012).  
The need for further 
research of the dissemination of di
stinct 
clones that are contained within a single 
!)!biological species
 would clarify the purpose of the energy costly
 process of mushroom 
and basidiospore production (Proffer et al., 1987). 
 In Basidiomycetes, basidiospores are involved in the primary infection process 
and are responsible for 
infection
 of new areas. The spre
ad of vegetative fungal mycelia
 aids in secondary infection, allowing for the spread of 
those 
produced from the primary 
infection process (Garbelotto, 2004). 
Sexual reproduction in 
Armillaria
 species is 
regulated by two mating
-type loci, containing multiple alleles. In order to be sexually 
compatible, single basidiospore isolates must have different alleles at both loci. 
Completion of the sexual cycle involves the formation of mushrooms that contain 
basidia lining the gills. Meiosis leads to the production of four haploid nuclei, each of 
which enters a basidiospore, 
completing
 the cycle. 
Armillaria
 species do not form 
asexual
 spores. Alternatively, they spread through
 the soil by growing as mycelia
 or 
rhizomorphs (Worrall, 2004).
 Pseudosclerotial plates aid in survival of the mycelium 
(Webster & Weber, 2010). 
 Armillaria 
species may spread through the soil either by 
rhizomorphs or by direct 
transfer of mycelium
. Successful colonization of root tissues may require the oxidation 
of phenolics. The browning of tissues is a result of the fungus oxidizing host phenols. 
However, the spread of pathogen growth may be limited t
o tissues under stress (Wargo 
& Shaw, 1985).
 Rhizomorphs are formed from individual hyphae that together produce 
a cylindrical structure (Yafetto et al., 2009). Like roots, rhizomorphs have a cap that 
reduces friction between the fungal structure and the s
urrounding soil. Rhizomorph 
production and colonization capabilities may be influenced by the presence of other 
fungi. A mutually negative effect on colonization was 
detected
 when A. cepistipes
 and !*!A. ostoyae 
were simultaneously co
-inoculated, which may be a result of interspecific 
competition. However, co
-inoculation induced rhizomorph production in 
A. cepistipes
, but had no effect on 
A. ostoyae
 (Prospero et al., 2006).
  A single isolate of
 Armillaria
 species 
can colonize acres of land. In northern 
Michigan, a 33
-acre genet of 
A. bulbosa 
was determined to be the largest living 
organism on earth. It is the secondary infection that is responsible for spreading among 
root systems of an entire forest for thousands 
of years (Garbelotto, 2004).
 A clone 
refers to all asexual derivatives from a common point of origin. In reference to mating in 
fungi, the term genet refers to one type of clone whose origin is associated with genetic 
exchange. Fungal genets include all de
rivatives of mitotic cell lineage originating in 
either fungi with dikaryotic or diploid mycelia or fungi with haploid mycelia. In fungi 
posse
ssing haploid mycelia,
 genets include all vegetative derivatives of the union of two 
different gametic nuclei. In 
fungi with dikaryotic or diploid mycelia, the mating of two 
different gametic nuclei forms a genet
 (Anderson & Kohn, 1995)
.   Virulence & Pathogenicity. 
 Armillaria
 species vary 
in hosts and pathogenicity; 
A. ostoyae
 is 
primarily a 
pathogen of conifers, 
A. mellea
 primarily a pathogen of hardwoods and 
A. gallica
 occurs 
most commonly as a saprophyte (Kim et al., 2006). 
Armillaria cepistipes
 behaves 
similarly to 
A. gallica
, which is considered a weak pathogen (Prospero et al, 2004). 
Armillaria obscura
 was found to be very pathogenic and 
A. bulbosa
 was only slightly 
pathogenic (Rishbeth, 1985).
 One-year-old almond trees were inoculated with 
A. 
mellea, A. gallica
 and A. tabescens
. Armillaria mellea
 was found to be the most virulent
 !+!on this particular hos
t and A. tabescens
 was the least virulent (Tsopelas & Tjamos, 
1997).  
 The conditions of Douglas
-fir and other conifer hosts were monitored after initial 
infection of 
A. ostoyae
. Armillaria
 ostoyae
 caused death in Douglas
-fir hosts seven 
years after infect
ion. This root rot pathogen caused mortality in nine years for other 
coniferous species (Morrison, 2011). In Japan, 
A. mellea, A. ostoyae, A. cepistipes
, and A. tabescens
 were classified as moderate to aggressive pathogens of conifers. 
Armillaria tabescens
 was rarely found compared to 
A. mellea, A. ostoyae
 and A. 
cepistipes
 (Hasegawa et al., 2011). 
Armillaria cepistipes, A. gallica, A. mellea
, and A. 
ostoyae
 are also commonly found in forests of central and southern Europe. Both 
A. 
mellea
 and A. ostoyae
 were classified as highly pathogenic on conifers. 
Armillaria 
cepistipes
, which mainly causes heart rot in conifers, has low virulence (
Ke
"a et al., 
2009). 
 Melanization contributes to increased virulence, resistance to microbial attack 
and aids in surviva
l of pathogens (Fogarty & Tobin, 1996). Melanin is 
produced from the 
oxidative polymerization of phenolic compounds, which usually result in dark brown or 
black pigments. Melanin pigmentation reduces th
e susceptibility of fungi to
 host defense 
mechanisms. 
Melanin also guards fun
gi from hydrolytic enzymes, UV 
radiation, heavy 
metals, abiotic stress, and toxic secondary metabolites (Taborda et al., 2007). Another 
factor influencing the pathogenicity of 
Armillaria
 species is their ability to produce 
rhizomorph
s, which is one method of spreading the disease to another host.
  There is a positive correlation between rhizomorph production and laccase 
activity (Worrall et al., 1986). Laccase activity is also involved in melanin production 
!,!(Fowler et al., 2011). Lacc
ase activity has been reported in 
Armillaria
 species, 
A. 
ostoyae, A. mellea, A. gallica
, and A. cepistipes
. Other white
-rot fungi are also known to 
have this phenol
-oxidizing enzyme. Laccase also plays a role in lignin biodegradation 
and oxidation of antifungal phenols (Robene
-Soustrade & Lung
-Escarmant, 1997). 
 Branching patterns of the rhizomorphs may reflec
t the virulence of the species. 
Rhizomorphs of species from the southern hemisphere, 
A. luteobubalina, A. fumosa, A. 
hinnulea, A. novae
-zelandiae
, and A. limonea
 all branch dichotomously. Rhizomorphs 
from species of the northern hemisphere branch both mono
podially and dichotomously. 
Armillaria mellea, A. borealis
 and A. ostoyae
 branched dichotomously. Monopodial 
branching was 
detected
 in A. gallica, A. cepistipes, A. gemina, A. calvescens, A. 
sinapine
, and A. nabsnona
. The northern dichotomously branching s
pecies were more 
virulent than species that branched monopodially (Morrison, 2004). 
  Management.
  The presence of enzymes, including chitinase and 
#-1,3-glucanase contribute to 
the protective barrier found in healthy trees and may aid in resistance to pat
hogen invasion by lysis of hyphal walls of 
A. mellea
 (Wargo, 1975). The responses 
triggered
 by wounding may aid in the identification of anatomical traits 
indicating
 resistance or 
susceptibility to 
A. ostoyae
 (Cleary et al, 2012). Trichoderma and some nema
todes 
have been found to be potential sources of biological control, but have shown limited 
success in a natural 
environment 
(Webster & Weber, 2010). 
Trichoderma
 species are 
known antagonists of some pathogenic fungi. Bark treated with 
Trichoderma atroviri
de can suppress Armillaria root rot. Strawberries with 
T. atroviride
 pre
-treated mulch 
!-!showed significantly lower infection of Armillaria root rot than those with untreated 
mulch (Pellegrini et al., 2014).
 Armillaria mellea
 is a pathogen of apple, walnut and kiwifruit. Mycelial growth of 
A. mellea
 was completely inhibited by the fungicide cyproconazole (Thomidis & 
Exadaktylou, 2012). The few additional chemicals known to inhibit 
Armillaria
 growth also 
include methyl bromide 
(phased out in 2005) and carbon disulphide, both chem
icals 
were able to kill mycelia
 present in decayed trees to a soil depth of approximately 1 m 
(Baumgartner et al., 2011). Chloropicrin has also been used to eradicate inoculum from 
soil in orchards (Worr
all, 200). Pinosylvin monothyl ether isolated from the fresh 
sapwood of 
Pinus strobus
 inhibited the growth of 
A. ostoyae
. However, pines are still 
attacked in the field setting despite the presence of these inhibitors (Mwangi et al., 
1990). 
Pinosylvin mono
thyl ether may potentially be used in management by utilizing 
these biological markers in breeding for resistance. 
Management must also be directed 
toward efficient detection and removal of diseased trees. Susceptible tree species 
should be avoided includi
ng Douglas-fir, Engelmann spruce and blue spruce (Worrall et 
al., 2004).  Determining whether a pathogen spreads by either primary infection or 
secondary infection is critical in understanding the epidemiology of the pathogen and 
utilizing proper managemen
t techniques (Garbelotto, 2004). 
      !%.!Resistance
.  Host susceptibility has been linked to low phenolic/sugar ratio among additional 
elements. Other factors include nutrition availability for the production of defense 
compounds and also the variation in 
chemical composition of the bark throughout the 
year.  It has been reported that high glucose concentrations in hosts are correlated with 
susceptibility to Armillaria root rot. The selection of resistant species should be made for 
low sugar concentrations 
(Myszewski et al, 2002).
 Western Larch tree
 roots form 
necrophylactic periderm with bands of phellem 
around lesions caused by 
A. ostoyae
. This wound
-response periderm may be 
responsible for the increased resistance 
detected
 in the western larch (Robinson &
 Morrison, 2001). Host enzymes, chitinase and glucanase may 
also 
be involved in host 
resistance. These enzymes are responsible for the lysis of fungal hyphal walls. 
Chitinase and 
#-1,3-glucanase were isolated from sugar maple (
Acer saccharum
), red 
oak (
Quercus rubra
), black oak (
Quercus velutina
), and white oak (
Quercus alba
) and 
were found to lyse hyphal walls of 
A. mellea
 (Wargo, 1975). 
       !!!!!%%!!!!!!!!!!!!!  !! !LITERATURE CITED 
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bark by 
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 and Heterobasidion annosum
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as a virulence factor of 
Paracoccidioides brasiliensis
 and other dimorphic pathogenic 
fungi: a minireview.
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E. (2012). Effectiveness of cyproconazole to control 
Armillaria root rot of apple, walnut and kiwifruit.
 Crop Protection
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K. (2012). Inferring dispersal patt
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Armillaria 
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Armillaria 
tabescens
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 J. B. (1988). 
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  Wargo, P. M. (1975). Lysis of the cell wall of 
Armillaria mellea
 by enzymes from forest 
trees.
 Physiological Plant Pathology
, 5, 99-105.  Wargo, P. M
., & Shaw III, C. G. (1985). Armillaria root rot: Th.
 Plant Disease
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 Introduction to Fungi
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New York, pp. 511
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spx. 
  Worrall, J. J., Chet, I., & Httermann, A. (1986). Association of rhizomorph formation 
with laccase activity in 
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 Journal of General Microbiology
, 132, 2527-2533.  Worrall, J. J., Sullivan, K. F., Harrington, T. C., & Steimel, J. P. (2004). Incidence, host 
relations and population structure of 
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campgrounds.
 Forest Ecology and Man
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Armillaria
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 Fungal Genetics and Biology
, 46, 688-694.             !!!!%*!LITERATURE REVIEW
  !!ROLE OF SECONDARY METABOLITES IN PLANT
 DEFENSE
  Primary Metabolism.
 Plant p
rimary metabolism refers to anabolic and catabolic processes that are 
required for cell viability. These 
processes 
include carbohydrate metabolism, the 
biosynthesis of amino acids and proteins, nucleotides, fatty acids, 
and lipids. Defense 
against pathogens require
s increased demands for energy, carbon skeletons and 
reducing equivalents (Bolton, 2009). Primary metabolism provides the metabolic 
precursors that are used in secondary metabolism. Resource availability may pla
y a role 
in the regulation of growth and defense
-related metabolism. The shikimate pathway 
directly or indirectly produces all phenols with most c
lasses using the aromatic amino 
acid 
phenylalanine as an intermediate 
(O§WALD
 ET AL., 2012
). 
  Secondary Metab
olism.
 Plant secondary metabolism includes a diverse range of compounds, enzymes 
and mechanisms of gene regulation of metabolites. Secondary metabolism involves 
compounds that are not essential for primary metabolism, but are needed for host 
survival in th
e environment (Lattanzio et al., 2008). All cells in an organism possess 
genes for the synthesis of secondary metabolites, but only a limited number express 
them (Seigler, 1998). 
  !%+!These metabolites may be released only when the cells are broken. 
Compartme
ntalization protects uninfected cells from the toxic compounds used in 
defense against pathogens. For example, when peppers were challenged against 
Colletotrichum coccodes
, the highest levels of total phenolic content were found in
 the bordering zone of healthy and 
infected tissue
. This zone of phenolic content indicates
 synthesis in
 tissues surrounding the site of infection (Mikulic
-Petkovsek et al., 2013).  A 
broad variety of pathogens produce
 tyrosinase, which is a 
phenol oxidase
 that is 
responsible for the biosynthesis of melanin as a contributing factor of virulence 
(Taborda et al., 2008). This multifunctional enzyme is inhibited by taxifolin (Miyazawa & 
Tamura, 2007). The virulence of pathogens partly depends on the pathogens a
bility to overcome host defenses (Casadevall & Pirofski, 2001). 
 Phenolic compounds are define
d as those with one or
 more hydroxyl group
s(s) on an aromatic ring. Phenolic compounds and polyphenols (a polymeric phenol) include 
all products arising from the 
shikimate
-phenylpropanoid pathway (
Lattanzio & Cardinali
, 2006).
 Plant phenolics may function as antioxidants by neutralizing free radicals. Some 
polyphenols are more easily oxidized than others because of the different types and 
locations of constituents.
 Flavonoids may possess properties for scavenging reactive 
oxygen species. The presence of an 
ortho
-hydroxylation on the B
-ring of the flavonoid 
molecule, free hydroxyl groups, a C2
-C3 double bond in the C-ring, or the presence of a 
3-hydroxyl group are in
dicative of antioxidant and antiradical capabilities (Burda & 
Oleszek, 2001). 
  !%,!The catechol
-type B ring in the skeletal structure of 
a flavonoid is the antioxidant 
active moiety. Since flavonoids such as quercetin, rutin and (+)
-catechin are known 
antioxidants and radical scavengers,
 they have also been linked to their inhibition of 
lipid oxidation. They possess antioxidant properties and also chelating properties of 
metal ions such as iron or copper
, which
 are known to catalyze many biological 
processes, having the ability to inactivate enzymes and proteins (
Le Nest
 et al., 2004).   Flavonoids
. Flavonoids are based on the skeleton structure containing two phenyl groups 
connected by a three
-carbon bridge, C
6 Ð C3 Ð C6, and can be further classified based 
on substituents (Bano et al., 2013). This carbon bridge often cyclizes to give rise a third 
ring termed the C
-ring (Cuyckens & Claeys, 2004). There is structural variation in the 
basic flavonoid skeleton due 
to the variation in hydroxylation, methoxylation, 
methylation, prenylation, benzylation, and glycosylation. Flavonoids include various 
groups such as chalcones, flavan
-3-ols, isoflavonoids, flavanones, flavones, flavonols, 
catechins, and anthocyaninidins (
Croteau & Lewis, 2000). Classes of flavonoids have 
been identified as both intermediates and as end products, including chalcones, 
flavanones, and flavan
-3-ols. Other classes of flavonoids have only been identified as 
end products in the shikimate pathway,
 which include anthocyanidins
 (Cushnie & Lamb, 
2005).  
   !%-!Phenylalanine ammonia
-lyase (PAL) is the first enzyme involved in the 
biosynthesis of phenylpropanoid compounds. PAL activity can be regulated by feedback 
control and accumulation of precursors (Ju et al., 1995). Feedback inhibition plays a 
role in control
ling the biosynthesis and metabolism of phenolic compounds. 
Intermediate products and end products of this pathway may repress or induce enzyme 
synthesis (Kosuge, 1969). Cinnamic acid inhibits the transcription of 
CHS, but p-coumaric acid stimulates the pr
oduction of CHS. 
PAL activity can be negatively 
regulated by exogenous application of 
trans
-cinnamic acid or by blocking downstream 
activity (Yin et al., 2012). 
Trans
-cinnamic acid regulates 
CHS 
at the transcriptional
 level. Therefore, cinnamic acid may ac
t as a regulator of phenylpropanoid biosynthetic genes 
(Mavandad et al., 1990).
 Chalcone synthase 
is part of the large superfamily of 
polyketide synthases. In the phenylpropanoid pathway of higher plants, chalcone 
synthase (CHS) is the first committed step
 of flavonoid biosynthesis. The CHS reaction 
products serve as the basic building blocks for secondary metabolism
 (Dare et al., 
2013). Chalcones, C
15H12O have a linear C
3-chain connecting the two 
phenyl rings, 
containing a double bond. In comparison, the C
3-chain is saturated in dihydrochalcones. 
Pre-flavonoid precursors are derived from carbohydrate metabolis
m and are condensed 
by chalcone
 synthase (CHS) (Halbwirth, 2010). Chalconaringenin (2Õ,4Õ,6Õ
, 4-tetrahydroxychalcone) has been determined to be one of the main phenolic components 
in cherry tomatoes (Slimestad & Verheul, 2005). However, there is a significant 
decrease in chalconaringenin concentrations during postharvest ripening (Slimestad & 
Ver
heul, 2005). 
 !&.!The cyclization of chalcones involves the reaction of the meta
-hydroxyl group 
with the '-carbon to produce aurones. Aurones, C
15H10O2 are responsible for yellow 
pigmentation in flowers (Vermerris & Nicholson, 2006) and are isomeric with flavo
nes 
(Seigler, 1998). Aurones contain a 5 
Ð membered C
-ring, instead of a 6 
Ð membered C
-ring more commonly found in other flavonoids. 
 The final flavanone structure is formed when chalcone isomerase (CHI) produces 
(2S)-flavanones (Fowler & Koffas, 2009). U
pon further enzymatic action, flavanones 
serve as the intermediate precursors to flavones, isoflavones, flavan 4
-ols, and 
dihydroflavonols (Halbwirth, 2010). Isoflavones are synthesized from flavanones by the 
co-action of 2
-hydroxyisoflavanone synthase (IF
S) and a dehydratase (IDH).  
Alternatively, position 3 of flavanones can be hydroxylated to produce dihydroflavonols, 
which is catalyzed by 2
-oxoglutarate
-dependent dioxygenase (FHT) (Halbw
irth, 2010). 
Flavanones contain
 various substitutions, e.g. hydroxy
, methoxy, on the A
- or B
- ring. 
Flavanones can also be isolated from both above and belowground parts of the plant 
(Khan & Dangles, 2014). 
 Flavones h
ave the backbone of 2
-phenylchromen
-4-one with a basic molecular 
formula of C
15H10O2, 
which is also the f
ormula for aurone and isoflavone structures.
  A C2-C3 double bond distinguishes flavones from flavanones (C
15H12O2); the flavones 
have a double bond at C2-C3. This conversion requires two types of enzymes, flavone 
synthase I (FNSI) and flavone synthase II 
(FNSII) (Halbwirth, 2010). This class of 
flavonoids is typically colorless
-to-yellow and soluble in both water and ethanol (Singh 
et al., 2014). Flavones are a biologically active class of flavonoids exhibiting antioxidant, 
!&%!antimicrobial and cytotoxic acti
vities. Flavones and flavonols are structurally v
ery similar 
and therefore flavonols may be referred to as 3
-hydroxyflavones (Das et al., 2014). 
 Dihydroflavonols, C
15H12O3 are biosynthetic intermediates of flavones (Balza et 
al., 1988). 
Trans
-dihydroflavonols can be synthesized from flavanones by flavanone 3
-#-hydroxylase (F3
-#-OH). Dihydroflavonols may be converted to flavonols by flavonol 
synthase (FLS) (Wilmouth et al., 2002). Flavonol synthase (FLS) catalyzes the 
introduction of a double b
ond between the C2-C3 positions of dihydroflavonols producing 
flavonols. Dihydroflavonols are also direct intermediates of leucoanthocyanidins. DFR 
reduces dihydroflavonols to leucoanthocyanidins, which serve as direct precursors of 
catechins and anthocyan
idins (Halbwirth, 2010). The most common dihydroflavonols 
include dihydroquercetin, dihydrokaempferol and dihydromyricetin (Marais et al., 2005). 
Dihydroflavonols, such as taxifolin, have antioxidative and antifungal properties (Gong 
et al., 2009). 
 Anthoc
yanins are a group of bio
logically active
 compounds that 
are 
responsible 
for the pigmentation of many plant parts including fruits, vegetables and flowers. The 
production of anthocyanins requires the action of flavonoid
-3-O- glucosyltransferase 
(UFGT) (Rah
im et al., 2014).  Other enzyme
s are required to synthesize 
anthocyanidins
, which include chalcone isomerase (CHI), 
flavanone 3
-hydroxylase 
(F3H), d
ihydroflavonol 4
-reductase (DFR), and finally anthocyanidin synth
ase (ANS) 
(Zhang et al., 2014).
 The site of
 the biosynthesi
s of flavonoids remains elusive. It
 is believed that they 
are formed in the cytosol requiring an unknown flavonoid transport mechanism from the 
formation site to the site of accumulation (Halbwirth, 2010). Flavonoids are most 
!&&!commonly prese
nt in glycosidic form
 and stored in vacuoles of stems, roots, flowers, 
and leaves (Cuyckens & Claeys, 2004).
  Phenolic Compounds in the Genus 
Prunus
.  Flavonols, flavanols, anthocyanins, isoflavones, and dihydrochalcones are 
commonly identified in Rosaceae
 (Ogah et al., 2014). 
In addition to flavonoids, 
coumarins have 
also 
been identified from 
Prunus
 species. Scopolin has been identified 
in the bark of 
Prunus verecunda
, P. cyclamina
 and P. maackii
 and herniarin was 
reported in the bark of 
P. mahaleb
. Herniarin has also been reported in the leaves of 
P. 
mahaleb
, P. pensylvanica
 and P. maximoqiczii
 (Santamour & Riedel, 1994).
  Flavonols that are found in sweet cherry (
Prunus avium
 L.) and tart cherry 
(Prunus cerasus
 L.) are in higher concentrations com
pared to other 
rosaceous fruits. 
Glycosylated quercetins and kaempferols are two flavonols
 commonly identified in 
sweet and sour cherries
 (Ogah et al., 2014). Additionally, the flavonols myricetin 3
-rutinoside, quercetin 3
-rutinoside and kaempferol 3
-rutin
oside have been found in the 
Ôsweet heartÕ and Ôsweet lateÕ sweet cherry cultivars of 
P. avium
. All of which were 
significantly increased by oxalic acid (OA) treatment (
Mart™nez
-Espl⁄
 et al., 2014). 
Cherry fruits are also known to contain high amounts of 
anthocyanins, which contribute 
to both fruit color and antioxidant capacity. Six Michigan tart cherry (
Prunus cerasus
 L.) 
selections were evaluated for their anthocyanin content; selection 25
-14 (20) contained 
the highest amount followed by selection Tamaris and 27
-10 (50), respectively. 
Montmorency cherries contained the lowest amount of anthocyanin content followed by 
Balaton (Siddiq et al., 2011).  
 !&'!Chlorogenic acid, neochlorogenic acid, (+)
-catechin, and (
-)-epicatechin are the 
major phenolic compounds found in apricot fruit, 
Prunus armeniaca
 (Schmitzer et al., 
2011). The leaves of 
Prunus persica
 contain caffeic acid,
 chlorogenic acid, kaempferol, 
and quercetin (Kazan 
et al., 2014). Procyanidins, c
yanidin 3
-rutinoside and C
-alkyl 
isoflavones have been recently identified in 
Prunus domestica
 (Khallouki et al., 2012). 
The fruit of 
Prunus avium
 have been found to be a ric
h source of neochlorogenic acid, 
p-coumaroylquinic acid and chlorogenic acid (Ballistreri et al., 2013). Kaempferol, 
naringenin and 
catechin have been isolated from almond 
Prunus amygdalus
 (Esfahlan 
et al., 2010).  
Prunus mume
 seed extracts contained 3
-O-caffeoylquinic acid, 5
-O-caffeoylquinic acid and 4
-O-caffeoylquinic acid. The
 crude extract had antifungal 
properties (Xia et al., 2011). The wood of 
P. campanulata
 contains a system of 
flavanones including naringenin, aromadendrin, isosakuranetin, sakurane
tin, pinocembrin, eriodictyol, taxifolin, prunin, among others (Hasegawa, 1958). 
  Plant Defense.
 Plants have evolved defense mechanisms including physical barriers and 
chemical protection. Antifungal metabolites can either be preformed or induced after 
infection (Grager & Harborne, 1994). Phytoanticipins are antimicrobial compounds that 
are present prior to pathogen attack. These low molecular weight 
compounds may also 
be produced from pre
-existing constituents (VanEtten et al., 1994). The presence of 
pref
ormed antifungal compounds has been correlated with the resistance 
detected
 in young rockmelon fruit
 (Cucumis melo
 L) cv. ÔC
oloradoÕ. Levels of these compounds 
decrease as the fruit matures (Kumar & McConchie, 2010). The durability of 
Larix 
!&(!leptolepis may be attributed to the toxic material found in the heartwood tissue including 
taxifolin, aromadendrin and quercetin (Sasaya et al.
, 1970). 
 Examples of phytoanticipins include avenacins, which are saponins found in 
roots of 
Avena sativa 
(Oat) and are re
sponsible for resistance to 
Gaeumannomyces 
graminis
 var. 
tritici
. However, oat plants are susceptible to 
G. graminis
 var. 
avenae
 due to the secretion of avenacinase, which detoxifies avenacins (Osbourn et al., 1994). 
Avenacin saponins also inhibit pathogen
 growth of 
Blumeria graminis f. sp. hordei
, Bipolaris oryzae
 and Magnaporthe oryzae
 (Inagaki et al., 2013). The following phenolic 
compounds have been identified as phytoanticipins from various host species including 
gallic acid and catechin in 
Acer
 spp., 
phloroglucinol, salicin and saligenin in 
Salix alba
 var. 
caerulea 
and ellagitannins in 
Castanea
 and Quercus
 spp. (Pearce, 1996). The 
phenolic acid, protocatechuic acid, is correlated with maximum pigmentation in scales. 
Unpigmented scales contain little or
 no protocatechuic acid (Link et al., 1929). Catechol 
and protocatechuic acid appear to be the chief toxic chemicals found in the outer scales 
of pigmented onions (
Allium cepa
) and are thought to contribute to the resistance to 
onion smudge caused by 
Colletotrichum circinans
. This type of resistance involving the 
accumulation of catechol is only found in pigmented onions and not in the susceptible 
white onions (Link & Walker, 1933). 
    !&)!Techniques for Phenolic Detection.
 Thin Layer Chromatography (TLC) can 
be used to separate phenolic compounds 
from crude extracts. A small amount of the extract is applied to the bottom of the TLC 
plate, which is co
ated with either silica gel or cellulose. 
The capillary action of the 
solvent moves the extract to the top of th
e plate. The Rf-value can be calculated for 
each compound, which can be used for characterization and identification. This value is 
the ratio of distance 
the compound has 
migrated divided by the migration value of the 
solvent, with a maximum value of one (
Vermerris & Nicholson, 2006). 
 Unknown compounds that are present together interfere with each other in 
chromatographs. HPLC
-MS and PDA detection can be used to separate and quantify 
those with similar UV spectra (
Justesen et al.
, 1998). High performance
-liquid chromatography, incl
uding reversed
-phase HPLC, 
can be used to quantify and purify 
individual compounds from a complex mixture (Temerdashev et al., 2011).
  Individual compounds can be identified by their retention times and UV spectra. 
High performance
-liquid chromatography coupled with diode array detection and 
electrospray ionization mass spectrometry (HPLC
-DAD/ESI
-MS) has been used to 
analyze the chemical
 properties of berries. Retention times and UV
-visible spectra were 
important for the identification of 
anthocyanins from 
complex mixtures in berries (Chen 
et al., 2014). UV spectrophotometry can also be used to detect polymeric forms of some 
flavonoids, i
ncluding dihydroquercetin (DHQ); absorption band at 328 nm corresponds 
to the monomeric form of DHQ and the 290 nm band was attributed to both monomeric 
and polymeric forms of DHQ (Vekshin, 2009). Determining flavonoid structures is 
challenging and require
s the use of analytical tools. ESI
-MS analyses of fragmentation 
!&*!patterns
 for flavonols and flavan
-3-ols are often analyzed in negative
-ion mode because 
of increased sensitivity compared to that of the positive
-ion mode (Crupi et al., 2014). 
 Mass spectrome
try is a technique that can be employed f
or the analyse
s of
 flavonoids. The purpose of MS
 is to detect charged molecular ions and fragments 
separated according to their molecular masses. Fragmentation patterns provide 
molecular masses and provide structura
l and stereochemical information (
Andersen & 
Markham
, 2005). The negative ion mode 
is usually
 more sensitive and selective 
compared to the positive ion mode for c
rude plant phytochemical analyse
s (Shu et al., 
2010).
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-169.  Wilmouth, R. C., Turnbull, J. J., Welford, R. W., Clifton, I. J., Prescott, A. G., & 
Schofield, C. J. (2002). Structure and mechanism of anthocyanidin synthase 
from
 Arabidopsis thaliana
. Structure
, 10, 93-103.  Xia, D., Wu, X., Shi, J., Yang, Q., & Zhang, 
Y. (2011). Phenolic compounds from the 
edible seeds extract of Chinese Mei (
Prunus mume
 Sieb. et Zucc) and their 
antimicrobial activity.
 LWT-Food Science and Technology
, 44, 347-349.  Yin, R., Messner, B., Faus
-Kessler, T., Hoffmann, T., Schwab, W., Hajire
zaei, M. R., ... 
& Sch−ffner, A. R. (2012). Feedback inhibition of the general phenylpropanoid and 
flavonol biosynthetic pathways upon a compromised flavonol
-3-O-glycosylation.
 Journal 
of Experimental Botany
, 63, 2465-2478. !''!Zhang, Y., Butelli, E., & Martin
, C. (2014). Engineering anthocyanin biosynthesis in 
plants.
 Current Opinion in Plant Biology
, 19, 81-90.                                           !'(!CHAPTER ONE
  ANTIFUNGAL BIOCHEMICAL COMPARISON OF 
PRUNUS
 SPECIES
   ABSTRACT
  The periderm
 tissues
 from seventeen 
Prunus 
species and common 
Prunus
 rootstocks w
ere
 screened for the ability to inhibit the growth of 
Armillaria
 species.
 Among the screened species and varieties, only 
Prunus maackii
 was able to 
significantly inhibit the growth of 
Armillaria 
ostoyae
, which is the most detrimental cherry 
root rot pathogen in Michigan. 
The P. 
maackii
 extracts from
 periderm, cambium, root, 
and wood tissue we
re analyzed by bioassay guided Thin Layer C
hromatography (TLC)
 techniques. Bas
ed on separation of extracts 
by TLC, the periderm from 
P. maackii
 appeared to have a much higher phenolic compound content in comparison to the other 
Prunus
 species screened
 based on the number and area 
of bands fluorescing and
 absorbing under the wave
lengths of 
365 nm. Since many 
phenolic compounds exhibit 
this behavior under UV, extracts were further characterized using che
mical reagents, 
which included d
iazotized 
p-nitroaniline
 (DPN, 
phenol detecting reagent) and vanillin (a 
flavanol detecting reagent).
 Periderm and cambial tissu
es from both mature and young 
P. maackii
 trees were screened to 
examine the distribution 
of phenolic compounds
 as 
trees
 advance
 in age. The periderm tissue was incorporated into YMPG nutrient
-medium to 
further 
screen for antimicrobial properties. 
The objective of this study was to 
partially 
characterize the compound(s) from the periderm of 
P. maackii
 that were 
inhibitory to 
Armillaria
 species. 
  !')!Introduction
 !The genus 
Prunus
 contains more than 400 species, including stone fruits, 
almonds and 
ornamentals (Osman et al
., 2012). 
Armillaria root rot, caused by 
Armillaria
 ostoyae
, is a 
major
 problem in cherry orchards in the northwest region of Michigan, 
particularly 
on sour cherry,
 Prunus cerasus
, variety 
Montmorency (Proffer and Jones, 
1988). 
Since 
Montmorency 
constitutes >95% of tart cherry production in Michigan, it is 
important
 that long-term management solution such as 
host 
resistance is found
. Aside 
from devastating cherry production in Michigan, Armillar
ia root rot caused by 
A. mellea
 is 
the second leading cause of peach tree mortality (Beckman and Pusey, 2001). 
Overall, 
Armillaria root disease 
is considered 
one of the most detrimental diseases of 
trees in the temperate regions of the world (
Worrall
, 2004). 
Armillaria
 species 
can be 
pathogenic, necrotrophic or mutualistic
 (Mihail, 2015). 
Fungicide drenches or fumigation 
treatments show little effects due to the difficulty of reaching the pathogens
 (Amiri & 
Schnabel, 2012).
 It is the white mycelium that destroys the phloem and cambium 
of the host. White 
mycelial fans bet
ween the wood and bark 
grow saprotrophically after death of the host 
(Webster & Weber, 2007). 
Wounds serve as an entrance for several pathogens. 
Necrophylactic periderm forms in response to wounding and is more resistant
 to infection. This type of periderm has major structural components that include lignin and 
suberin (Briggs, 1986). These polymers aid in resistance to pathogens. Resistance is a 
result of the plant pathogens inability to degrade lignin and suberin, which
 serve as 
both a physical and chemical 
barrier to the diffusion of nutrients. Diffusion and physical 
!'*!barriers concentrate antimicrobial compounds at the site of infection (Bostock & 
Stermer, 1989).   
  Rhizomorph production
 and the colonization of wood allow
 the pathogen to spread through the soil forming infection centers that can cover 
large areas
 (Szewczyk
 et al, 2014).
 Due to the devastating effects of this disease, finding a long
-term solution 
is critical. Host resistan
ce is one potential means of management. Preliminary screening 
using 
the colonization of
 Armillaria
 species on
 twig segments has suggested that 
P. maackii 
has the potential for resistance to 
A. ostoyae
. In vitro
 work included
 the screening of twigs from
 various 
Prunus
 species
. Armillaria
 ostoyae
 was able to 
completely colonize 
the cambial tissue 
of all Prunus
 spp. 
screened 
with the exception of 
P. maackii
 (Warnstrom, 2011
). 
This reduced colonization of 
A. ostoyae
 on P. maackii
 twig segments may be a resu
lt of antimicrobial compounds contained in the periderm 
tissue. 
 Higher plants produce
 diverse chemical compounds with defensive properties 
against 
pathogens. These chemicals 
include various
 low-molecular
-weight natural 
products (Dixon, 2001). Some of the 
most well studied defensive compounds are the 
phytoalexins, which are antimicrobial compounds that are synthesized after the plant 
tissue is exposed to pathogen infection
 occurring first at the attempted infection site 
within single host cells (Nicholson &
 Hammerschmidt, 1992)
. Phytoalexins are known to 
have defensive capabilities
 and can be either fungitoxic or fungistatic (Ingham, 1972).
   !'+! Phytoalexins accumulate in quantities that exceed the concentration needed to 
inhibit pathogen growth
 (Ribera & 
ZuŒiga, 2012). Piceatannol was found to inhibit the 
growth of 
Colletotrichum falcatum
. This phytoalexin was only synthesized as a response 
to pathogen infection, therefore uninfected 
sugarcane 
stalks 
(Saccharum
 spp.) 
did not contain piceatannol or it was found in very low levels (Brinker & Seigler, 1991). 
Camalexin is a nitrogen and sulf
ur containing phytoalexin from 
Arabidopsis thaliana
 and Camalina sativus
, which is synthesized in response to various plant pathogens 
(Glawischnig, 2007).
 Young rice 
(Oryza sativa
 L.) 
seedlings produce the diterpene 
momilactone B 
as a phytoalexin and as a potential allelochemical to inhibit the growth of 
neighboring plants (Kato
-Noguchi et al
., 2002).
 Additional
 examples of phytoalexins 
include medicarpin from alfalfa, 
Medicago sativa
, and pisatin from pea
s, Pisum sativum
 (Iriti & Faoro, 2009).
  Phytoanticipins are preformed antimicrobial compounds that are constitutively 
present or produced in response to pathogen elicitors from 
preexisting precursors 
(VanEtten et
 al., 1994). Some
 phytoanticipins 
include saponins, cyanogenic glycosides 
and glucosinolates
 (Piasecka et
 al., 2015). Antifungal glucosinolate derivatives are 
found in significant quantities localized in the roots of Bras
sicaceae members (Schreiner 
& Koide, 1993). 
The avenacin
s are 
antifungal saponin
s found in oat roots and determine
 resistance to the take
-all disease of wheat pathogen 
Gaeumannomyces graminis
 var. tritici
. This soil pathogen is able to infect wheat but not oats. However, 
Gaeumannomyces graminis
 var. avenae 
produces avenacinase, which detoxifies 
avenacin and therefore can infect both cereals (
Osbourn et
 al., 1994).
 Phytoalexins
 require
 induced 
transcription
al and/or translational 
activities while phytoanticipins are 
!',!constitutively present or
 produced from preexisting constituents (
Gonz⁄lez
-Lamothe et al., 2009). 
 The objective
s of this research 
include
 screen
ing a variety of 
Prunus 
species for 
antifungal activity in the periderm
. Thin Layer C
hromatography (TLC) bioassays 
will be used 
to examine the biological activity and complexity of the antifungal component(s) in 
the crude periderm extract of 
P. maackii
. TLC bioassays will also be used to screen for 
heritable
 antifungal compounds
 in P. maackii
 hybrids
. DPN and v
anillin reagents will be 
use
d for the comparison of
 phenolic content in various tissues from
 young and mature
 P. maackii
 trees
 and in other 
Prunus
 species.
  Materials and Methods
 !!Young (<
 5 years) 
P. maackii
 were purchased from
 Nature Hills Nursery, Omaha, 
NE.
 Tree
s were planted
 in pots,
 using Baccto¨ premium potting mix,
 located 
in the MSU greenhouse courtyard. Mature 
Prunus
 twigs, from 
P. maackii
, P. mahaleb
 and P. 
serotina
 were collecte
d at the NW Michigan Horticulture Research Station near 
Traverse City, MI and stored at 
-20ûC until used for analysis. 
Mature twigs were ground 
using a chipper
-shredder to produce mulch. The periderm was removed from the mulch 
for samples of wood. Dr. Amy 
Iezzoni
 and Audrey Sebolt
 of the MSU Department of 
Horticulture provided the following materials for testing: 
MSU test rootstock samples of 
Cass, Clare, Lake, and Clinton
. Samples of current cherry roots
tocks include Mazzard, 
mahaleb and 148-1 (Gisela
 6). Breeding selection samples, having 
P. maackii
 in their 
ancestry include R1 (1), R2 (1), 25 11 (15), PO1 (01), P11 (2), P12 (2), and 26e 20 (50)
. !'-!The 14 samples used are shown
 with their assigned number
s and other identifiers
 (Table 1.1), which are alternat
ive names mainly related to the field location the samples 
were collected from.
 Periderm, cambial and root tissues were also collected from a 
mature 
P. maackii 
tree
 on Mich
igan State UniversityÕs campus, accession number 
97P028*02, 
samples were kindly 
provided by Dr. Frank Telewski. Three extraction 
methods were evaluated to determine the best method for extract
ing the antifungal compounds in different
 Prunus
 species.
  Table 1.1. Samples
 used
 to screen
 for Armillaria
 growth inhibition 
 Number NAME
 OTHER IDENTIFIER 
(mainly field location)
 1 Cass
  2 Clare
  3 Lake
  4 Clinton  5 Mazzard
 NY54
 6 mahaleb  7 R1(1)
 24 3 (1)
 8 R2(1)
 24 2 (1)
 9 25 11 (15) 
 MB 2 (6)
 10 P01(01)
 MB 6 (30)
 11 P11 (2)
 MB 6 (50)
 12 P12 (2)
 MB 7 (15)
 13 26e 20 (50)
 26 B(13)
 14 148-1 (Gisela 6)
 MB 2 (29
-33)  1-4 =  MSU test rootstock
 5,6 and 14 =  Current cherry rootstocks 
 7-13 = B
reeding selections that have 
P. maackii
 in their ancestry
    !(.!Method 1:
 The periderm was removed
 from the twigs and macerated with
 a razor 
blade. One half gram
 tissue samples were
 placed in clean vials containing 12.5 mL of 
80% acetone. The ti
ssue was allowed to extract for
 three days at room temperature. 
After the initial extraction, 
tissues were ri
nsed twice with 5 mL of 100% acetone. All of 
the acetone extracts for each sample were combined for further analysis.
 The acetone 
extracts were concentrated to dryness by rotary evaporation at 37.2ûC. The residue 
remaining after evaporation of the solvent 
was dissolved 
in 2.5 mL of 80% acetone 
yielding a final concentration of 0.2
0 g tissue/mL
. The samples were stored at 
-20ûC. 
  Method 2:
 The periderm was removed from the twigs and macerated with a razor 
blade. One half g tissue 
samples 
were
 added to 62.5 mL of water 
in 125 mL flask
s. These sample
s were
 autoclaved for 35 minutes and then allowed to cool to room 
temperature. Once cooled, the tissue 
was filtered with Whatman #1 filter paper and was 
rinsed with 20 mL of water (2 x 10 mL rinses).
 The extracts were stored at 
-20ûC
 prior to 
lyophilization. The lyophilized material was dissolved in 80% 
acetone 
to a final concentration of 0.
10 g of tissue per m
L.   Method 3:
 The peri
derm was removed from the twigs and
 macerated by a razor 
blade. Samples containing 0.50
 g of tissue 
were
 placed into vials containing 20 mL of 
distilled water. The vials were
 place in a water bath at 99ûC 
for 2 hours. The water 
extracts were 
filtered and 
transferred to clean
 storage container
s and an additional 
10mL of distilled water
 was added back to the vials containing periderm tissue.
 The vials were 
placed back into the water bath for an additional hour to collect any additional 
!(%!compounds
 still contained
 in the periderm. The remaining supernatant was combined 
with the initial extraction and transferred to a separate container. The extracts were 
stored at 
-20ûC
 and then subjected to lyophilization. 
The lyophilized material was 
dissolved in 80% acetone to a final concentration of 0.1
0 g of tissue per mL. 
 Thin Layer C
hromatography (TLC)
 was used to separate compounds from a 
mixture
. TLC plates (20 x 20 cm) were purchased from Analtech. 
A small amount of the 
extract is applied approximately 2 cm from the bottom of a TLC plate, which is 
a glass 
plate coated with either 
cellulose or silica gel
. Cellulose plates were
 used for polar 
compounds 
and developed in water. Silica plates were used for non
-polar compounds 
and were
 developed in 90:10 chloroform 
Ð methanol 
(v/v)
. The TLC plate is placed in a 
glass container with appro
ximately 1
00 mL of solvent
. A zone was carved out at the 12 
cm mark
 to allow a separation length of 10 cm
. Plates were allowed to air dry in a fume 
hood and then were placed in a vacuum desiccator overnight for removal of any 
residual solvent. 
Aceton
e (0.2
0 g/mL), autoclave
 (0.10 g/mL) and hot water (0.10 g/mL) extract
s were used for TLC analyses. 
Since acetone was found to be the most efficient
 solvent for extraction
, it was used 
for extraction in
 all additional assays
 unless otherwise 
specified
.  To create a TLC bioassay, 
within 24 hours
 of development, the plates were 
evenly sprayed with a 
dense spore
 and mycelia
 suspension of 
Cladosporium
 cucumerinum
 in 
) strength potato
-dextrose broth
 and then placed in an incubation 
chamber for three days. The 
darkly pigmented 
hyphae of C. cucumerinum
 covered the 
entire surface of the TLC 
plate with the exception of 
areas containing antifungal 
compounds
 (Klarman & Sanford, 1968)
. !(&!For phenolic compound detection, a 
ChromaDoc
-It TLC imaging system
 with a Digi 105 color camera (12
-megapixel) 
was used
 to take pictures of TLC plates under UV
 light (365 nm)
 and under white light to record bioassay results
. Phenolic compounds 
were 
also 
detected on TLC plates using two reagents: diazotized 
p-nitroaniline
 (DPN) 
assay 
(Somaroo et al., 1973) and the v
anillin assay
 (Sarkar & Howarth, 1976). 
The vanillin reaction used to assay for tannin content in Sorghum grain was found to be 
extremely temperature dependent (Price et al., 1978).
 This assay is 
semi
-specific 
to a range of both mo
nomeric and polymeric flavanols, 
dihydrochalcones
 and anthocyanins
 (Sarkar & Howarth, 1976
). 
Additionally, in some cases 
this assay showed reactivity with 
some non
-flavanol compounds including phenolic acids, such as cinnamic acid and 
flavonols including kaempferol and myricetin (Sun et al., 1998). 
 Diazotized 
p-nitroaniline (DPN) is one of the various spray reagents used for the detection of 
phenolic compounds including syringic, 
p-coumaric and sinapic acids (Somaroo et al., 
1973). 
 Twigs (approx. 2
 cm in diameter) were collected for periderm extraction. The 
periderm was removed with a razor blade
 and was 
ground
 using a Krups¨ coffee 
grinder
. The periderm tissue was added to the YMPG medium prior to autoclaving.
 After 
autoclaving, t
he periderm amended
-medium was poured into the petri plates
 (150
 x 15 mm) and then inoculated
 with plugs (
5mm) of 
A. ostoyae
. Plugs were taken from the 
margin of actively growing cultures.
 The cultures were grow
n for three weeks and then 
the radial growth (in
 cm) was
 measured and recorded. 
The ChromaDoc
-It TLC imaging 
system was also used to take pictures of the Petri dishes containing 
Armillaria
 growth
. This system
 contains 
a white light setting
, which was used while taking pictures of 
!('!pathogen growth on
 media. The radial growth of 
pathogens 
was 
recorded, using 
the ruler tool in 
Photoshop. Measurements were analyzed using 
TukeyÕs HSD in the 
statistical computing program, R
 (R Core Team, 2014
).   Results
  Both c
ellulose
 and silica
 gel TLC plates were used
 to screen for antifungal 
com
pounds. TLC analyses of the extract (0.20 g/mL) from 
P. maackii
, P. serotina
 and P. 
mahaleb
 were used to determine preliminary information on the presence and chemical 
nature of the antifungal compounds. 
The best band resolutio
n was detected when 
extracts 
were 
applied to silica gel TLC plates and developed in a chloroform 9:10 v/v 
solvent (Figure 1.1). 
 !((!  Figure 1.
1. Separation
 of P. maackii
, P. serotina
 and P. mahaleb
 samples
 visualized 
with UV (365
 nm). Samples were developed on silica TLC plates using chloroform 
- methanol 9:1 (v/v)
 (top) and on cellulose TLC plates using water as the solvent 
(bottom)
.     !()!When extracts were applied to cellulose plates and developed in distilled water, 
the unknown compounds appeared as streaks instead of individual bands. 
For both 
plates shown in Figure 1.1, lanes 1, 2 & 3 contain 1, 2 & 3
µg equivalent of P. maackii
 periderm
 tissue
 (respectively)
. Lanes 4, 5 & 6 contain 1, 2 & 3µg of 
P. mahaleb
 periderm tissue, and lanes 7, 8 & 9 contain 1, 2 & 3µg of 
P. serotina
 periderm tissue. 
The Rf value can be used to characterize unknown compounds, based on their 
migration in various s
olvents. 
 The extracts were
 first examined und
er ultraviolet light (
365 nm). The unknown 
compounds can be characterized based 
on their R
f values and on their ability to
 absorb 
or fluoresce UV light. After the extracts are visualized on TLC plates under UV light,
 TLC bioassays can be done
 for the characterization of
 biological activity. 
TLC plates are 
sprayed with a spore and mycelia suspension of 
C. cucumerinum
 and any antifungal compounds 
present are 
visualized as white areas on the dark
ly pigmented TLC plate when the bioassay is complete.
 For both 
Figures 1.2 & 
1.3, lanes 1
-9 contain 
P. maackii
 periderm tissue. Lanes 
1-3 are samples from the acetone extraction, lanes 4
-6 are sample
s from the hot water 
extraction
 and lanes 7
-9 are samples from the autoclave hot water extraction. The same 
order follows for extraction types 
in lanes 10
-18 containing 
P. serotina
 periderm tissue 
and lanes 10
-18 containing extracts from 
P. 
mahaleb
. For both cellulose and silica gel 
TLC plates 
Ð solvent systems, 
P. maackii
 was the only species shown to have 
significant antifungal activity.
  !(*!  TLC analyse
s of
 extracts 
revealed that 
P. maackii
 was the only species that 
contained 
significant amo
unts of antifungal
 compounds 
as determined by bioassays
.  The separation
 with water of 
P. maackii
 periderm extract
 applied to a cellulose plate
 revealed three zones of antifungal activit
y, as shown by the white
 area
s against a da
rk background of fungal 
mycelia
 and spores
 (Figure 1.2)
. The bioassay results show that 
antimicrobial compounds migrate together when applied to a cellulose plate. 
In the acetone extracts, the major zone was found between R
f values of 
0.59 and 0.83 with a smaller zone at the orig
in (.07)
. Separation of extracts prepared with hot water or by 
autoclaving also showed zones of inhibition. However, the upper z
one was found over 
a smaller R
f range.  
Additionally, t
he activity at the origin was reduced
 in hot H2O and autoclaved H
2O extracts
. The extracts were also applied to
 silica gel TLC 
plates and were developed in a
 CHCl3: MeOH
 (v/v) 
solvent. The more nonpolar solvent 
revealed
 several zones of 
inhibition in the periderm extract from 
P. maackii
 (Figure 1.3). Similar to the results 
using cellulose plates, the activity was greatest in the acetone extracts. 
The antifungal compounds seem to be correlated with the unknown compounds that absorb purple 
under UV (365 nm)
 with Rf values of 0.08, 0.51, 0.7
4, and 0.90. The separation of 
acetone extracts from 
P. serotin
a on silica gel 
showed
 a small amount of activity at an 
Rf  of 0.80.  The separation of 
P. 
mahaleb
 extracts on silica gel 
showed compounds with
 antifungal activity
 with Rf values of 0.64 and 0.85. 
However, the activity was much less 
than what was observed with 
P. maackii
.    !(+!TLC results
 indicate
 that all three
-extraction
 methods were able to extract 
compounds from 
Prunus
 periderm tissues
.  However, 
increased white areas 
in the lanes 
containing acetone extracts suggest
 that this solven
t was 
the most efficient
 in antifungal compound extraction
. The zones of antifungal activity
 were smaller when periderm 
tissue was extracted with
 hot H2O and autoclave
 H2O, possibly because the 
hot water 
and autoclave extraction procedures may have
 destroyed
 some of the compounds.
               !(,! Figure 1.
2. The periderm 
extracts from 
P. maackii, P
. serotina
 and P. mahaleb
 were 
developed on cellulose TLC plates, in a water solvent and sprayed with 
C. cucumerinum
. Four microliters of acetone extract (
0.20g/mL) was used and eight 
microliters of hot water extract (
0.10g/mL) and autoclave extract (
0.10g/mL) were 
applied to the TLC plate.  Figure 1.
3. The extracts from 
P. maackii, P
. serotina
 and P. mahaleb
 were 
applied to silica TLC plates, 
developed in chloroform 
Ð methanol (9:1 v/v) and sprayed with 
C. cucumerinum
. Four microliters of acetone 
extract (
0.20g/mL), eight microliters of hot 
water extract (
0.10g/mL) and autoclave extract (
0.10g/mL) were applied to the TLC 
plate.   !(-!In order to determine if the antifungal properties
 were
 related to the age of the 
tree, 
extracts from 
five young trees 
were screened for antifung
al compounds and were 
compared with 
mature 
P. maackii
 trees.
 Periderm and cambia
l tissues were removed 
from 
twigs collected from young and mature trees for the 
comparison of chemical 
content. Samples 
from
 both age groups showed 
significant antifungal activity
. The young trees
 were not exposed to 
any soil containing 
Armillaria
 spp.
, suggesting t
he presence of phytoanticipins, which are 
constitutively present 
antimicrobial compounds
. However,
 trees may have been 
exposed to other mi
crobes
 naturally present in the 
growing environment
.  Dr. Amy Iezzoni, and Audrey Sebolt,
 kindly
 provided samples of the 
P. maackii 
x Clare
 hybrid seedling, which is part of the sweet and sour cherry breeding program. 
The periderm 
crude extract 
of P. 
maackii
 was 
compared to a 
P. maackii
 x Clare hybrid 
seedling
 to see if any antifungal compounds were inherited in the cross
 (Figure 1.4
). Crude extracts were first visualized under UV light (365 nm) and later sprayed with 
C. cucumerinum
 for TLC bioassay re
sults. 
Lane 1 shows the UV detection (365 nm) of 
compounds contained in the crude extract of 
P. maackii
 periderm tissue. Lane 3 shows 
the bioassay results of the same compounds in lane 1. Lane 2 contains periderm from 
the hybrid 
P. maackii
 x Clare. Lane 4 
shows the bioassay results of lane 2. Based on 
TLC bioassay results, the antifungal compounds did not appear to be inherited in this 
particular hybrid seedling. 
The periderm of 
P. maackii 
(PM) contained 
significant 
antifungal activity and the 
P. maackii
 x Clare hybrid seedling (PM x) did not show 
antifungal activity. 
 !).! Figure 1.
4. UV 
detection (365 nm) and TLC bioassay results for the 
crude 
periderm 
extract 
of P. maackii
 (PM) and 
P. maackii
 x Clare hybrid seedling (PM x)
.  All samples 
were 
0.20g/mL, 10 µL were applied to each lane. Silica TLC plates were used and 
developed in chloroform 
Ð methanol (9:1 v/v).
     !)%!It is critical that the source 
antimicrobial activity 
is also located in the roots as 
well. The periderm 
tissue 
from roots was examined
 to detect 
whether the inhibition was 
also found in the periderm below ground. Samples were collected from roots 
approximately 6 inches below ground.
  Antimicrobial 
activity was greater 
in the above ground peride
rm, but the roots did
 exhibit antifungal activity. This is significant since 
Armillaria
 tends not to do well in the above ground parts of trees. The fact that the 
antimicrobial compounds are present in the above ground periderm, suggests that this 
would make it difficult for t
he pathogen to cause disease
 above the soil line
. The phenolic content from the shoot periderm (P), cambium (C), root periderm (R) and wood 
(W) were compared under UV 
light (365 nm) and then sprayed with 
C. cucumerinum
 (Figure 
1.5). Lanes 1
-4 contain different 
older 
tissues collected from mature 
P. maackii
 trees; lane 1 contains periderm, lane 2 contains cambial extract, lane 3 contains root 
extract and wood extract was applied to lane 4. Antifungal compounds were detected in 
all tiss
ues screened.
 !)&! Figure 1.
5. UV 
detection (365 nm) and TLC bioassay of 
P. maackii
 periderm tissue (P), 
cambial tissue (C), peri
derm from roots (R), and 
wood (W)
. All
 samples were 
0.20g/mL, 
10 µL were applied to each lane. Silica 
TLC plates were used and developed in 
chloroform 
Ð methanol (9:1 v/v). 
 !)'!For phenolic compound detection, t
he crude extracts from 
P. maackii
 periderm 
were chemically compared to extracts from 
P. serotina
 and P. mahaleb
 (negative 
controls).  Crude extracts f
rom 
P. serotina
 (lanes 1, 4 & 7), 
P. mahaleb
 (lanes 2,5 & 8) 
and P. maackii
 (lanes 3, 6 and 9) were applied to silica gel TLC plates and developed in 
a chloroform
-methanol 9:1 v/v 
solvent 
(Figure 1.6
). Lanes 1
-3 of the TLC plate were 
sprayed with diazotized 
p-nitroaniline (DPN) reagent, lanes 4
-6 were sprayed with 
a vanillin reagent and lanes 7
-9 were sprayed with 
C. cucumerinum
 for 
the detection of 
antifungal compounds. 
 Based on the number of bands,
 the DPN reagent revealed that 
P. maackii
 contained 
higher phenolic content compared to the negative controls, 
P. serotina
 and P. mahaleb
. The DPN reagent detected four bands in 
P. serotina
 extract. The first band 
was detected directly above the origin, R
f  0.12, the second band at R
f  0.23, third at R
f 0.38, and a very faint band at R
f 0.66. The vanillin reagent only detected one band in 
the P. serotina
 extract at R
f 0.12. Seven
 bands from the 
P. mahaleb
 extract were 
detected using the DPN assay
 with the following Rf  values 0.12, 0.34, 
0. 37, 0.40, 0.44, 0.50, and 0.65. The vanillin reagent detected 
six
 of the seven
 bands 
with Rf  values 
0.12, 0.34, 0.40, 0.44, 0.50, and 0.65. The DPN 
assay detected eleven bands 
in the periderm 
extract of 
P. maackii
 with Rf  values 
0.09, 0.13, 0.33, 0.44, 0.51, 0.55, 0.62, 0.68, 0.75, 0.89, and 0.95 and the vanillin assay detected eight bands at 
0.13, 0.33, 0.44, 0.51, 0.62, 0.68, 0.89, and 0.95.   !)(! Figure 1.
6. Extracts 
from 
P. serotina, P. mahaleb
 and P. maackii
 were
 compared. 
Extracts were
 applied to a silica TLC plate
 (0.20 g/mL)
, developed in chloroform 
Ð methanol (9:1), 
sprayed with the DPN reagent 
(left)
, vanillin reagent 
(middle) and C. cucumerinum
 (right). 
 !      !))!Periderm and cambial
 extracts 
from young 
trees 
and from 
older tissues collected 
from mature trees 
were compare
d under UV light (365 nm) in 
lanes 1,2,5, & 6, and by 
using a vanillin spray reage
nt in lanes 3,4,7, & 8 (Figure 1.7
). The number of 
compounds, 
spot intensity and R
f values were 
compared. 
In both mature and young tree 
samples
, there were more bands in the periderm, detected under UV light and by the 
vanillin reagent, compared to
 cambial tissues. 
The extract from older tissues collected 
from mature
 trees 
contained 
higher phenolic content in 
cambial tissues than the young 
trees based on the number of bands detected under UV (365 nm). 
  Figure 1.
7. UV 
detection (365 nm) and v
anillin reagent 
comparison of 
periderm tissue 
from 
mature and young 
P. maackii
 trees. Crude extracts were applied to s
ilica TLC 
plates 
(0.20 g/mL) and 
were developed in chloroform
-methanol (9:1 v/v).
  ! !)*!TLC bioassays are not a practical option for 
growth inhibition screening of 
Armillaria 
species because of slow mycelial growth. 
Assays to test the inhibitory effects 
of the periderm and other tissues were conducted by incorporating plant tissues into 
culture media.
 Periderm incorporated bioassays (left to right) using tissue from 
P. 
maackii, P. serotina
 and P. mahaleb
 were compared to the 
control 
samples that 
contained only YMPG nutrient
-medium. The radial mycelial growth was recorded from 
all samples 
after three weeks
 of incubation
. Complete growth inhibition was only 
detected whe
n P. maackii
 periderm tissue was added to the YMPG 
(10g/L YMPG), 
while extracts from the other species did
 not inhibit growth (Figure 1.8
).  Additional samples of 
Prunus 
species and rootstocks, provided by Dr. Iezzoni 
and Audrey Sebolt, were screened for their ability to inhibit the growth of 
A. ostoyae
. Periderm from
 an additional 13 rootstocks plus 
P. mahlaleb
 (negative control)
 and P. 
maacki
i (positive control)
 were scr
eened for antifungal properties
. At the concentration 
of 10 g/L YMPG, o
nly periderm from 
P. maackii
 inhibited growth of 
A. ostoyae
 (Figure 
1.9).  
To ensure that the
 other rootstocks 
didnÕt have the potential to inhibit, the amount 
of periderm in 
the media
 was increased to 17 
g/L YMPG
 and cultures were grown for 
two weeks. 
Even with 
additional periderm tissue added to the samples, 
only the periderm from 
P. maackii
 was 
able to completely inhibit the growth of 
A. ostoyae
 (Figure 
1.10). Crystals app
eared only 
on the surface of
 P. maackii
 periderm
-amended medium, 
approximately two weeks after preparation (discussed in later chapters)
.   !)+! Figure 1.
8. The growth of 
A. ostoyae
 on periderm
-amended media (10 g/L YMPG) of 
P. maackii
 (1)
, P. serotina
 (2)
 and P. mahaleb
 (3)
. The control growth is shown on YMPG 
nutrient
-medium (4).
 Cultures
 were grown for 3 wk.
     Figure 1.
9. Survey 
of Prunus
 species and 
rootstocks (10 g periderm tissue/L YMPG) for 
their ability to 
inhibit the growth of 
A. ostoyae
. Samples 
were grown for 3 wk.
 .!./)!%!%/)!&!&/)!Growth of 
Armillaria ostoyae 
(cm) 
Rootstock Type 
!),! Figure 1.
10. Survey 
of Prunus
 species and 
rootstocks (17 g/L YMPG) for their ability to 
inhibit the growth of 
A. ostoyae
. Samples 
were grown
 for 2 wk.
           .!./&!./(!./*!./,!%!%/&!Growth of 
Armillaria ostoyae 
(cm) 
Rootstock Type 
!)-!Discussion  Armillaria
 species 
can inf
ect over 700 species of plants 
including fruit, nut and 
vine crops.
 Armillaria root rot was previously controlled by soil fumigation with methyl 
bromide (Larue et al., 1962), but it was phased out because of its harmful effects on the 
environment. F
inding a resistant species is critical for proper management of this 
disea
se (Solta
ntoyeh et al., 2014). Pears are considered to be immune or highly 
resistant to 
Armillaria
 infection. Thus, it is recommended that pear 
may serve as
 an alternative crop to repla
nt in infected sites (Dalili et
 al., 2010). However pears are 
susceptib
le to infection caused by 
Erwinia amylovora
 in states that have significant fire 
blight pressure
. Crop rotation is often utilized to reduce pathogen inoculum, but this 
means of control is not practical for forest or fruit trees. The removal of infected roo
ts 
and stumps is one management strategy that successfully reduc
es inoculum levels 
(Morrison et
 al., 2014). 
Additionally, the orchard may be left fallow for a number of years 
to allow for the breakdown of wood and roots, which may harbor the pathogen. 
 The periderm serves as a structural barrier and varies in texture, color and 
thickness depending on the species (Dickison, 2000).
 New periderm tissue is produced 
each year, which accumulates as trees mature. Periderm tissue is non
-living, compared 
to the livi
ng vascular 
and cambial tissue. 
In both young and mature trees, the 
accumulation of additional layers results in thicker
 periderm tiss
ue, which may 
contribute to increased concentrations
 of tannins, phenolics, waxy substances, etc. 
 Periderm tissue from se
veral
 Prunus
 species and rootstocks were screened for
 phenolic 
compo
unds, which
 can range from single
-aromatic ring
s to complex tannins
. Phenolic 
!*.!compounds
 are
 often involved in host defense against UV radiation a
nd plant pathogens (Ferretti et
 al., 2010). The periderm of 
P. maackii
 contained a greater 
number of
 UV- fluorescing and absorbing bands
. Vanillin and DPN reagents were used 
to further characterize these compounds
 as phenolics
. These reagents
 also
 detected 
the grea
test
 number of bands
 in the periderm extract from 
P. maackii
 compared to 
extract from 
P. mahaleb
 and P. serotina
.  The incorporation of periderm tissue into culture media was used to screen for 
growth inhibition of 
A. ostoyae
. It is likely that the antifungal compounds detected in 
the TLC bioassay are extracted from the periderm during the autoclaving process and 
contribute to antimicrobial activity. Complete g
rowth 
inhibition of A. ostoyae
 was 
detected at 10 g /
L YMPG. Cryst
als appeared on the surface of the 
P. maackii
 periderm
-amended medium approximately
 two weeks after the date of preparation.. 
Results suggest that t
he extract slightly decreases in antimicrobial activity as the 
periderm
-amended medium ages, indicating that some antifungal compounds crystallize 
out of the medium.  Plants need to balance the costs and benefits of 
defense responses in the 
absence of enemies because employing these me
chanisms is costly to the host 
(Thaler 
et al, 2012). 
Future research could include examining 
P. maackii
 as a 
model host 
system 
for the
 balance 
of constitutive
ly present
 antifungal compounds with any 
inducible phytoalexins present. 
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                            !!**!CHAPTER TWO
  IDENTIFICATION OF FLAVONOIDS FROM 
PRUNUS MAACKII
 TISSUES
       !ABSTRACT
  Prunus maackii
 was identified as a potential source of resistance to 
Armillaria 
species. Previous research 
using 
Thin Layer C
hromatography
 (TLC) 
bioassays 
detected the presence of antifungal 
compounds that are most likely phenolic in
 P. maackii
 periderm extract. 
Prunus maackii
 periderm 
collected from
 both young trees 
and older tissues from 
mature
 trees completely inhibit
ed the growth of 
A. ostoyae
 when added to the YMPG nutrient
-medium. Prunus maackii
 contained twice the amount of 
dried extract compared to 
P. mahaleb 
and P. serotina
. High-performance liquid 
chromatography 
coupled with mass spectrometry (HPLC
-MS) was used to characterize 
the unknown compounds by comparing molecular 
masses and retention times. Dry
-column chromatography was used to separate and purify the crude extract.  X
-ray 
crystallography was used to ident
ify the unknown compounds. Alnusin and two 
stereoisomers 
of alnustinol were identified from
 the crude periderm extract
 and were 
found to be antifungal, possibly contributing to the broad
-spectrum antimicro
bial activity 
of the P. maackii
 periderm extract
. Dihydrowogonin was identified from
 the crude extract 
of P. maackii
 leaves. These flavonoids are unique compared to those commonly found 
in other 
Prunus
 species because of the absenc
e of substituents on the B
-ring.
  !*+!Introduction
  Phytoanticipins are preformed compounds that create the first chemical barrier in 
some host
-microbe interactions. Distinction between phytoalexins and phytoanticipins is 
based on when they are produced. Phytoanticipins are constitutively present whereas 
phytoalexins accumulate after exposure to pathogens (VanEtten et al., 1994). 
Phytoalexins are synthesized as a result of attempted pathogen infection. 
Phytoanticipins may be stored in vacuoles in their glucosylated forms, which increases 
solubility and prote
cts the host from the toxic effects of the aglycone. The aglycones are 
released through hydrolysis by 
#-glucosidases (Morant et al., 2008). 
  Research reported in the previous chapter demonstrated the presence of 
antifungal activity through bioassays using
 periderm extracts prepared with 80% 
acetone, hot water and with water 
used for extraction during the
 autoclaving
 process
. The antifungal compounds were partially characterized and determined to be phenols, 
most likely flavonoids. Among the 
Prunus 
species 
and rootstocks screened, 
P. maackii
 was the only 
Prunus
 species that showed significant antifungal activity.
  There are various chemical forms of phytoanticipins, which include terpenoids, 
dienes and phenolic compounds (Aharoni et al., 2005). 
Phytoanticipi
ns 
also 
include 
saponins, cyanogenic glycosides and glucosinolates (Iriti & Faoro, 2009). The seeds of 
Prunus spinosa
 contain cyanogenic glycosides including amygdalin, prunasin and 
sambunigrin (Kumarasamy et al., 2003). Avenacins, terpenoid sapnonins, hav
e potent antifungal activity and have been shown to play a role in resistance of oats to certain 
varieties of the
 Gaumanannomyces graminis
, which is often referred to as the take all 
fungus. 
 !*,!Take
-all is a disea
se of cereal plant roots
.  Evidence supportin
g the role of
 avenacins
 in host 
defense comes from surveys of 
Avena
 spp.
 that show little variation in 
avenacin content with exception of a diploid species (
Avena longiglumis
), which lacks 
avenacin A
-1. This species is significantly more susceptible to infection by 
G. graminis
 than other 
Avena
 spp. (Osbourn, 2003). Avenacin B
-1, also absent or present in trace
-amounts in 
A. longiglumis
, was found to be of secondary importance 
compared to 
avenacin A
-1 in terms of aiding in resistance (Osbourn et al.,1994). Saponin
-deficient 
mutant varieties of 
A. strigosa
 were unable to produce 
#- amyrin, which is required for 
the production of avenacins (Trojanowska et al., 2001). Oat species t
hat lack the 
saponin avenacin are more susceptible to the take
-all fungus 
Gaumanannomyces 
graminis
 var. 
tritici
 (Osbourn et al., 1994).
 Avocado fruit contains long
-chain dienes that suppress 
Colletotrichum 
gloeosporioides
 growth in the epidermis of young f
ruits (Shin et al., 2014). When 
avocado fruits were inoculated with 
C. gloeosporioides
, the concentration of the 
preformed diene, 1
-acetoxy
-2-hydroxy
-4-oxo-heneicosa
-12,15-diene, was 
nearly 
doubled. Mechanical wounding also enhanced the concentration of th
e diene in the peel and flesh of avocado. However, wounding only enhanced concentrations over a 24
 h period compared to 3
-4 days with 
inoculated fruits (Prusky et al., 1990). 
The concentration of antifungal 
dienes decrease
 as the fruit 
matures
 (Prusky et a
l., 1983). 
Disease resistance in strawberry cv. Elsanta fruit and flowers against 
Botrytis cinerea
 is also
 dependent on concentrations of 
preformed antifungal compounds (Terry et al., 
2004). 
 !*-!Additional examples of phytoanticipins include prenylated genist
ein, which may 
function as a phytoanticipin in lupin seedling development (Dixon & Ferreira, 2002). 
Andean lupin seeds (
L. mutabilis
) are a potential source of isoflavones, including 
genistein (Ranilla et al., 2009). Constitutive antifungal resorcinols in 
mango fruit 
(Mangifera indica
) inhibit the development of black spot by 
Alternaria alternata
 (Adikaram et al., 2010).  In response to peeling, mango fruits contained 5
-substituted 
resorcinols in the outer layer of flesh in similar concentrations found in s
usceptible 
ripened peels. Fruits that were peeled or ripened contained approximately half the 
amount of resorcinols compared to the concentration found in resistant unripened peels 
(Droby et al., 1987).
  Objectives of this research include
 the isolation 
of phytoanticipins present in 
P. 
maackii
 periderm and leaves. Suitable single crystals collected from purified fractions 
will be used for x
-ray crystallograp
hy analyses, which will provide 
structure 
identification.
 These structures will be used to further 
elucidate the unique 
phenylpropanoid pathway found in this ornamental species. 
         !+.!Materials and Methods
  Young (< 5 years) 
P. maackii
 trees 
were purchased from 
Nature Hills Nursery, 
Omaha, NE, and 
planted in large pots
, using Baccto¨ premium potting 
mix, and
 grown 
in the MSU greenhouse courtyard. As needed, periderm and cambial tissues were 
removed from the young twigs and
 were
 used for 
chemical 
analyses. 
Mature 
Prunus
 twigs (2
-3 cm in diameter) were collected at the NW Michigan Horticulture Research 
Station near Traverse City, MI and stored 
(-20ûC
) until used for analysis. 
Older p
eriderm 
tissue was also collected from a mature 
P. maackii
 tree, accession number 97P028*02, 
on Michigan State UniversityÕs campus, samples were kindly provided by Dr. Frank 
Telewski.
 The periderm was removed from the twigs and macerated with a razor blade. 
Thirty grams of periderm tissue were collected from 
P. maackii, 
P. mahaleb
 and P. serotina
 and placed in 500 mL of 80% acetone. 
 These samples were allowed to extract for three days at room temperature. After 
the initial extraction, the tissue was rinsed twice with 100 mL of 100% acetone. All of the 
acetone extracts for each sample were combined and concentrated to near dryness by 
rotary 
evaporation at 37ûC. The samples were further concentrated to complete dryness 
by lyophilization. In dry weight, 4.5 grams of extract were collected from the thirty grams 
of P. maackii
 periderm tissue, 1.92 grams from 
P. serotina
 and 1.72 grams from 
P. mahaleb
. Samples were stored in a
 freezer (
-20ûC
) until needed. Leaves were collected and extracted on June 4
th, 2013. Three grams
 of fresh 
leaves were collected from 
P. maackii
 and placed in 100 mL of 80% acetone.
 Green 
buds were collected and extracted on 
June 5
th, 2015. These samples were allowed to 
!+%!extract for three days at room temperature. The extract was filtered 
using Whatmanª 
grade 1 
filter paper and the solvent was evaporated using a rotary evaporator. Cry
stals 
were formed upon evaporation and were removed from the crude extract. The crude 
extract was re
-suspended in 8 mL of 80% methanol. Samples were stored at 
-20ûC.
   High-performance liquid chromatography coupled with mass spectrometry 
(HPLC
-MS) 
requires
 a very small amount of analyte. Samples
 contained 
approximately 
10 mg periderm tissue
/mL 80% methanol and 10µL
 was used for analyses. Solvent A 
was 0.1% Formic Acid (FA) in H
2O and Solvent B was acetonitrile. Run time was set to 
12 minutes. An Ascentis ¨ 
Express 10 cm C18 column was used for analyses.
  Dry column
-chromatography was 
used to separate 
individual compounds in the 
crude 
periderm
 extract
. Silica gel was purchased from Jade 
Scientific Inc.
 and was used 
to pack the column for the
 collect
ion and purification of compounds. 
Six grams of crude 
extract w
ere
 dissolved in methanol and mixed with 10 grams silica gel. The
 column was 
made from nylon tubing purchased from Sorbtech and packed with silica gel. The tubing 
was 4 cm in diameter and appro
ximately 12 cm long. Glass wool was purchased from 
Alltech¨ and was placed in the bottom of the column, with holes punched through the 
surface to allow air to escape as the solvent moved through the dry silica gel.  Sand 
was placed on the top of the 
column
 so th
at the solvent wouldnÕt disrupt the silica gel as 
it was added
. Chloroform: m
ethanol, 90:10 (v/v) was used as the solvent. A hand
-held UV light (Spectroline, Westbury) was used to detect and locate the different UV 
absorbing bands from the periderm e
xtract contained in the column. Based on UV 
detection (365 nm), the column was cut into sections according to different 
fluorescing/absorbing bands. Each isolated fraction from the column was placed in 80% 
!+&!methanol for the re
-elution of compounds. The sili
ca gel was centrifuged out of the 
samples using a Beckman Coulterª Microfuge¨ 22R centrifuge.
  All fractions from the column, in add
ition to crude extract 
collected 
from
 the leaves, were concentrated to dryness by the use of a rotary
-evaporator (KDscientific, 
Holliston). 
The dried 
powder 
product
 from the crude extract 
of leaves and individual 
fractions from the periderm extract were dissolved in 1 mL of c
hloroform (J.T. Baker, 
Austi
n) 
contained in scintillation vials (Sigma
-Aldrich Co., St. Louis). One drop of 
toluene (J.T. Baker, Austin) was added 
to the samples 
prior to capping the scintillation 
vials, giving an approximate concentration of 95% chloroform. Crystals were formed 
after 3 days of slow
-evaporation. Dr. Richard Stap
les, Michigan State University 
Department of Chemistry and Chemical B
iology, provided x
-ray crystallography 
analyses.
  Thin Layer C
hromatography
 assays were 
used to confirm the antimicrobial 
activity of three 
fractions collected
 using 
dry
-column 
chromatography and identified
 using
 x-ray crystallography
. Five microliters of fractions were applied to a silica TLC
-plate and developed with a chloroform
-methanol, 90:10 (v/v) solvent. Within 24 hours of 
developing the TLC plates, the plates were evenly sprayed with a
 dense fungal 
suspension of 
Cladosporium
 cucumerinum 
in 
) strength potato
-dextrose broth and then 
placed in a moist incubation chamber for three days to allow fungal growth. 
  !!!!!!+'!Results
 !Preliminary HPLC
-MS analyses of 
the crude extract from 
older periderm tissues 
collected from a 
mature 
P. maackii
 tree, 
detected over five different compounds 
present, with flavonoid structural characteristics.
 The peaks and shoulders can be used 
to identify the class of the unknown compound.  Dihydroflavonols, flavanones and 
isoflavones can be readily distinguished from flavones and flavonols by their UV spectra 
due to having little or no conjugation between the A
- and B- rings. 
 In methanol, dihydroflavono
ls show a major peak in the range of 270 
Ð 295 for 
band II, with only a shoulder or low intensity peak for band I (Mabry et al., 1970).
 An unknown compound
 with a molecular weight of 301
, in negative ion mode, was in
 much 
higher concentration than the rest
 of the other 
unknown 
compounds (Figure 2.1). 
This 
unknown compound
, 301 m/z, 
was 
present in 
samples from 
both young trees (<
5 years 
old) and more mature trees 
(>12 years)
 with a retention time of 4.6 minutes
 (Figure 2.2). 
 MS/MS analysis of the 301m/z 
peak produced 225.05, 213.06, 168.00, 197.06, 
183.03, and 242.05 m/z fragmentation, 
indicating how the flavonoid structure was 
broken apart
 (Figure 2.3)
. The UV spectrum of the 
unknown 
compound with the 
301-m/z peak is also charac
teristic to that of flavonoids, having a 
major peak at 293.9 nm 
with a shoulder peak in the region of 340
-360 nm (Figure 2.4). 
 !+(! Figure 2.
1. HPLC
-MS 
analysis of 
periderm extract from 
older tissues collected from 
a mature 
P. 
maackii
 tree
. Potential names were assigned based on retention times and 
masses. 
  !+)! Figure 2.
2. HPLC
-MS 
comparison of crude 
periderm 
extract; samples were collected 
from a
 young P. maackii
 tree
 (top) and
 older tissues were collected from a mature tree
 (bottom).
 !+*! Figure 2.
3. MS/MS 
of unknown compound with 301 m/z
 from periderm tissue of 
P. 
maackii
.  !++! Figure 2.
4. The UV spectrum of the unknown 
compound with 301 m/z
 from the periderm 
tissue of 
P. maackii
.                !+,!Based on UV detection (365 nm) and polarity, three
 semi
-purified
 fractions were 
collected from the 6 g of crude
 P. maackii
 periderm
 extract
. Dry
-column 
chromatography was used to separate 
out the three fractions from the 
crude 
periderm 
extract
. All fractions
 collected
 from the periderm were re
-eluted from the silica gel with 
80% methanol and TLC analysis was used to confirm 
the purity of fractions based o
n the number 
of bands present under UV light
.  Compound X was the most non
-polar compound present in the silica gel; 
compound Y was the second most non
-polar compound present. All semi
-purified 
fractions were submitted for X
-ray crystallography analyses, but only three samples 
contained suitable single
 crystals; 0.39 g of compound X from fraction 4 and 0.28 g of 
compound Y from fraction 3. The third sample from fraction 2 was identified, as a 
stereoisomer of compound Y. 
A modified version of the bioassay was done to see if any 
antifungal activity is pre
sent in any of the isolated fractions. 
All fractions collected were 
only partly purified.
 Fraction 2, 3 and 4 showed antimicrobial activity to 
C. cucumerinum
 (Figure 2.5). 
  !+-!  Figure 2.
5. Modified 
bioassay of isolated fractions from the periderm of 
P. maackii
.   X-ray crystallography identified the unknown compounds as 3,5,7
-trihydroxy
-6-methoxyflavone (alnusin) and 3,5,7
-trihydroxy
-6-methoxyflavanone (alnustinol). Lane 1 
contains semi
-purified 
alnusin and alnustinol. Lane 2 has more purified alnustinol, 
compared to lane 1. Lane 3 contains mostly alnusin, wi
th small amounts of alnustinol 
(Figure 2.6).
  !,.! Figure 2.
6. UV 
detection (365 nm) use
d to determine the purity of
 three biologically 
active fraction
s 2, 3 and 4 collected 
from 
crude 
P. maackii
 periderm extract
.   3,5,7-trihydroxy
-6-methoxyflavone (alnusin) and 3,5,7
-trihydroxy
-6-methoxyflavanone (alnustinol) were obtained during the 
P. maackii
 periderm extraction 
process. 
A single crystal was used for the identification of 3,5,7
-trihydroxy
-6-methoxyflavone
, The common name for this flavonoid is alnu
sin
 (Figure 2.7).
 An 
additional crystal was used to identify 3,5,7
-trihydroxy
-6-methoxyflavanone,
 which is 
commonly called alnustinol (Figure 2.8). The crystal used to identify alnustinol also 
showed potential hydrogen bonding properties (Figure 2.9). Although the only difference 
between the two compounds in Figure 2.7 and
 Figure 2.8 is a double bond 
contained in 
!,%!the C-ring, the two crystals 
used for identification have 
different shapes. 
A third crystal 
was used to identify a
 stereoisomer of 3,5,7
-trihydroxy
-6-methoxyflavanone (
Figure 
2.10) and its potential hydrogen b
onding properties (Figure 2.11). 
  Crystal Data for 
3,5,7-trihydroxy
-6-methoxyflavone (Compound X):
 C16H12O6, M =300.26, triclinic, 
a = 7.2248(14) †, 
b = 7.3142(14) †, 
c = 12.588(2) †, 
' = 84.755(2)¡, 
# = 79.644(2)¡, 
* = 82.434(2)¡, 
V = 647.1(2) †3, 
T = 173.15, space group P
-1 (no. 2), 
Z = 2, µ(MoK
'
) = 0.119, 5928 reflections measured, 2396 unique (
Rint = 0.0445) which 
were used in all calculations. The final 
wR2 was 0.0948 (all data) and 
R1 was 0.0462 
(>2sigma(I)).
   Figure 2.
7. Single 
crystal used for x
-ray c
rystallography analysis and the corresponding 
structure of 3,5,7
-trihydroxy
-6-methoxyflavone. 
     !,&!Crystal Data for 
3,5,7-trihydroxy
-6-methoxyflavanone (Compound Y):
 C16H14O6, M =302.27, monoclinic, 
a = 13.751(2)
 †, 
b = 5.4210(8)
 †, 
c = 36.666(6)
 †, 
# = 100.204(2)¡, 
V = 2690.0(7)
 †3, T = 173.15, space group C2 (no. 5), 
Z = 8, µ(MoK
'
) = 0.115, 11109 reflections measured, 4681 unique (
Rint
 = 0.0559) which were used in all 
calculations. The final 
wR2 was 0.0948 (all data) and 
R1 was 0.0513 (I > 2
\s(I)). 
   Figure 2.
8. Single 
crystal used for x
-ray c
rystallography analysis and the corresponding 
structure of 3,5,7
-trihydroxy
-6-methoxyflavanone.
 !,'! Figure 2.
9. Potential 
hydrogen bonding of 
3,5,7-trihydroxy
-6-methoxyflavanone
 identified using x
-ray crystallography
.  Crystal Data for 
3,5,7-trihydroxy
-6-methoxyflavanone (stereoisomer of Y):
 C16H16O7, M =320.29, triclinic, a = 7.4815(8) †, b = 9.7111(11) †, c =
 10.6107(12) †, 
'
 = 76.6330(10)¡, 
#
 = 72.8300(10)¡, 
* = 87.6590(10)¡, V = 716.28(14) 
†3 , T = 173.15, space group P
-1 (no. 2), Z = 2, µ(MoK
'
) = 0.118, 11802 reflections 
measured, 2613 unique (Rint = 0.0309) which were used in all calculations. The final 
wR2 was 0.2258 (all data) and R1 was 0.1079 (>2sigma(I)). 
    !,(!One single crystal was also used for the identification of a stereoisomer of 3,5,7
-trihydroxy
-6-methoxyflavanone (Figure 2.10). The only difference between the 
compounds in Figure 2.8 and 2.10 is d
isorder at the C1 and C2 atom positions and a 
water molecule of co
-crystallization from the solvent. 
Thus, the bonds at C1 and C2 are 
the same in both stereoisomers, but have different configurations. 
Despite being 
stereoisomers, the crystals used for iden
tification appeared 
differently when compared.
   Figure 2.
10. Single 
crystal used for x
-ray c
rystallography analysis and the 
corresponding structure of the stereoisomer, 3,5,7
-trihydroxy
-6-methoxyflavanone.
  !,)! Figure 2.
11. Potential 
hydrogen bonding of 3,5,7
-trihydroxy
-6-methoxyflavanone 
(stereoisomer)
 identified using x
-ray crystallography
. ! Crystals were also formed
 when leaves were placed in
 80% acetone
 and extracted for 3 or more days
. The crystals were 
collected
 from 
the crude extract of 
P. 
maackii
 leaves 
and one suitable crystal was isolated 
and used for the 
identification of 
(S)-5,7-dihydroxy
-8-methoxy
-2-phenyl chroman
-4-one (Figure 2.12).
 This flavonoid is 
commonly called dihydrowogonin.
 Hydrogen
-bonding properties were also identified in 
dihydrowogonin molecules (Figure 2.13). 
    !,*!Crystal Data for (S)
-5,7-dihydroxy
-8-methoxy
-2-phenyl chroman
-4-one: C17.75
H16O5, M =309.30, monoclinic, 
a = 9.74520(10)
 †, 
b = 23.7471(3)
 †, 
c = 13.3595(2)
 †, 
# = 104.4600(10)¡, 
V = 2993.72(7)
 †3, T = 172.99, space group P2
1 (no. 4), 
Z = 8, µ()
 = 0.835, 23919 reflections measured, 9001 unique (
Rint
 = 0.0496) which were used in all 
calculations. The final 
wR2 was 0.1824 (all data) and 
R1 was 0.0609 (I > 2
\s(I)).
   Figure 2.
12. Single 
crystal used for x
-ray c
rystallography analysis and the 
corresponding structure of (
S)-5,7-dihydroxy
-8-methoxy
-2-phenyl chroman
-4-one. !,+! Figure 2.
13. Potential 
hydrogen bonding of (
S)-5,7-dihydroxy
-8-methoxy
-2-phenyl chroman
-4-one identified using x
-ray crystallography
. !!!!!!!!!!!!! !,,!X-ray crystallography identified the structures of two unknown compounds from 
crude 
P. 
maackii
 periderm
 extract.
 Samples containing a
lnusin (compound X) and 
alnustinol (co
mpound Y) were analyzed by TLC 
and were 
screened for antifungal 
activity
. Samples of alnusin and alnustinol were compared under UV light (365 nm). 
Based on the presence of 
additional bands, the samples containing alnusin and 
alnustinol were only partly purified.  Both of these fractions contained more alnusin, but 
the latter 
sample named alnustinol 
contained both 
alnusin and alnustinol 
in more 
comparable amounts. 
 After The 
TLC plates were
 compared under UV light, 
the same plates were used 
for the bioassay detection of 
antifungal compounds. TLC plates were 
sprayed with a 
dense 
C. cucumerinum
 spore and mycelia
l suspension. 
 Both flavonoids showed 
antifungal activity; w
hite areas on the TLC plate 
are a result of 
growth
 inhibition of C. cucumerinum
, indicating the 
presence of antifungal compounds
 (Figure
 2.14). 
  !,-! Figure 2.
14. UV 
detection (365 nm) and bioassay of semi
-purified fractions both 
containing alnusin (19
5mg/mL 80% MeOH), approximately 80% pure on left lane; right 
lane contains approximately 40% alnustinol (140mg/mL 80% MeOH).
 !!  !-.!To visualize which compounds crystallized, the crystals 
collected during the 
extraction process 
were dissolved in 100% methanol for TLC analysis. 
Based on UV 
light (365 nm) detection, most all of the compounds contained in the 
crude
 P. maackii
 periderm extract 
were capable of forming crystals
. The crystals that were collected 
during the extraction pr
ocess
 (lane 1) 
were compared in Figure 2.15 to the compounds 
present in 
the crude
 periderm
 extract (
lane 2). Bioassay results indicated that the 
antimicrobial activity was significant in both samples of crystal extract 
(lane 3) 
and cru
de periderm extract
 (lane 4) (Figure 2.15
). 
   !-%! Figure 2.
15. Most 
of the compounds, including antimicrobial metabolites, present in the 
crude periderm extract of 
P. maackii
 were able to form crystals. 
 !!!!!!!!!!!-&!The bands of compounds under UV 
light (365 nm) are shown in the top TLC 
plate with the bioassay results shown in
 the bottom plate in Figure 2.16
. A black and 
white filter was applied to the bioassay to better visualize the antifungal regions.
 Methanol extract from the crystals was applied in lane 1, crude leaf extract was applied 
in lane 2 and the crude extract of 
P. maackii
 buds was applied in lane 3. All
 three 
samples contained antifungal compounds, which were detectable und
er long
-wave UV 
light. The extract 
from 
P. maackii
 leaves 
appeared to contain antifungal compounds with 
higher R
f values, indicating that some of the compounds are considerably non
-polar. 
 !-'! Figure 2.
16. Antimicrobial 
compounds present in the leaves and buds of 
P. maackii
.       !-(!Some of the compounds took on crystalline form while in the 80% acetone crude 
extract
 of P. maackii
 leaves. 
The crystals were dissolved in methanol and applied in 5, 
10 and 15 µL in the left 3 lanes 
and 5, 10 and 15 µL of crude 
extract
 from leaves
 was 
applied in the right three lanes of the
 TLC plate shown in Figure 
2.17. Dihydrowogonin
 would be expected to have
 similar chemical properties
 to that of alnusin and alnustinol 
because
 of their similar substituent patterns. F
lavanones with a 5
-OH group lacking B
-ring 
hydroxyl groups 
have been documented to 
appear deep purple under UV (Mabry et 
al.,1970). Further purif
ication of the
 extract
 from leaves
 is needed to identify the exact R
f value and
 to determine
 if there is any 
biological activity of dihydrowogonin. The
 expected R
f value (shown in red box) 
is based on 
migration
 values similar
 to alnusin and 
alnustinol
.         !-)! Figure 2.
17. 5, 10 and 15 µL of crystal
 extract containing dihydrowogo
nin (left) 
compared under UV light (
365 nm) to the crude extract 
(right) of 
P. maackii
 leaves 
(40mg/mL 80% acetone). 
Estimated R
f value of dihydrowogonin is in 
the red box. 
   !!-*!Discussion  This is the first report of alnusin and alnustinol in the genus 
Prunus
. This is also 
the first report of crystallization and antimicrobial activity for both compounds. Alnusin 
and alnustinol were 
first isolated from the male flower of 
Alnus sieboldiana
, but not from 
the closely related species 
Alnus pendula
 (Suga et al., 1972). Both compounds were 
identified later from the leaf and stem resin produced by 
Baccharis bigelovii
 (Arriaga
-Giner, 1986) 
and Chromolaena chaslaea
. Alnusin
 has been identified in trace amounts 
in Gnaphalium microcephalum
 and from the leaf exudates of 
Anaphalis margaritacea 
(Wollenweber et al., 1996). Alnustinol has also been reported in 
Cassinia quinquefaria
 (Wollenweber, 199
3). Alnusin and alnustinol are somewhat unique with 6
-methoxylation 
and lack of B
- ring substitution (Asakawa, 1971). 
 Numerous flavonoids and other phenolic compounds have been identified in 
various tissues of 
Prunus 
species. The leaves of 
Prunus persica
 contain various 
phenolic compounds including prunasin and amygdalin and the bark was reported to 
contain persicogenin, naringenin, aromadendrin, 
and eriodictyol (Backheet et al., 2003). 
Genistin, prunin, isosakuranin, chrysin
-7-glucoside, and dihydrowogoni
n-7-glucoside 
were isolated from a 
Prunus
 heterograft,
 Prunus avium
 as the scion and 
Prunus 
cerasus
 as the rootstock. These compounds were found to move from their original 
compartments and were concentrated in the scion, which may result in slight or delayed 
incompatibility (Feucht & Treutter, 1991). Dihydrowogonin
 has been isolated from 
Adenostoma spa
rsifoliaum
 and from the heartwood and bud exudate of 
Prunus
 species 
(Wollenweber, 1996). 
 !-+!Prunet
in 5-glucoside, tectochrysin 5
-glucoside and pinostrobin 5
-"-D-glucoside 
distinguish 
P. cerasus
 from 
P. avium
. Genistein 5
- glucoside (Geibel et al., 1990), 
apigenin 5- glucoside, genkwanin 5
- glucoside
, and neosakuranin are minor 
components of both species (Geibel & Feucht, 1991). 
Prunus cerasus
 is more resistant 
to the perennial canker pathogen 
Cytospora persoonii
, in comparison to the 
susceptibility of 
P. aviu
m. Flavonoid aglycones are present in trace amounts in 
unwounded bark of 
Prunus 
spp.
 suggesting that
 hydrolysis of flavonoid aglycones from 
their glucosides may be involved in resistance. Stock solutions of naringenin (2.5 mM) 
and chrysin (1.0 
Ð 2.5 mM) inhibited the growth of 
C. persoonii
 (Geibel, 1995).
 Two stereoisomers of alnustinol were identified i
n the periderm of 
P. maackii
. The biological significance of stereoisomers needs to be further investigated
. Stereoisomers have the same chemical bonds, but different configurations
, and cannot
 be interconverted without breaking covalent bonds (Nelson et a
l., 2008). Hydrogen 
bonding involves electrostatic interactions between electronegative atoms, which 
involve weaker interactions compared to covalent bonding. 
Further research is needed 
to determine if hydrogen bonding has any influence on the biological a
ctivity of 
flavonoids. 
 Phenols that form intermolecular hydrogen bonds are typically solid at room 
temperature (Vermerris & Nicholson, 2007), which allows for crystalline solid samples. 
Based on
 comparing
 experimental 
data, the periderm amended
-medium from 
P. 
maackii
 (24+ hours) 
that contained crystals on the surface had slightly less antimicrobial 
activity compared to freshly made medium
 that did not have crystals. These results 
!-,!suggest that the 
compounds
 crystallized
 out of the agar, which could resu
lt in a loss of 
available compound.
  The primary metabolite phenylalanine serves as a precursor to flavonoids found 
in plant tissues including dihydroflavonols, flavones, flavanones, and flavonols. 
Phenylalanine ammonia lyase (PAL) is found in the parenchy
ma cells of many plants 
and catalyzes the first step of flavonoid biosynthesis (Seigler, 2012). 
 Chalcone isomerase requires naringenin chalcone as a substrate for the formation of 
the flavanone naringenin, which leads to the formation of dihydroflavonols,
 flavonols 
and flavan-3,4-diols (Figure 2.19). Additional examples of possible flavonoids that 
contain B
-ring substituents include 
kaempferol (flavonols), apigenin (flavone) and 
dihydroquercetin (dihydroflavonols). 
The biosynth
esis of most flavonoids invol
ves p-coumaric acid derivatives that are hydroxylated at the 4Õ
-position of the B-ring
. Those 
that lack this hydroxylation pattern on the B
-ring are comparatively rare (Seigler, 2012). 
 The possible phenylpropanoid pathway that may be found in 
P. maackii
 likely involves a 
variety of secondary metabolites lacking B
-ring substituents. The lack of B
-ring 
substituents is one unique characteristic of the flavonoids contained in 
P. maackii
 periderm 
tissue indicating that it is likely that the phenylpropanoid pa
thway includes 
derivatives
 from cinnamic acid (Figure 2.18
). The absence of B
-ring substituents in 
flavonoids, from 
crude 
P. maackii
 perider
m extract
, may influence antimicrobial 
properties.
    !--!The biosynthetic pathway of flavonoids lacking B
-ring 
substituents, including 
alnusin and alnustinol, is predicted to involve pinobanksin and pinocembrin derivatives 
with a cinnamic acid origin (Figure 2.20
). 
These particular flavonoids have two 
hydroxylations
 on the A-ring
, without B-ring substitutions
, and therefore are not derived 
from the common 
p-coumaric acid precursor and are considered comparatively rare. 
Cinnamic acid and cinnamoyl
-CoA give rise to flavonoids lacking B
-ring substitutions. 
Pinocembrin is a known antifungal flavanone found in leaf gland
s of cottonwood (Shain 
& Miller, 1982). Pinobank
sin also lacks B
-ring substituents
 and may be an additional precursor in the biosynthesis of alnusin and alnustinol. Additional identification of 
structures in the crude extract would give insight to which ge
nes and enzymes are 
required to synthesize
 antimicrobial
 flavonoids
.   Figure 2.
18. Diagram 
of the possible phenylpropanoid pathway found in 
P. maackii.
 !%..! Figure 2.
19. Diagram 
of possible 
biosynthetic pathways
 of flavonoids 
containing
 B-ring 
substituents
.  !%.%! Figure 2.
20. Diagram 
of the possible
 biosynthesis of 
alnustinol and alnusin. 
   !%.&!The primary goal of future research will be to elucidate the gene expression 
correlated with c
innamoyl-CoA-ligase, flavonoid
-6-hydroxylases and flavonoid
-6-methoxylases. Additionally, further characterization of enzymes is needed. Enzymatic 
modifications are responsible for the conversion of different classes of flavonoids. The 
cor
relation of gene expression and enzymes such as flavone synthase, flavanol 
synthase, flavanone reductase
, and dioxygenase will help el
ucidate the general 
biosynthetic pathway
 of flavonoids found in 
P. maackii
. Based on TLC bioassays, there 
are 
several 
other antifung
al compounds present in various 
P. maackii
 tissues that 
remain unidentified.
              !%.'!           LITERATURE CITED
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, Springer Netherlands, pp. 1
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; Bruker Analytical X
-ray 
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  Arriaga
-Giner, F. J., Wollenweber, E., & Hradetzky, D. (1986). New flavonoids from the 
exudate of Bacch
aris bigelovii
 (Asteraceae).
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 Alnus sieboldiana
 (Betulaceae) ii. The 
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 Prunus persica
 (L.) Batsch 
(Meet Ghamr) peach local cultivar in Assiut region.
 Bulletin 
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al Harmaceutical
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  Dixon, R. A., & Ferreira, D. (2002). Genistein.
 Phytochemistry
, 60, 205-211.  Droby, S., Prusky, D., Jacoby, B., & Goldman, A. (1987). Induction of antifungal 
resorcinols in flesh of unripe mango fruits and its relation to latent infection by
 Alternaria 
alternata
. Physiological and Molecular Plant Patholo
gy, 30, 285-292.  Feucht, W., Dirr, U., Treutter, D., & Santos
-Buelga, T. (1997). Leaching properties of 
antimicrobial
 Prunus
 phenols.
 Zeitschrift fr Pflanzenkrankheiten und 
Pflanzenschutz
, 104, 370-379.  Geibel, M. (1995). Sensitivity of the fungus
 Cytospora persoonii
 to the flavonoids 
of Prunus cerasus
. Phytochemistry
, 38, 599-601.  Geibel, M., & Feucht, W. (1991). Flavonoid 5
-glucosides from
 Prunus cerasus
 bark and 
their characteristic weak glycosidic 
bonding. Phytochemistry
, 30, 1519-1521.  !%.)!Iriti,
 M., & Faoro, F. (2009). Chemical diversity and defence metabolism: how plants 
cope with pathogens and ozone pollution.
 International Journal of Molecular 
Sciences
, 10, 3371-3399.  Kumarasamy, Y., Cox, P. J., Jaspars, M., Nahar, L., & Sarker, S. D. (2003).
 Cyanogenic 
glycosids from 
Prunus spinosa
 (Rosaceae).
 Biochemical systematics and ecology
, 31, 1063-1065.  Mabry, T. J., & Markham, K. R. &M. B. Thomas 1970. 
The Systematic Identification of 
Flavonoids
. Springer
-Verlag, New York, pp. 165
-166.  Morant, A. V., J¿rgensen, K., J¿rgensen, C., Paquette, S. M., S⁄nchez
-P”rez, R., 
M¿ller, B. L., & Bak, S. (2008). 
#-Glucosidases as detonators of plant chemical 
defense.
 Phytochemistry
, 69, 1795-1813.  Nelson, D. L., Lehninger, A. L., & Cox, M. M. (2008).
 Lehninger principles of 
biochemistry
. Macmillan. New York, p. 15
  Osbourn, A. E., Clarke, B. R., Lunness, P., Scott, P. R., & Daniels, M. J. (1994). An oat 
species lacking avenacin is susceptible to infection by
 Gaeumannomyces 
graminis
 var.
tritici
. Physiological and Molecular Plant Pathology
, 45, 457-467. Osbourn, A. E. (2003). Saponins in cereals.
 Phytochemistry
, 62, 1-4. O. V. Dolomanov, L. J. Bourhis, R. J. Gildea, J. A. K. Howard 
& H. Puschmann, OLEX2: 
a complete structure solution, refinement an
d analysis program. J. Appl. Cryst. (2009). 
42, 339-341. Prusky, D., Karni, L., Kobiler, I., & Plumbley, R. A. (1990). Induction of the antifungal 
diene in unripe avocado fruits: effect of inoculation with
 Colletotrichum 
gloeosporioides
. Physiological and 
Molecular Plant Pathology
,37, 425-435.  Prusky, D., Keen, N. T., & Eaks, I. (1983). Further evidence for the involvement of a 
preformed antifungal compound in the latency of
 Colletotrichum gloeosporioides
 on unripe avocado fruits.
 Physiological Plant 
Pathology
, 22, 189-195.  Ranilla, L. G., Genovese, M. I., & Lajolo, F. M. (2009). Isoflavones and antioxidant 
capacity of Peruvian and Brazilian lupin cultivars.
 Journal of Food Composition and 
Analysis
, 22, 397-404.  SAINT V 7.68A 
Software
 for the 
Integration of CCD Detector System
 Bruker Analytical 
X-ray Systems, Madison, WI (2010).
  SADABS V2.008/2 Program for absorption corrections using Bruker
-AXS CCD based 
on the method of Robert Blessing; Blessing, R.H. Acta Cryst. A51, 1995, 33
-38.  !%.*!Seigler, 
D. S. (2012).
 Plant Secondary Metabolism
. Springer Science & Business 
Media
. Kluwer Academic Publishers, Boston, pp. 4 
Ð 173.  Shain, L. & Miller, J.B. (1982). Pinocembrin: an antifungal compound secreted by leaf 
glands of eastern cottonwood.
 Phytopatholog
y, 72, 877-880.  Sheldrick, G.M. "A short history of SHELX".
  Acta Cryst
. A64, 2008, 112
-122.  Shin, M., Umezawa, C., & Shin, T. (2014). NATURAL ANTI
-MICROBIAL SYSTEMS| 
Antimicrobial Compounds in Plants.
 Encyclopedia of Food Microbiology (Second 
Edition)
, 920-929.  Suga, T., Iwata, N., & Asakawa, Y. (1972). Chemical constituents of the male flower 
of Alnus pendula
 (Betulaceae).
 Bulletin of the Chemical Society of Japan
,45, 2058-2060.  Terry, L. A., Joyce, D. C., Adikaram, N. K., & Khambay, B. P. (2004). Pre
formed 
antifungal compounds in strawberry fruit and flower tissues.
 Postharvest Biology and 
Technology
, 31, 201-212.  Trojanowska, M. R., Osbourn, A. E., Daniels, M. J., & Threlfall, D. R. (2001). 
Investigation of avenacin
-deficient mutants of
 Avena strigo
sa. Phytochemistry
,56, 121-129.  VanEtten, H. D., Mansfield, J. W., Bailey, J. A., & Farmer, E. E. (1994). Two classes of 
plant antibiotics: Phytoalexins versus" Phytoanticipins".
 The Plant Cell
, 6, 1191.  Vermerris, W. & Nicholson, R. (2006). 
Phenolic Com
pound Biochemistry
. Springer, 
Netherlands, pp. 40
-43.  Wollenweber, E., Fritz, H., Henrich, B., Jakupovic, J., Schilling, G., & Roitman, J. N. 
(1993). Rare flavonoid aglycones from
 Anaphalis margaritacea
 and two Gnaphalium species.
 Zeitschrift fr 
Naturforschung C
, 48, 420-424.  Wollenweber, E., Henrich, B., Mann, K., & Roitman, J. N. (1996). Lipophilic exudate 
constituents of some Rosaceae from the Southwestern USA.
 Zeitschrift fr 
Naturforschung C
, 51, 296-300.      !%.+!CHAPTER THREE
 !THE ANTIMICROBIAL SPECTRUM OF PERIDERM TISSUE FROM 
PRUNUS 
MAACKII
   ABSTRACT
 !The periderm of 
Prunus maackii
 contains compounds that are inhibitory to the 
growth of 
Armillaria ostoyae
. Periderm from
 young trees 
and older tissues 
collected 
from mature trees 
were
 screened for antimicrobial properties. To determine if this 
activity is effective against other plant pathogens, additional 
Armillaria
 species, other 
true fungi and one Oomycete were tested
. Yeast
-malt-peptone-glucose (
YMPG
) medium supplemented with 
P. maackii
 periderm was used to test for antifungal activity 
against 
A. mellea, A. gemina
 and A. gallica
 in addition to A. ostoyae
. Growth of all 
Armillaria
 species 
screened 
was inhibited.
 The periderm was also antimicrobial to 
several other plant pathogens including 
Rhizoctonia solani
 and Sclerotinia sclerotiorum
. A series of bioassays were performed to examine the range and potency of 
antimicrobial activity in the periderm. A dose
-response of 
P. maackii 
periderm was 
examined to further characterize
 the threshold of antifungal 
activity. The plugs 
containing
 mycelia of
 A. ostoyae
 (warren)
 previously grown on Petri dishes containing 
periderm were
 transferred to plain YMPG
-nutrient medium to determine wh
ether the 
periderm amended
-medium was fungistatic or fungitoxic.  Thes
e tests indicated that the 
medium was fungistatic at a concentration of 12 g periderm/L YMPG and displayed 
fungitoxic properties.
 Preliminary bioassays using leaves and mulch from
 P. 
maackii
 also 
detected
 antimicrobial properties. 
 !%.,!Introduction
 !Plants have developed a variety of defense mechanisms in response to 
pathogens. Potential pathogens may trigger the biosynthesis of antimicrobial 
compounds, pathogenesis
-related (PR) proteins, and reactive oxygen species, among 
other defense responses (Ahu
ja et al., 2012). 
Antimicrobial compounds that are 
constitutively present, or produced from pre
-existing precursors, are known as 
phytoanticipins (VanEtten et al., 1994). 
R-genes play a role in the mediation of disease 
resistance levels by controlling the 
amount of phytoalexins produced and the speed at 
which they accumulate (Hammerschmidt, 1999). Phytoalexins are low molecular weight 
antimicrobial compounds that accumulate in response to pathogen infection (Ku
%, 
1995). Phytoalexins must be present at the r
ight time and place to be effective 
(Hammerschmidt, 2011). 
 The plant family Rosaceae is composed of approximately 90 genera and 3,000 
species (Potter et al., 2007) and includes apricot, sweet & sour cherry, almond, apple, 
blackberry, rose, and strawberry 
(Hummer & Janick, 2009). Plants belonging to the 
family Rosaceae have been studied extensively using targeted analytical assays, to 
investigate the secondary metabolites and corresponding pathways (Shulaev et al., 
2008).
 Flavonols are found in high concentrations in cherry, such as quercetins and 
kaempferols (Ogah et al., 2014). Herniarin has been identified in the leaves of 
P. 
mahaleb, P.
 pensylvanica, 
P. maximowiczii
, and P. maackii
. Herniarin has also been 
identified in the bark of 
P. mahaleb
 and P. pensylvanica
. The bark of 
P. verecunda
, P. cyclamina
 and P. maackii
 contained scopolin (Santamour & Riedel, 1994).  
Flavonoids 
identified from the bark of 
P. 
cerasus
 include naringenin, prunin, sakuranetin, sa
kuranin, 
!%.-!prunetin,
 and tectochrysin,
 all of which slowed mycelial growth of 
Cytospora persoonii
 at 1 mM concentration (Geibel, 1995). Isosakuranetin, dihydrokaempferide and naringenin 
have been isolated from the heartwood extract of 
P. domestica
 (Parmar et al., 199
2). 
The wood of P. verecunda
 contained genistein, prunetin, pinocembrin, and taxifolin 
(Hasegawa & Shirato, 1957). Eriodictyol, genkwanin and sakur
anin have been isolated 
from 
P. donarium
 var. 
spontanea
 (Hasegawa & Shirato, 1955
).  Scopoletin, herniarin, t
axifolin, and naringenin 
are some phenolics that have 
been classified as 
antimicrobial.
 The isoflavones genistein is antifungal and also inhibits 
soybean lipase and peroxidase activity. The flavanone pinocembrin is antimicrobial to 
Candida albicans
, Saccha
romyces cerevisiae
 and Cryptococcus neoformans
 at concentrations of .0001 mg/ml 
- .003 mg/ml and to 
Bacillus subtilis
 at concentrations of 
.003 mg/ml.  The flavanone eriodictyol has antibacterial activity against 
Pseudomonas 
maltophilia
 and Enterobacter 
cloacae
 (Harborne & Baxter, 1993). 
An infection site 
caused by 
Cochliobolus
 heterostrophus
 on resistant maize cultivar B73 Htrhm results in 
the accumulation of anthocyanin pigments in the margin area of lesions. This 
accumulation of anthocyanins is absent 
in the susceptible genotype (Ht) and therefore 
may play a role in microbial defense 
(Nicholson & Wood, 2001).
 The objectives of 
this research
 include
 further characterizing
 the antifungal compounds found in various 
P. maackii
 tissues. 
Samples
 of periderm tissue were
 taken 
from y
oung trees 
and older tissues were collected from 
mature trees
. These samples
 will be screened for constitutive
ly present
 antimicrobial compounds to further support 
the presence of phytoanticipins. 
Screening other 
Armill
aria
 species in addition to non
-host pathogens
 will further 
characterize
 the spectrum of antimicrobial properties
. The !%%.!threshold of fungistatic and 
fungitoxic activity 
will be characterized
 through dose 
response experiments. 
Petri dishes containing 
P. maackii
 mulch
 and leaves in YMPG 
nutrient medium
 were
 inoculated with non
-host
 pathogens as a preliminary 
screen for 
additional antimicrobial compounds.
  Materials and Methods
  Twigs from young (< 5 years) 
P. maackii
 trees, purchased from Nature Hills 
Nursery, Omaha, NE, 
were harvested as needed from potted trees
 (Baccto¨ premium 
potting mix) grown in the MSU greenhouse courtyard. Twigs (approx. 4 cm in diameter) 
from 
P. maackii, P. serotina
 and P. mahaleb
 were collec
ted for periderm extraction from 
the NW Michigan Horticulture Research Station near Traverse City, MI. 
Twigs collected 
from mature trees were ground using a chipper shredder to produce mulch. 
Older 
periderm
 tissue
 was 
also 
collected from a mature 
P. maacki
i tree on Mich
igan State 
UniversityÕs campus
, accession number 97P028*02. S
amples were kindly provided by 
Dr. Frank Telewski. 
Leaves were collected from 
young P. maackii
 trees 
on June 4
th, 2013 and were stored at 
-20ûC.
 Periderm tissue
 was removed with a razor blade from 
P. maackii
 branches
. Periderm and leaves
 were
 ground to a fine powder 
using
 a Krups¨ coffee grinder. 
Mature twigs were ground using a chipper
-shredder to produce mulch. These 
tissue 
types were
 added to the YMPG medium w
ith a final concentration of 12 g of tissue/L of 
YMPG. This 
periderm
-amended medium was autoclaved at standard time (15 
minutes/L). 
The medium was poured into the P
etri 
dishes
 (60 x 15 mm) and then 
!%%%!inoculated 
with Armillaria
 species and non
-host pathogens
, within 24 hours of 
preparation
, with a 4.8 mm cork borer. The cultures were allowed to grow for three 
weeks
 for 
Armillaria
 species and one week for non
-host pathogens.
 Radial growth, in 
cm,
 was measured and recorded. 
 To further characterize
 the antifungal activity
 present in the periderm tissue
, this 
bioassay used 
samples containing 
decreased
 amounts of periderm
. The first
 dose
-response experiment had samples 
containing
 an initial 25%, 50% and 75% reduction in 
grams of 
P. maackii
 periderm per L of YMPG. The second dose
-response experiment 
starts with 12 
g periderm/L YMPG and ends with
 2 g periderm/
L YMPG, cons
isting of 
0.5-gram/L YMPG increments
. A third dose
-response experiment starts with 12 
g/L YMPG and ends with
 0.4 g/L YMPG.
 Petri dishes containing 
P. maackii
 periderm
-amended media were inoculated with 
A. ostoyae
.  Data were analyzed using TukeyÕs 
HSD test, 
p = 0.05.
   Armillaria
 species used for inoculation for growth inhibition screening are shown 
in Table 3.1. The 35-5 isolate of 
A. gemina
 was 
collected from CamelÕs Hump, VT. The
 ISRA
-5 isolate was collected
 from a hardwood forest in Ionia C
ounty, MI. The collection 
information for
 the Kloody T1T isolate of 
A. gallica
 is 
undetermined. Three isolates of 
A. 
melle
a were used for inoculation. The M. Thomas isolate was found on plum in Van 
Buren
 County, MI. The 97-1 isolate was found on red maple in Provincetown, MA. The 
third 
A. mellea
 isolate, 49
-5, was collected in
 the Boston area from an undetermined 
host. All th
ree isolates of 
A. ostoyae
 were isolated from tart cherry trees in the Grand 
Traverse area. The 4
-58 isolate was from Grand Traverse Co
unty, MI and both CT3T1 
and warren originating from Leelanau 
County, MI.
  !%%&!Media containing periderm from 
P. maackii
 was inoculated with isolates from four 
species of 
Armillaria: Armillaria ostoyae, A. gemina, A. mellea,
 and A. gallica
. This 
experiment was repeated three times. Data were analyzed using TukeyÕs HSD test, 
p = 0.05.  Table 3.1. Armillaria
 species used to evaluate the antimicrobial activity of 
P. maackii
 periderm 
   Fungistatic and fungitoxic assays were 
designed to determine the
 toxicity of the 
P. maackii
 periderm
-amended medium. The medium containing 
periderm (12 g/L 
YMPG) was prepared and inoculated with 
Armillaria
 species. The cultures were grown 
for three weeks and the radial growth was recorded.  Once the radial growth data were 
collected, the plugs used t
o inoculate the 
periderm amended
-medium were
 transferred 
to plain YMPG medium.  This would determine whether the compo
unds 
in P. maackii
 periderm tissue are fungistatic (
pathogen is viable
) or if it they are fungitoxic (
pathogen !%%'!is non-viable). This exper
iment was repeated three times, 10 Petri 
dishes/species. 
Data were analyzed using TukeyÕs HSD test,
 p = 0.05.
 The antifungal content was compared between
 samples from
 young and mature 
trees. The young trees were purchased from Nature Hills Nursery, Omaha, NE and 
immediately planted in Baccto
¨ Premium potting mix. Periderm tissue was collected 
from five young trees. For each tree, 
13 Petri dishes
 containing 
periderm
-amended medium were inoculated with 
A. ostoyae
. This experiment was done once. Data were 
analyzed using TukeyÕs HSD test, 
p = 0.05.
 Twigs were collected from m
ature 
Prunus
 trees
 grown 
at the NW Michigan 
Horticulture Research Station near Traverse City, MI and stored at 
-20ûC until used for 
analysis. 
Prunus maackii
 periderm samples were collected from seven different trees 
and screened 
for antimicrobial properties
. For each mat
ure tree sample
, 18 Petri dishes
 containing periderm
-amended medium were inoculated with 
A. ostoyae
. This 
experiment was done once. Data were analyzed using TukeyÕs HSD test,
 p = 0.05.
 To examine the spectrum of an
tifungal activity, 
the periderm
-incorporated media
 was inoculated with several plant pathogens from three different phyla:  Basidiomycota, 
Ascomycota and Oomycota (Table 3.2). 
The non-host pathogens used to 
further 
characterize antimicrobial activity 
include
 Sclerotinia sclerotiorum, Botrytis cinerea, 
Rhizoctonia solani,
 and Phytophthora capsici
. All of these pathogens have a 
broad host 
range. 
Cladosporium cucumerinum
 and Colletotrichum orbiculare
 are pathogens of 
cucumber. 
Fusarium sambucinum
, F. Samb
ucinum
 TBZ
-resistant and
 Alternaria solani
 are pathogens of potato 
and Cochliobolus carbonum
 is a pathogen of Maize. 
 !%%(!Periderm from 
P. maackii
 was i
ncorporated into YMPG medium (
12 grams/L of 
YMPG
) and
 inoculated with 
P. capsici, S
. sclerotiorum, 
F. sambuc
inum, F. sambucinum
 (TBZ
-resistant), 
R. solani, C
. orbiculare, 
and C. cucumerinum
. This experiment was 
repeated three times
, 17 Petri dishes/species were inoculated
. Data were analyzed 
using TukeyÕs HSD test, 
p = 0.05.
   Table 3.2. Pathogens of non
-Prunus 
species used to evaluate antimicrobial activity of 
P. 
maackii
 periderm.
   The ChromaDoc
-It TLC imaging system was employed f
or recording the 
bioassay results for Petri dishes containing inoculated periderm
-amended media. This 
system 
contains white light, which was used to take pictures of 
mycelial 
growth
. Radial growth was
 rec
orded, using Photoshop, and
 analyzed by TukeyÕs HSD, 
p = .05, in the 
statistical computing program, R (R Core Team, 2014).
  !%%)!Results
 !To determine the threshold of antifungal activity, the standard screening 
concentration for 
Armillaria 
growth inhibition was 
reduced to
 dilute samples containing 
6g/L and 3g/L (Figure 3.1). The growth of 
A. ostoyae
 on media containing the different 
concent
rations of periderm tissue was compared to growth on plain YMPG nutrient 
medium (control growth). Even at a dilute concentration of 3g/L, the periderm
-amended medium was able to significantly inhibit the mycelial growth of 
A. ostoyae
. All 
concentrations we
re able to
 significantly
 inhibit the growth of 
A. ostoyae
. Complete 
growth inhibition was 
detected 
at 12 g /L YMPG and significant inhibition was 
detected
 at 6 g/L YMPG. Since the 3 g /L still inhibited the growth of 
A. ostoyae
, periderm 
concentrations wer
e reduced further to better identify the threshold of inhibition. 
 The second dose
-response experiment started 
with the standard concentration of 
12 g /L of YMPG
 and was diluted by 0.50 gram/L, ending with a final concentration of 2 
gram
s/L of YMPG. All of
 the plugs containing
 Armillaria 
mycelia 
were transferred to 
YMPG medium to see if they were viable after b
eing exposed to 
P. maackii
 periderm
. The medium containing high
 concentrations 
of P. maackii
 periderm 
had fungitoxic 
properties.
  !%%*! Figure 3.
1. Dose
-response 
of P. maackii
 periderm on 
the growth of 
A. ostoyae
 (warren 
isolate
).  Table 3.3. Dose
-response
 of P. maackii
 periderm
-amended media on the growth and 
viability of 
A. ostoyae
 (warren isolate).
     0!0!.!.!./)!%!%/)!&!&/)!'!'/)!(!1234526!789:;<!
'!=!2>!?@5"A@5BCD!
89:;!
*!=!2>!?@5"A@5BCD!
89:;!
%&!=!2>!?@5"A@5BCD!
89:;!
"#$%&'!$(!
!"#$%%&"$&'()*(+&,'
)*+,!
!%%+!To further examine the dose
-response of 
P. maackii
 periderm
-amended medium, additional concentrations were screened for the ability to inhibit the growth of 
A. ostoyae
 (Table 3.3). When YMPG contained 2.60
-12.00 g of periderm tissue/L YMPG, the 
average radial growth of 
A. ostoyae
 was less than 0.50 cm after three weeks 
of growth.
 The threshold of antifungal activity to 
A. ostoyae
 appears to be around 2.40
-2.60 g of peri
derm tissue/L YMPG. 
 There was a significant decrease in antifungal act
ivity in periderm
-amended media containin
g less than 2.60g/L YMPG. Media
 containing 0.40
-0.20g/L YMPG had 
Armillaria
 average radial growth slightly less than that of the control growth 
on plain YMPG medium. The strength of fungitoxic activity decreased as periderm tissue 
concentrations decreased. At standard screening
 concentration, the periderm
-amended media was fungitoxic to most 
Armillaria
 samples, resulting in plugs containing non
-viable mycelia. 
 Percentages of viability were recorded when 
plugs were
 transferred to plain 
YMPG medium.
  There was a
n increase in 
A. ostoyae
 mycelial growth 
on media containing
 2.40 Ð .60 g of periderm tissue
 /L YMPG. 
When the periderm
-amended media was di
luted to 0.40 Ð 0.20 g/L, the growth was similar to that of the control.  The 
periderm amended
-media was fungitoxic to approximately 70% of the 
plugs containing 
Armillaria
 mycelia
 when grown on media containing 
12.00 Ð 8.00 g/L YMPG. The 
fungitoxic 
properties
 of the periderm
 decreas
ed to approximately 40% of 
A. ostoyae
 plugs when inoculated on 
media containing 
7.80 Ð 4.00 g of periderm
/L YMPG
. The fungitoxic threshold 
was detected
 when plugs containing 
Armillaria
 mycelia were grown 
on media containing 3.80 
Ð 2. 60g/L YMPG. This concentration was only fungitoxic to 
!%%,!5% of the isolates; h
owever, fungistatic properties are still 
detected 
at this 
concentration. The average radial 
mycelial 
growth 
of A. ostoyae
 increased
 when grown 
on media containing
 2.60 - 0.60 g of periderm tissue
/L YMPG concentrations. 
 An additional dose
-response experiment was setup to further characterize the 
threshold of antifungal activity. Similar to the previous dose
-response experiment, this 
experiment started with the standard screening concentration of 12g/L and consisted of 
2g incremental decreases, but ends with 0.4g/L YMPG (Figure 3.2). Complete inhibition 
of A. ostoyae
 growth was detected when the pathogen was 
grown on periderm
-amended media containing 12 
Ð 6.0g/L YMPG, with an exception of a few samples 
showing very slight growth (less than approximately 0.25 cm). 
 The growth of 
A. ostoyae
 was less than 0.25 cm when grown on
 medium containing
 4.0 g/L YMPG. There was significant growth inh
ibition when the pathogen was grown on medium containing 2
.0 g/L, however, average radial growth for this 
concentration exceeded 1 cm, which is still significantly inhibited compared to control 
growth. Growth of 
A. ostoyae
 was comparable to control growth on YMPG 
when the periderm
-amended media contained less than 0.40g/L YMPG. Thus, the threshold for 
antifungal activity in this dose
-response experiment is expected to be between 2.0 
Ð 0.40 g/L YMPG. 
Complete inhibition was 
detected when 
the pathogen was grown on 
media containing 
increased
 amounts of periderm tissue (
12 g/L Ð 4.0 g/L) 
with the exception of a few plugs of 
A. ostoyae 
that showed slight viability
.  !%%-! Figure 3.
2. Dose
-response
 (12 g 
Ð 0.4 g/L) of 
P. maackii
 periderm on the growth of 
A. ostoyae
 (warren isolate
). TukeyÕs HSD, 
p = 0.05. 
* = Significantly different from the 
control.
 !!To further test the spectrum o
f antifungal activity, 
samples containing 
periderm
-incorporated 
YMPG 
were
 inoculated with additional 
Armillaria
 species
. Preliminary 
screening for the ability of three 
Prunus
 species to inhibit 
growth of 
A. ostoyae
, warren 
isolate, 
showed that 
P. maackii
 was the only 
antifungal species (Figure 3.3). This 
preliminary screen was done using older
 periderm
-amended media. As previously 
discussed, the age of the medi
a slightly affects toxicity.
 Since pathogen growth 
inhibition was only detected when isolates were grown on 
P. maackii
 periderm
-amended media, additional experiments only used 
P. maackii
 as a positiv
e control and 
.!.!0!.!0!0!.!./)!%!%/)!&!&/)!'!'/)!(!%&!=CD!
%.!=CD!
,/.!=CD!
*/.!=CD!
(/.!=CD!
&/.!=CD!
./(!=CD!
1EFGHED!
"#$%&'!$(!
!"#$%%&"$&'()*(+&,
!)*+,!
-$.*/.&#0&1$.!$(!2/#13/#+45+/.3/3!6/310!
!%&.!plain YMPG nutrient
-medium as 
a negative control.
 Additional screening included 3 
isolates of 
A. ostoyae
, 1 isolate of 
A. gemina
, 3 isolates of 
A. mellea
, and 2 isolates of 
A. gallica
.Armillaria ostoyae
 isolates 
grown on periderm
-amended medium (12 g/
L YMPG) 
were compared to 
those grown on plain YMPG nutrient
-medium. Complete growth 
inhibition of all A. ostoyae
 isolates 
was detected 
when grown on 
P. maackii 
periderm
-amended medium  (Figure 3.4). 
The periderm
-amended media was able to significantly 
inhibit 4-58, CT3T1 and warren isolates of 
A. ostoyae
. The growth of 
A. gemina
, isolate 
35-5, was significantly inhibited when grown on 
P. maackii
 periderm
-amended medium (Figure 3.5). The majority of the samples from 
all three i
solates of 
A. mellea
 were 
completely inhibited, with the exception of a few samples showing slight growth. The 
periderm
-amended medium completely inhibited 100% of the samples of isolate 97
-1 (Figure 3.6). 
Most samples of 
A. gallica
, isolates ISRA
-2 and Kloody T1T, were 
completely inhibited
 when grown on the medium containing periderm from 
P. maackii
 (Figure 3.7)
.  Most plugs containing the isolates were not abl
e to establish any growth 
on medium containing 
P. maackii
 periderm. 
 All 
Armillaria
 isolates 
screened 
were sensitive to the 
P. maackii
 periderm 
Ð amended medium. Isolates were screened for viability after exposure to the periderm. 
Armillaria mellea
 was the most sensitive to the 
P. maackii
 periderm
-amended medium. The peride
rm tissue
 from 
P. seroti
na stimulated the growth of 
A. mellea
 and A. gallica
 and periderm from 
P. mahaleb
 stimulated the growth of 
A. ostoyae
, A. gemina
 and A. 
gallica
. These results were taken from isolates grown on media that was within a week 
old. Fresh med
ia was used in Figur
es 3.4 
Ð 3.7.  !%&%! Figure 3.
3. The growth of 
A. ostoyae
 (warren), 
A. gemina
 (35
-5), 
A. mellea
 (49
-5), and 
A. gallica
 (ISRA
-5) on 
periderm
-amended media (12 g/
L YMPG)
. * = Significantly 
different from the control. TukeyÕs HSD, 
p = 0.05.    0!0!0!0!.!./)!%!%/)!&!&/)!'!'/)!(!!"#$%%&"$&'
()*(+&,'
!"#$%%&"$&'
-,#$.&'
!"#$%%&"$&'#,%%,&'
!"#$%%&"$&'-&%%$/&'
"#$%&'!$(!
!"#$%%&"$&')-,.$,)'
)*+,!
0"1.1)'#&2%&,3'
0"1.1)'#&&/4$$'
0"1.1)'),"(*$.&'
I234526!
!%&&! Figure 3.
4. The growth of 
A. ostoyae
 isolates on 
P. maackii
 periderm
-amended medium (12 g/
L YMPG)
. * = Significantly different from the control. TukeyÕs HSD, 
p = 0.05. 
   0!0!.!.!./)!%!%/)!&!&/)!'!'/)!(!(/)!:@5"A@5BJ!(K),!
1234526J!(K),!!
:@5"A@5BJ!
1G'G%!1234526J!1G'G%!
:@5"A@5BJ!
LM55@3!
1234526J!
LM55@3!
"#$%&'!$(!
!"#$%%&"$&'()*(+&,'
17$80&/7!)*+,!
6/319+:!;7$80&/!
!%&'! Figure 3.
5. The growth of one isolate of 
A. gemina
 on P. maackii
 perider
m-amended medium (12 g/
L YMPG).
 * = Significantly different from the control. TukeyÕs HSD, 
p = 0.05.   Figure 3.
6. The growth 
of A. mellea
 isolates 
on P. maackii
 periderm
-amended medium (12 g/
L YMPG)
. * = Significantly different from the control. TukeyÕs HSD, 
p = 0.05.
 0!.!./)!%!%/)!&!&/)!'!'/)!:@5"A@5BJ!')K)!
1234526J!')K)!
"#$%&'!$(!
!"#$%%&"$&'/,#$0&'
17$80&/!)*+,!
6/319+:;7$80&/!
0!.!0!.!./)!%!%/)!&!&/)!'!'/)!:@5"A@5BJ!9/!
GN2BMO!
1234526J!9/!
GN2BMO!
:@5"A@5BJ!
-+K%!
1234526J!-+K%!
:@5"A@5BJ!
(-K)!
1234526J!(-K)!
"#$%&'!$(!
!"#$%%&"$&'#,%%,&'
17$80&/7!
)*+,!
6/319+:;7$80&/!
!%&(!  Figure 3.
7. The growth of 
A. gallica
 isolates
 on P. maackii
 periderm
-amended medium (12 g/
L YMPG)
. * = Significantly different from the control. TukeyÕs HSD, 
p = 0.05. 
 !!Results suggest that older media was found to be less fungitoxic and therefore
 periderm
-amended media should be inoculated within one day of preparation. 
More 
plugs containing mycelia 
were able to colonize the older periderm
-amended media, thus 
the average radial growth is slightly higher when isolates are grown on media that is 
older than 24 hours. However, even when grown on media older than 24 h, the growth 
of A. ostoyae
 is significantly inhibited compared to control growth
 on YMPG
.  The slight 
decrease in antifungal activity could be partly attributed to the crystal format
ion on the surface of the media
 (Figure 3.8). This could be a possible indication that some of the 
antimicrobial compounds have crystallized out of 
the media 
resulting in decreased 
toxicity due to less chemicals coming into contact with plugs containing mycelia. 
 0!0!.!./)!%!%/)!&!&/)!'!'/)!(!:@5"A@5BJ!PQHRK&!
1234526J!PQHRK&!
:@5"A@5BJ!S622AT!
G%G!1234526J!S622AT!G%G!
"#$%&'!$(!
!"#$%%&"$&'/&%%$.&'
17$80&/7!
)*+,!
6/319+:;7$80&/!
!%&)! Figure 3.
8. Crystal 
formation on
 the surface of
 P. maackii
 periderm
-amended medium.           !%&*!To determine whether the periderm
-amended media was fungistatic or fungitoxic, 
all plugs containing 
Armillaria
 isolates were transferred from Petri dishes containing 
periderm tissue to those with only YMPG nutrient
-medium. 
The figures below show the 
growth of transferred plugs of 
A. ostoyae
, A. gemina
, A. mellea
, and A. gallica
. The toxic 
effects 
of the periderm 
were shown to significantly inhi
bit the growth of all 
Armillaria
 species
 screened, even after being 
transfe
rred to YMPG nutrient
-medium.  The growth of 
A. ostoyae
 isolates 
was recorded when isolates 
4-58, CT3T1 and warren 
were
 transferred from 
P. maackii
 periderm
-amended media to YMPG
 (Figure 
3.9). Some species were able 
to establish more growth
 than others. The periderm
-amended medium was fungitoxic to 100% of
 samples containing
 mycelia of isolate
 4-58. It was also fungitoxic to most samples of CT3T1 and
 warren, with the exce
ption of a few samples/isolate
. The growth of all three isolates of 
A. ostoyae
 was significantly inhibited 
after being transfer
red
 to YMPG me
dium. The growth of 
A. gemina
, isolate 35
-5, when transferred from periderm
-amended medium to plain YMPG medium remained 
significantly inhibited (Figure 3.10).
 The growth of 
A. mellea
 isolates 
M. Thomas, 97
-1 and 49-5, transferred from the 
periderm
-amended medium onto YMPG nutrient
-medium was recorded
. All three 
isolates were significantly inhibit
ed after being transferred from media containing 
periderm tissue
 (Figure 3.11)
. The periderm
-amended medium was fungitoxic to 
samples 
including those of M. Thomas and 49
-5. However, there were a few samples 
that were able to re
-establish growth when transferred to plain YMPG, showing average 
radial growth between 0.5 
Ð 1 cm. The medium was fungitoxic to 100% of the 97
-1 !%&+!samples. 
The P. m
aackii
 periderm
-incorporated medium was
 100% fungitoxic to isolate 
97-1 of A. mellea
.  The periderm
-amended media was fungitoxic to 
some plugs containing mycelia 
of Kloody T1T and
 ISRA
-1. The samples that established growth were still significantly 
inhibited compared to samples that had not been exposed to 
P. maackii
 periderm. 
The growth of 
A. gallica
 isolates, ISRA
-5 and Kloody T1T
, was inhibited 
even when transferred from periderm
-amended medium to YMPG medium
 (Figure 3.12)
. The Kloody T1T isolate was 
more sensitive to the periderm
-incorporated med
ia compared to 
ISRA
-5 isolate
.     !%&,! Figure 3.
9. Growth 
of A. ostoyae
 isolates when transferred from 
P. maackii
 periderm
-amended medium to plain YMPG nutrient
-medium. * = 
Significantly
 different from the 
control. TukeyÕs HSD, 
p = 0.05.
   0!0!.!./)!%!%/)!&!&/)!'!'/)!"#$%&'!$(!
!"#$%%&"$&'()*(+&,
!17$80&/7!)*+,!
6/319+:!;7$80&/!
!%&-! Figure 3.
10. Growth 
of A. gemina
, isolate 35
-5, when transferred from 
P. maackii
 periderm
-amended medium to plain YMPG
 nutrient
-medium. * = 
Significantly
 different 
from the control. TukeyÕs HSD, 
p = 0.05.
   0!.!./)!%!%/)!&!&/)!:@5"A@5BJ')K)!
1234526J')K)!
"#$%&'!$(!
!"#$%%&"$&'/,#$0&'
17$80&/7!)*+,!
6/319+:!;7$80&/!
!%'.! Figure 3.
11. Growth 
of A. mellea
 isolates when transferred from 
P. maackii
 periderm
-amended medium to plain YMPG nutrient
-medium. * = 
Significantly
 different from the 
control. TukeyÕs HSD, 
p = 0.05.  
   0!.!0!.!./)!%!%/)!&!&/)!'!'/)!"#$%&'!$(!
!"#$%%&"$&'#,%%,&'
17$80&/7!)*+,!
6/319+:;7$80&/!
!%'%! Figure 3.
12. Growth 
of A. gallica
 isolates when transferred from 
P. maackii
 periderm
-amended medium to plain YMPG nutrient
-medium. * = 
Significantly
 different from the 
control. TukeyÕs HSD, 
p = 0.05.
  Thus, growth inhibition of 
Armillaria
 species was maintained even after being 
removed 
from the periderm
-amended medium. The fungitoxicity varied among isolates 
of Armillaria
 spp. The medium containing 
P. maackii
 periderm was fungitoxic to a 
percentage of all isolates. 
  Older periderm
 tissue
s were collected 
from several mature 
P. maackii
 trees and 
were 
screened for antifungal activity against 
A. ostoyae
. All of the trees 
screened
 were 
able to significantly inhibit pathogen growth. After this screen was done,
 it was found 
that the periderm
-amended media needed to be inoculated within 24 hours
 of preparation. The media used to screen for
 antifungal activity
 in young and mature trees
 was approximately one week old.
 0!0!.!./)!%!%/)!&!&/)!'!:@5"A@5BJ!PQHRK%!
1234526J!PQHRK&!
:@5"A@5BJ!S622AT!G%G!
1234526J!S622AT!G%G!
"#$%&'!$(!
!"#$%%&"$&'/&%%$.&'
17$80&/7!)*+,!
6/319+:!;7$80&/!
!%'&!Five young (< 5 years old) 
P. maackii
 trees were screened for their ability to 
inhibit the growth of 
A. ostoyae
 (Figure 3.13). 
The periderm
-amended media was older 
than 24 hours 
when used. 
Older media (over 24 h) was als
o used to screen 
older 
tissues collected from 
eight mature (>
12 years) 
P. maackii 
trees. Despite the slight 
decrease in toxicity d
ue to the age of the media, all P. maackii
 trees
 screened
 were able 
to significantly inhibit the growth of 
A. ostoyae
 (Figure 3.14). 
The antifungal compounds 
were also confirmed using TLC and HPLC
-MS analyses. 
Samples from t
he same 
mature trees were later used for 
fungistatic and fungitoxic bioassays. When inoculation 
is done within 24 hours of preparation, 
the growth of
 A. ostoyae
 is 
completely inhibited
. Armillaria ostoyae
 growth 
is shown 
on media containing periderm from young (A) 
and mature (B) 
P. maackii
 trees and on plain YMPG medium (C) 
(Figure 3.15
). This 
assay was also done using older media. The top three Petri dishes in the A group 
(periderm from young trees) show slight viability of 
A. ostoyae
.  When these 
experiments were repeated with fresh media there was an increase in fungitoxic activity, 
most plugs showing no growth at all. Pictures of samples A, B and C were taken after 3 
weeks of growth. Crystals appeared on the surface of the periderm from mature trees. 
There is also a slight difference in the color of the media. Crystals later appeared o
n the surface of media containing young periderm; perhaps this is an indication of a difference 
in concentration of some yellow colored flavonoids.
   !%''! Figure 3.
13. Periderm 
tissues from five young 
P. maackii
 trees were screened for their 
ability to inhibit the growth of 
A. ostoyae
.  All trees were able to significantly inhibit 
pathogen growth. TukeyÕs HSD, 
p = 0.05.
 0!0!0!0!0!.!./)!%!%/)!&!&/)!'!'/)!(!#1 #2 #3 #4 #5 control 
Growth of 
Armillaria ostoyae 
(cm) 
Young 
Prunus maackii 
trees 
!%'(! Figure 3.
14. Screening
 for antifungal activity
 in older tissues collected from 
mature 
P. 
maackii
 trees
. All trees were able to significantly inhibit pathogen growth. TukeyÕs HSD, 
p = 0.05.
   Figure 3.
15. Periderm
-amended media from both young and mature 
P. maackii
 trees 
were able to significantly inhibit 
A. ostoyae
 growth. 
Samples
 were grown for three 
weeks. TukeyÕs HSD, 
p = 0.05.
    0!0!0!0!0!.!0!0!.!./)!%!%/)!&!&/)!'!'/)!#1 #2 #3 #4 #5 #6 #7 #8 control 
Growth of 
Armillaria ostoyae 
(cm) 
Older tissues collected from mature 
P. maackii 
trees 
!%')!To further classify the toxicity of the 
P. maackii
 periderm, non
-host pathogens 
were used for inoculation. Additional antimicrobial screening used 
P. maackii
 periderm
-incorporated media
 inoculated with four Ascomycetes: 
Cladosporium cucumerinum
, Colletotrichum orbiculare
, Fusarium sambucinum
, and Scleroti
nia sclerotiorum
, one Basidiomycete, 
Rhizoctonia solani
 and one Ooymcete, 
Phytophthora capsici
. The periderm
-amended media significantly inhibited all pathogens 
screened 
(Figure 3.16
). 
Complete inhibition was detected in samples containing
 P. capsici, S
. sclerotiorum, C
. cucumerinum
, and C. orbiculare
. Slight growth was 
detected
 in samples containing 
plugs of mycelia from 
F. sambucinum
, F. sambucinum 
TBZ
-resistant
 and A. solani
. Rhizoctonia solani
 and B. cinerea
 were the most tolerant, colonizing the grea
test areas 
among species screened, 
however samples were still significantly inhibited when 
compared to control growth.
 The amount of inhibition was not equal among the different species screened. 
Alth
ough the periderm
-amended media was able to significantl
y inhibit all pathogens 
screened, 
R. solani
 and B. cinerea
 appeared
 to be the most tolerant.
 The periderm
-amended media completely inhibited the growth of 
P. capsici
, S. sclerotiorum
, C. cucumerinum
, and C. orbiculare
. Significant
 growth
 inhibition was 
detected 
in all other 
species 
used for screening, which include
 A. solani
, F. sambucinum
 (wild
-type) and
 thiabendazole
 (TBZ
)- resistant isolates. TBZ
-resistant isolates of 
F. sambucinum
 are 
those resistant to the fungicide (TBZ). 
For comparison, s
alicylald
ehyde when applied as 
a volatile
 is known to inhibit TBZ
-resistant 
F. sambucinum
 at gas levels of 20 µg/mL or 
lower. Media containing cinnamaldehyde and thymol (0.1% v/v) also inhibits growth of 
TBZ
-resistant isolates (Vaughn & Spencer, 1994). 
 !%'*!  Figure 
3.16. The periderm
-amended medium from 
P. maackii
 was inoculated with 
several non
-host
 pathogens. Isolates were grown for two weeks. TukeyÕs HSD test, 
p = 0.05.           .!%!&!'!(!)!*!+!52$6(/*(.$&')(%&.$'
!%*,".&"$&')(%&.$'
7(*"+*$)'/$.,",&'
8(/2%$(3(%1)'/&"3(.1#'
8(%%,*(*"$/21#'("3$/1%&",'
91)&"$1#')&#31/$.1#'
9:')&#31/$.1#';7<'",)$)*&.*'
8%&=(>)("$1#'/1/1#,"$.1#'
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;52U4N!7IB<!23!:R9!
;52U4N!7IB<!23!89:;!
!%'+!Discussion  Phytoanticipins play a role in host resistance in many different host
-pathogen interactions. 
The peel of an unripe avocado fruit contains th
e preformed antifungal 
cis, 
cis
-1-acetoxy
-2-hydroxy
-4-oxo-heneicosa
-12,15-diene, which is toxic to 
Colletotrichum 
gloeosporioides
. The concentration of this diene decreases as fruits mature (Prusky et 
al., 1983).
 Additionally, 
catechol in onion, epicatechin in avocado, chlorogenic acid (widely 
distributed), caffeic acid (widely distributed), 3
-hydroxyacetophenone in carnations, 5
-pentadecyl resorcinol in mango, and pinocembrin in cottonwood are all known phenolic 
phytoanticipins
 (Hammerschmidt & Hollosy, 2008). The phenolic composition changes 
seasonally in phloem tissues of sweet cherry, 
Prunus avium
 L. (Schwalb & Feucht, 
1999). The first rise of flavanols in the phloem tissues of sweet cherry occur at the end 
of winter 
with tra
nsiently increased temperatures. Just before bud breaking in early 
spring, a second increase in flavanols was observed. Flavanol maximum occurred 
during mid
-July and coincided with decline of shoot growth. The fourth flavanol peak in 
shoot phloem was obser
ved during the fall season when there is a back flow of 
carbohydrates from the leaves (Schwalb & Feucht, 1999). For this reason, future 
research
 should include sampling of 
P. maackii
 tissues
 each month
 to examine any 
seasonal effect on phenolic concentration.
 The viability of 
A. ostoyae
 isolates increased as 
periderm concentration 
decreased.
 There were c
onstitutive
ly present
 antifungal compounds in media made 
from all trees. It was found that the periderm
-amended media should be inoculated 
!%',!within 24 hours of preparation. To further examine the spectrum of antimicrobial activity, 
non-host
 pathogens were used to inoculate 
P. ma
ackii
 periderm
-amended media. The periderm was fungistatic to all pathogens tested.  Mulch was made from the twigs of 
P. 
maackii
 to screen
 for antimicrobial activity in
 the periderm, cambial and wood tissues
.  The mulch
 extract was able to significantly in
hibit A. solani, C
. orbiculare, C
. carbonum
, and C. cucumerinum
 and completely inhibit one
 Trichoderma
 spp.  Leaves 
were also screened for antimicrobial activity. Preliminary data shows that the leaf
-amended media completely inhibited 
the growth of 
C. orbiculare
 and C. cucumerinum
.  Samples containing periderm from young trees and older periderm tissues from 
mature 
P. maackii
 trees had antimicrobial activity. The periderm tissue showed the most 
broad
-spectrum inhibition to both 
Armillaria 
species and no
n-Prunus
 pathogens 
compared to other tissues screened. These antimicrobial compounds are likely 
phytoanticipins since they are consistently extracted from all extracts. The periderm 
extract was fungitoxic to 
a percentage of all 
Armillaria
 isolates. As the 
periderm 
concentrations decreased, growth and viability of the isolates increased.
  In addition to serving as a resistant rootstock,
 P. maackii
 may also provide a 
unique phenylpropanoid pathway involving the biosynthesis of flavonoids lacking B
-ring 
substituents. The structure of the antimicrobial compounds can provide valuable 
information for plant engineering. This ornamental species could serve as a model 
organism for the production of
 phytoanticipins. With this information, the 
phenylpropanoid pat
hway in other plants could be manipulated to produce more of 
these constitutively present antimicrobial compounds.
   !%'-! In co
nclusion, 
P. maackii
 periderm tissue
 is antimicrobial to a wide variety of 
pathogens. Assays using periderm
-amended media show that c
ompounds in the 
periderm tissue are fungitoxic. Certain enzymes can provide valuable information for 
the engineering of resistant crops. For example, 
genistein serves as a common precursor in 
phytoalexin and phytoanticipin biosynthesis in legumes. Isoflavone synthase (IFS) is 
a key
 enzyme 
in isoflavone production and can 
be utilized
 for engineering purposes. 
When chalcone isomerase is overexpressed in 
Arabidopsis
, the expression of IFS leads 
to a 3-fold increase in flavonols (Dixon & Ferreira, 2002). This type of enzymatic 
research would 
provide
 needed information 
in regard to 
flavonoid biosynthesis
 in P. maackii
. Future research should also include
 the characterization and 
identification of all 
unknown compounds from all tissue extracts
, including those tissues that have not ye
t been examined including fruit
 and heartwood tissue
s.             !%(.!           APPENDIX
            !%(%!APPENDIX
   !Figure A.
1. Growth 
comparison of 
A. solani, C. orbiculare, C. carbonum
, and C. cucumerinum
 on P. maackii
 mulch
-amended medium and YMPG nutrient
-medium. 
        !%(&!!Figure A.
2. Growth 
of non-host pathogens on 
P. maackii
 mulch
-amended medium. .!%!&!'!(!)!*!+!"#$%&'!$(!20&'$</.7!)*+,!
;52U4N!23!9V6INK
RB@3A@A!9@A"VB!
;52U4N!23!89:;!
!%('!!Figure A.
3. Growth 
of non-host pathogens on 
P. maackii
 leaf-amended medium.  .!%!&!'!(!)!*!+!"#$%&'!$(!20&'$</.7!)*+,!
;52U4N!23!D@M>KRB@3A@A!
9@A"VB!
;52U4N!23!89:;!
!%((!!Figure A.
4. HPLC 
chromatogram of the semi
-purified fraction collected from 
P. 
maackii
 periderm tissue using dry
-column chromatography. Alnustinol showed a retention time 
of approximately 6.77 (301 ion) and alnusin with a retention time of approximately 8.44 
(299 ion) in negative ion mode. 
 !%()!!Figure A.
5. HPLC 
chromatogram of the semi
-purified fraction collected from 
P. maackii
 periderm tissue containing alnustinol with a retention time of approximately 6.83 and 
alnusin (nearly 80% pure) with a retention time of approximately 8.46 (299 ion) in 
negative ion mode.  !%(*!!Figure A.
6. Crude 
extract of 
P. maackii
 wood tissue containing alnustinol with a 
retention time of approximately 6.79 and alnusin with a retention time of approximately 
8.45 in negative ion mode.       !%(+!!Figure A.
7. Crude 
extract of 
P. maackii
 bud tissue containing alnustinol with a retention 
time of approximately 6.74 and alnusin with a retention time of approximately 8.43 in 
negative ion mode.      !%(,!!Figure A.
8. Crude 
extract of 
P. maackii
 cambial tissue containing alnustinol with a 
retention time of approximately 6.78 and alnusin with a retention time of approximately 
8.44 in negative ion mode.     !%(-!!Figure A.
9. Crude extract 
of P. maackii
 periderm tissue containing alnustinol with a 
retention time of approximately 6.76 and alnusin with a retention time of approximately 
8.43 in negative ion mode.       !%).!!Figure A.
10. Crude 
extract of 
P. maackii
 exposed root tissue containing alnustinol with a 
retention time of approximately 6.77 and alnusin with a retention time of approximately 
8.43 in negative ion mode.     !%)%! !Figure A.
11. Crude 
extract of 
P. maackii
 non-exposed root tissue containing alnustinol 
with a retention time of approximately 6.77 and alnusin with a retention time of 
approximately 8.43 in negative ion mode.
     !%)&!!Figure A.
12. Crude 
extract of 
P. maackii
 leaves containing alnustinol with a retention 
time of approximately 6.81 and alnusin with a retention time of approximately 8.63 in 
negative ion mode.          !%)'!TLC Bioassay Protocol
    1. 20 - 60 µL of sample extracts (.20g periderm/5mL 80% acetone) are applied 
approximately 1 cm from the bottom of a TLC plate (typically 20 x 20 cm; Silica 
gel G). Plates are scored to stop solvent migration, using the pointed end of a 
metal spatula, at appr
oximately 12 cm from the bottom of the plate. 
  2. The TLC plates are developed with chloroform
-methanol 90:10 (v/v). 
  3. TLC plates are photographed under long
-wave (365 nm) or short
-wave (254 nm) 
ultraviolet light, if UV absorbing/fluorescing compounds are th
ought to be present. 
  4. Plugs taken from the outer region of mycelial growth from an established culture 
on PDA can be used to start cultures of 
Cladosporium cucumerinum
. Alternatively, cultures can also be started using a loop of mycelia and spores that 
are spread onto the PDA plate. Cultures are grown on PDA for approximately 7
-11 days prior to use.
  5. Add half
-strength potato
-dextrose broth (PDB). Full strength requires the 
suspension of 24 g HiMedia¨ premade PDB powder/L distilled water and only 12 
g/L is 
required to make half
-strength PDB. The mycelia and spores can be 
!%)(!scraped from the surface of the medium contained in the Petri dishes using a 
plastic spatula. The suspension is filtered through 2 layers of cheesecloth. 
  6. Humid chambers are created using t
wo large baking dishes of the same size, the 
dishes should be large enough to contain a 20 x 20 cm TLC plate allowing for 
extra space to keep the edges of the top and bottom dishes away from the plate. 
One end of the baking dish, towards the top of the TLC
 plate (labeling section), is 
propped up by approximately 2 inches to avoid accumulation of moisture 
droplets. The other dish can be used as the cover (see below). 
  7.  Distilled water should be brought to a boil prior to adding. Enough hot water 
should be a
dded to cover 
+ of the bottom
-baking dish, leaving the propped end 
water-free. After adding water, the top
-baking dish is used as a cover. The inside 
cover should be wiped down 3
-4 times, removing all moisture, prior to spraying 
plate.  8. TLC plates are spra
yed with a suspension of 
C. cucumerinum 
spores
 (usually 3 
Ð 3.5 x 106 spores/ml) in half
-strength potato
-dextrose broth.
  9. Small beakers, 50 
Ð 100mL, can be inverted to keep the plate above water (4 
beakers/plate).
   !%))!  10.  After the plates are sprayed with the 
C. cucumerinum 
suspension
, they are 
immediately placed in the humid chamber. Masking tape should be used to 
completely seal the edges where the inverted baking dish (used as the top) is 
placed around the edge of the bottom
-baking dish, sealing the two dishes 
together creating a ch
amber. 
  11. Plates should remain in the chamber for 3 days.
  12.  White areas on a grey
-black background of fungal spores and mycelia on the   
TLC plate indicate antifungal compounds. 
                   !%)*!            !LITERATURE CITED
                       !%)+!LITERATURE CITED
   Ahuja, I., Kissen, R., & Bones, A. M. (2012). Phytoalexins in defense against 
pathogens.
 Trends in Plant Science
, 17, 73-90.  Geibel, M. (1995). Sensitivity of the fungus 
Cytospora persoonii
 to the flavonoids of 
Prunus cerasus
. Phytochemistry
, 38, 599-601.  Dixon, R. A., & Ferreira, D. (2002). Genistein.
 Phytochemistry
, 60, 205-211.  Hammerschmidt, R. (1999). Phytoalexins: what have we learned after 60 years? 
Annual 
Review of Phytopathology
, 37, 285-306.  Hammerschmidt, R. (2011). Phytoalexins at the right place and time. 
Physiological and 
Molecular Plant Pathology
, 76, iii-iv.  Hammerschmidt, R., & Hollosy, S. I. (2008). Phenols and the Onset and Expression of 
Plant Disease Resistance.
 Recent Advances in P
olyphenol Research
, 1, 211-227.  Harborne, J. B., & Baxter, H. (1993).
 Phytochemical Dictionary. A Handbook of 
Bioactive Compounds from Plants
. Taylor & Francis Limited, London, Washington, DC, 
pp. 359-421.   Hasegawa, M., & Shirato, T. (1955). 
Flavonoids of various 
Prunus 
species. IV. The 
flavonoids in the wood of 
Prunus donarium
 var. 
spontanea
. Journal of the American 
Chemical Society
, 77, 3557-3558.  Hasegawa, M., & Shirato, T. (1957). Flavonoids of various 
Prunus 
species. V. The 
flavonoids in
 the wood of Prunus verecunda
. Journal of the American Chemical 
Societ
y, 79, 450-452.  Hummer, K. E., & Janick, J. (2009). Rosaceae: Taxonomy, Economic Importance, 
Genomics. In
 Genetics and genomics of Rosaceae
. Springer, New York, pp. 1
-17.  Ku
%, J. (1995
). Phytoalexins, stress metabolism, and disease resistance in 
plants.
 Annual Review of Phytopathology
, 33, 275-297.  Nicholson, R. L., & Wood, K. V. (2001). Phytoalexins and secondary products, where 
are they and how can we measure them? 
Physiological and 
Molecular Plant 
Pathology
, 59, 63-69.  Ogah, O., Watkins, C. S., Ubi, B. E., & Oraguzie, N. C. (2014). Phenolic Compounds in 
Rosaceae Fruit and Nut Crops.
 Journal of Agricultural and Food Chemistry
, 62, 9369-9386.  !%),!Parmar, V. S., Vardhan, A., Nagarajan, G. R., & Jain, R. (1992). Dihydroflavonols from 
Prunus
 domestica
. Phytochemistry
, 31, 2185-2186.  Potter, D., Eriksson, T., Evans, R. C., Oh, S., Smedmark, J. E. E., Morgan, D. R., 
Kerr, 
M., Robertson, K. R., Arsenaul
t, M., Dickinson, T.A., 
& Campbell, C. S. (2007). 
Phylogeny and classification of Rosaceae.
 Plant Systematics and Evolution
, 266, 5-43.  Prusky, D., Keen, N. T., & Eaks, I. (1983). 
Further evidence for the involvement of a 
preformed antifungal compound in the latency of 
Colletotrichum gloeosporioides
 on unripe avocado fruits.
 Physiological Plant Pathology
, 22, 189-198.  R Core Team, 2014. R: A Language and Environment for Statistical
                   Computing. R Foundation for Statistical Computing. Vienna, Austria. Retrieved from 
http://www.R-project.org
. 4 Aug. 2015. 
  Santamour, F. S., & Riedel, L. G. (1994). 
Distribution and inheritance of scopolin and 
herniarin in some 
Prunus 
species.
 Biochemical Systematics and Ecology
, 22, 197-201.  Schwalb, P., & Feucht, W. (1999). Changes in the concentration of phenolic substances 
in the bark during the annual developmen
t of the cherry tree (
Prunus avium
 L.).
 Advances in Horticultural Science
, 71-75.  Shulaev, V., Korban, S. S., Sosinski, B., Abbott, A. G., Aldwinckle, H. S., Folta, K. M.,
 Iezzoni, A., Main, D., Arœs, P., Dandekar, A. M., Lewers, K., Brown, S. K., Davis, T. M., 
Gardiner, S. E., Potter, D.,
 & Veilleux, R. E. (2008). Multiple models for Rosaceae 
genomics.
 Plant Physiology
, 147, 985-1003.  VanEtten, H. D., Mansfield, J. W., Ba
iley, J. A., & Farmer, E. E. (1994). Two classes of 
plant antibiotics: phytoalexins versus" phytoanticipins". 
The Plant Cell
, 6, 1191.  Vaughn, S. F., & Spencer, G. F. (1994). Antifungal activity of natural compounds 
against thiabendazole
-resistant 
Fusariu
m sambucinum
 strains. 
Journal of Agricultural 
and Food Chemistry
, 42, 200-203.            !%)-!CHAPTER FOUR
  PRUNUS MAACKII
 FUTURE RESEARCH
   Knowledge of the common flavonoids found in 
Prunus
 and the identification of 
unique compounds, including non
-substituted B
-ring flavonoids, will help identify 
common precursors along with unique ones. The extraction of flavonoids is contingent 
on the type of plant and the biologically
 active compounds con
tained in the tissues 
(Khoddami et al., 2013). An HPLC/MS/MS machine, with a large fraction
-collecting 
column, may be used to isolate the individual compounds based on retention
-time
s and their UV
-spectra. Once this is done, the individual compounds can be
 submitted for 
NMR and MS/MS analyses. Depending on each compounds crystallization properties, 
single crystal analyses may be employed as well. 
 In addition to the identification of
 unknown compounds, tissues also need to be 
examined for possible precursor
s. The chalcone isomerase (CHI)
 enzyme
 further 
synthes
izes naringenin from a chalcone. Naringenin
 is the core flavonoid structure 
(flavanone) that leads to the production of other commonly found flavonoids by 
enzymatic modification e.g., flavones, isoflavo
noids and flavanones (McNulty et al., 
2009).
  Thus, 
initial enzyme assays should include those commonly found 
in the flavonoid biosynthetic 
pathways,
 e.g., PAL, CHS/CHI and FHT assays (Halbwirth  et al., 
2009). 
For example, 
PAL 
gene expression would be screened 
in vitro
 because 
the PAL
 enzyme is a regulator of
 phenolic biosynthesis (Pina and Errea, 2008). The expression 
of this enzyme is critical because it represents the conversion of a primary metabolite
 !%*.!(phenylalanine) into a 
secondary metabolite (cinnamate)(Halbwirth et al., 2009). 
O-methyltransferases (OMTs) are
 particularly interesting from a plant pathology aspect. 
This class of enzymes is involved in the produc
tion of lignin precursors and
 the biosynthesis of phytoalexins.
 Expression of these enzymes has been shown to increase 
upon pathogen invasion. Gene expression can be 
examined by RNA blot analysis. In 
addition, the Southern blot analysis
 can
 be carried out upon DNA preparation (Suelves 
and Puigdomenech, 1998).
 Since al
nusin and alnustinol are somewhat unique with the 6
-methoxylation and 
lack of B
-ring substitution, it is likely that these flavonoids are synthesized from 
cinnamate, via 4
-hydroxycinnamate. For this reason, unique enzymes will likely be 
found. One precurso
r could be pinocembrin, which is an antifungal compound found in 
the leaf glands of cottonwood (Shain & Miller, 1982). This particular flavonoid has two 
hydroxylations without B
-ring substitutions. Pinocembrin is 
structurally 
similar to the 
commonly found flavonoids, e.g. naringenin and eriodictyol, but it is not synthesized 
from 
p-coumaric acid, but rather cinnamic acid
. This compound can also be converted 
to pinobanksin by hydroxylation of the ketone.  Thus
, it is likely that
 pinobanksin is also 
present in the crude extract.
 Enzymatic modifications are responsible for the conversion of flavanones (such 
as pinocembrin) to dihydroflavonols (such as pinobanksin). Many other products are 
produced from enzymatic reactions, which in
clude tannins, flavonols, flavan
-3-ols, and 
proanthocyanidins. The correlation of gene expression and enzymes such as flavone 
synthase, flavanol synthase, flavanone reductase
, and dioxygenase will help elucidate 
the general biosynthesis of flavonoids found
 in P. maackii
. Alnusin and alnustinol are
 !%*%!found in high concentration
 in P. maackii
 periderm tissue
, and either absent or in very 
low concentration in other 
Prunus
 spp., therefore 
the primary goal will be to elucidate 
the gene expression correlated with cinnamyl
-CoA-ligase, 6 hydroxylases and 6
-methoxylases. 
 The expression of different genes should be compared 
among those commonly 
found in susceptible spp. and compared to the potential
ly resistant 
P. maackii
. Once the 
biosynthetic flavonoid backbone enzymes have been identified, these enzymes should 
be compared to those found in 
P. mahaleb
 and P. serotina
. The differences in enzyme 
activity and specificity will be of great use to identi
fy the regulation of secondary 
metabolites with unique antimicrobial actvity
. Since fungicide drenches and fumigation treatments show little effects against 
Armillaria
 species (Amiri & Schnabel, 2012), finding a resistant rootstock is critical. It 
has been
 reported that pears are somewhat resistant (Dalili et al., 2010) and can serve 
as an alternative crop. However this is not an option in regions where fire blight is 
prevalent. It is possible that 
P. maackii
 can serve as a resistant rootstock. The cost and
 benefits of employing secondary metabolite biosynthesis in the absence of pathogens is 
important in host fitness (Thaler et al, 2012), therefore 
this 
should be investigated in 
P. 
maackii
 to give insight on the balance of phytoanticipin production.
  The ultimate goal of 
P. maackii
 research is to identify the antifungal compounds 
and to potentially use this ornamental species as a resistant rootstock. The recent 
identification of two antifungal compounds suggests that flavonoids without B
-ring 
substitu
ents may contribute to
 the strong antifungal activity 
detected
 in the crude 
P. 
!%*&!maackii
 periderm
 extract
. This species may contain a model pathway for the 
engineering of resistant 
Prunus
 species.
 !!!       !!!!!!!!!!!!!!!!!!!!!!!!!!%*'!!!!!!!!!!!!!!!!!    LITERATURE CITED
                        !%*(!LITERATURE CITED
   Amiri, A., & Schnabel, G. (2012). Persistence of propiconazole in peach roots and 
efficacy of trunk infusions for Armillaria root rot control.
 International Journal of Fruit 
Science
, 12, 437-449.  Dalili, S. A. R., Nanagulyan, S. G., Alavi, S. V., & Razavi, M. (2010). Investigation of the 
wood destroying activity of 
Armillaria mellea
 on horticultural and forest plants species. 
Australian
 Journal
 of Crop
 Science
, 4, 209-215.   Halbwirth, H., Waldner,
 I., Miosic, S., Ibanez, M., Costa, G., & Stich, K. (2009). 
Measuring flavonoid enzyme activities in tissues of fruit species.
 Journal of Agricultural 
and Food Chemistry
, 57, 4983-4987.  Khoddami, A., Wilkes, M. A., & Roberts, T. H., 2013. Techniques for a
nalysis of plant 
phenolic compounds. 
Molecules
, 18, 2328-2375.  Pina, A., 
& Errea, P., 2008. Differential induction of phenylalanine ammonia
- lyase gene 
expression in response to 
in vitro
 callus unions of 
Prunus
 spp. 
Journal of Plant 
Physiology
, 165, 705-714.  McNulty, J., Nair, J. J., Bollareddy, E., Keskar, K., Thorat, A., Crankshaw, D. J., & Ejim, 
L., 2009. Isolation of flavonoids from the heartwood and resin of 
Prunus avium
 and some preliminary biological investigations. 
Phytochemistry
, 70, 2040-2046.  Shain, L. & Miller, J.B. (1982). Pinocembrin: an antifungal compound secreted by leaf 
glands of eastern cottonwood.
 Phytopathology
, 72, 877-880.  Suelves, M., & Puigdomenech, P. (1998). Specific mRNA accumulation of a gene 
coding for an 
O-methyltransferase in almond (
Prunus amygdalus
, Batsch) flower 
tissues.
 Plant Science
, 134, 79-88.  Thaler, J. S., Humphrey, P. T., & Whiteman, N. K. (2012). Evolution of jasmonate and 
salicylate signal crosstalk.
 Trends in Plant Science
, 17, 260-270.