ENHANCEMENT OF BIOLOGICAL CONTROL OF … · I would like to thank the Toves and Calimlim families...
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ENHANCEMENT OF BIOLOGICAL CONTROL OF ANTHURIUM BLIGHT CAUSED BY Xanthomonas axonopodis pv. dieffenbachiae A THESIS SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAW AI'I IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN TROPICAL PLANT AND SOIL SCIENCES MAY 2008 By PETER 1. TOVES Thesis Committee: Anne Alvarez, Chairperson Richard Criley Adelheid Kuehnle Hector Valenzuela
Transcript of ENHANCEMENT OF BIOLOGICAL CONTROL OF … · I would like to thank the Toves and Calimlim families...
ENHANCEMENT OF BIOLOGICAL CONTROL OF ANTHURIUM BLIGHT CAUSED BY Xanthomonas axonopodis pv. dieffenbachiae
A THESIS SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAW AI'I IN PARTIAL FULFILLMENT OF THE
REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE
IN
TROPICAL PLANT AND SOIL SCIENCES
MAY 2008
By PETER 1. TOVES
Thesis Committee:
Anne Alvarez, Chairperson Richard Criley
Adelheid Kuehnle Hector Valenzuela
We certify that we have read this thesis and that, in our opinion, it is satisfactory in scope
and quality as a thesis for the degree of Master of Science in Tropical Plant and Soil
Sciences.
THESIS COMMITIEE
Chairperson
12ubJJ. ~ 4J..eLIM A t.1I. Q L
b. I",,J..
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To my grandmothers, Josefina G. Toves and Rosario Villamor
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ACKNOWLEDGEMENTS
I would like to thank the Almighty for blessing me with the opportunity to
achieve this degree. Thank you to my committee members who have provided resources,
guidance, and suggestions to complete my research. My sincerest gratitude to Dr. Anne
Alvarez for giving me the opportunity to not only learn about plant pathology, but also
about life itself.
I would like to thank my fellow lab mates for all the support, the late hours, and
most especially the fun times. Special thanks to Wendy Sueno for helping me through
the more difficult times during my graduate work. I thank Dr. Tessie Amore for all her
help, patience, and expert advice on anthurium micropropagation. I would like to thank
Drs. Mark Wright and Ian Pagano for helping me with the statistics. Thanks to Grayson
Inouye for providing space to run our field experiments in Hilo. Thank you to the
secretaries from TPSS and PEPS, but most especially to Ms. Susan Takahashi for looking
out for me and keeping me on track with all my graduate requirements. I thank Emily
Lloyd for reviewing my thesis at the last minute.
I would like to thank the Toves and Calimlim families for all the moral support
and encouragement through the duration of my research.
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ABSTRACT
Anthuriums are Hawaii's signature cut flower, and optimal growth and protection
of anthurium plants are crucial to the Hawaiian floriculture industry. Anthurium blight,
caused by Xanthomonas axonopodis pv. dieffenbachiae (Xad), is the most destructive
disease of anthurium worldwide. Beneficial bacteria have been identified for use as
biological control agents (BCAs) against Xad and these strains have also been shown to
stimulate growth of micro-propagated plants. Optimization of transplanted microplant
growth was examined. Anthurium microplants grew better with the combination of
inorganic fertilizer combined with BCAs than when grown in either fertilizer or BCAs
alone. Biostimulation was observed on all anthurium cultivars treated with the beneficial
strains.
Populations of beneficial bacteria decline after foliar application on anthurium
plants. Studies were focused on improving the efficacy of the BCAs with carbon sources
that sustain beneficial bacterial populations on plant surfaces without stimulating
pathogen growth. Valine and isoleucine were identified as amino acids that enabled
growth of beneficial bacteria while inhibiting growth of the pathogen in vitro. In
greenhouse and field studies, treatments with valine combined with the BCAs reduced
disease incidence by 12 to 21 % compared to treatments with BCAs alone.
Key words: Biological control agents, beneficial bacteria, feedback inhibition, amino
acid
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TABLE OF CONTENTS
ACKNOWLEDGEl\1ENTS ....................................................................... .iv
ABSTRACT ........................................................................................... v
LIST OF TABLES .................................................................................. .ix
LIST OF FIGURES .................................................................................. xi
LIST OF ABBREVIATIONS ..................................................................... xiii
CHAPTER 1: Literature Review .................................................................. 1
Anthurium ..................................................................................... 1
Economic importance ....................................................................... 2
Production practices ......................................................................... 3
Special practices for greenhouse grown anthuriums ........................ ... 5
Major diseases ................................................................................ 6
Fungal diseases ....................................................................... 6
Bacterial diseases ................................................................... 6
Previous studies on anthurium blight ....................................................... 8
Disease control of blight .......................................................................................... 8
Cultural practices for control ofblight... ..................................................... 8
Breeding anthuriums for resistance to anthurium blight ........................... l 0
Genetic Engineering for Resistance to Anthurium Blight ................... 10
Tissue cultured anthurium ....................................................... 11
Biological control of anthurium blight ......................................... 11
Feedback inhibition of amino acid pathways ........................................... 12
Literature cited .............................................................................. 13
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CHAPTER 2: Use of Selected Amino Acids to Enhance Effectiveness of Beneficial
Bacteria in Biocontrol of Anthurium Blight ............................................ 20
Abstract ...................................................................................... 20
Introduction .................................................................................. 21
Materials and Methods ..................................................................... 23
Bacterial strains and inoculum preparation ..................................... 23
Bacterial growth on various carbon sources ................................... 24
Greenhouse studies ................................................................ 25
Field Studies ....................................................................... 27
Results ........................................................................................ 28
Bacterial growth on various carbon sources ................................... 28
Greenhouse studies ................................................................ 31
Field studies ........................................................................ 31
Discussion ................................................................................... 32
Literature Cited ............................................................................. 44
CHAPTER 3: Optimizing Growth of Anthurium Microplants with Mineral Nutrients
and Beneficial Bacteria .......................................................... .48
Abstract ..................................................................................... .48
Introduction ................................................................................ .48
Materials and Methods ..................................................................... 49
Plant materials and growth conditions .......................................... 49
Bacterial strains and inoculum preparation .................................... 49
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Effects of fertilizer treatments and beneficial bacteria on anthurium
microplants ......................................................................... 50
Bacterial growth on various mineral solutions ................................ 51
Effects of mineral solutions on anthurium microplants ...................... 52
Results ....................................................................................... 53
Effects of fertilizer treatments and beneficial bacteria on anthurium
microplants ......................................................................... 53
Bacterial growth on various mineral solutions .......................................... 54
Effects of mineral solutions on anthurium microplants ...................... 54
Discussion .................................................................................... 54
Literature Cited ............................................................................. 61
APPENDICES
A. Susceptibility of Anthurium antioquense 'Cotton Candy' to anthurium
blight and bioprotection by beneficial bacteria ......................................... 62
B. Bioprotection of five cultivars of .anthurium microplants ................................ 65
C. Comparison of anthurium microplants treated with beneficial bacteria and two
inoculum levels of Xanthomonas axonopodis pv. dieffenbachiae ........... ......... 69
D. Feedback inhibition of valine, leucine, and isoleucine biosynthesis ...................... 74
E. Overall conclusions ............................................................................. 75
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LIST OF TABLES
Tables
2-1 Relationship between viable cell counts and turbidity (measured in Klett units)
for bacterial strains used in growth curve studies ....................................... 35
2-2 Oxidation and growth of beneficial bacteria on carbon sources not utilized by
Xanthomonas axonopodis pv. dieifenbachiae ... ....................................... 36
2-3 Growth of beneficial bacteria and Xanthomonas axonopodis pv. dieifenbachiae
on amino acids supplied as sole carbon sources in a standard mineral base ........ 37
2-4 Growth of beneficial bacteria and Xanthomonas axonopodis pv. dieJlenbachiae
on a standard mineral base plus glucose (10 mglml) supplemented with amino
acids (1 mglml) or other products of amino acid synthetic pathways (AASP) ..... 38
2-5 Growth of beneficial bacteria and Xanthomonas axonopodis pv. dieJlenbachiae
on a standard mineral base plus glucose (10 mglml) and combinations of selected
amino acids (1 mglml) ....................................................................... 39
3-1 Effect offertilizer treatments on growth of three anthurium cultivars ............... 57
3-2 Survival ofUH 780 treated with reduced nutrient applications 30, 50, and 80 days
after transplant ............................................................................... 57
3-3 Mean parameter measurements for UH 780 plants treated with biological control
agents (BCAs) and inorganic fertilizer .................................................. 58
B-1 Disease severity of Anthurium andreanum microplants treated with beneficial
bacteria and inoculated with Xanthomonas axonopodis pv. dieJlenbachiae ... ..... 67
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LIST OF TABLES
Tables
C-l Disease incidence and severity ofanthurium plants challenged withXanthomonas
axonopodis pv. diejJenbachiae at 108 CFU/mI ............................................ 72
C-2 Disease incidence and severity ofanthurium plants challenged withXanthomonas
axonopodis pv. dieffenbachiae at 109 CFU/mI ............................................ 73
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LIST OF FIGURES
Figures
2-1 Growth of Gut 6 (Herbasprillum rubrlsulbalbicans) andXanthomonas
axonopodis pv. dieffenbachtae (Xad) in standard mineral base (SMB) containing
1% glucose (Glc), and various combinations of valine (Val), isoleucine (lIe),
glutamic acid (Glu), and glutamine (Gin) ................................................ .40
2-2 Effects of 0.1% valine and 0.1% isoleucine treatments on the progression offoliar
infection ofanthurium 'Ozaki' by Xad-Lux under greenhouse conditions ........ .41
2-3 Effects of 0.1 % valine and 0.1 % isoleucine treatments on the progression of foliar
infection ofanthurium 'Ozaki' by Xad-Lux under greenhouse conditions ........ .41
2-4 Effects of valine treatments on progression offoliar infection of anthurium
'UH780' by Xad-Lux under greenhouse conditions .................................... .42
2-5 Effects of 0.1 % valine and 0.1 % isoleucine treatments on the progression of foliar
infection of anthurium 'Ozaki' by Xad under field conditions ......................... 42
2-6 Effects of 0.1 % valine treatments on the progression of foliar infection of
anthurium 'Ozaki' by Xad under field conditions ..................................... ..43
3-1 Effect of beneficial bacteria (BeAs) on growth parameters of four varieties of
Anthurlum andreanum ........................ .............................................. 59
3-2 Growth of anthurium beneficial bacteria and Xanthomonas axonopodis pv.
dieffenbachiae in selected mineral solutions (Standard mineral base, Miracle-Gro,
and modified Hoagland solution) and D-glucose ...................................... 60
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LIST OF FIGURES
Figures
3-3 Comparison of plant height, canopy width, and leaf length and width of
Anthurium UH 780 treated with modified '.4 strength Hoagland solution and '.4
strength Standard Mineral Base (SMB) ................................................... 60
A-I Effects of beneficial bacterial treatments on the progression of disease incidence
of Anthurium antioquense cultivar 'Cotton Candy' by Xanthomonas axonopodis
pv. die.ffenbachiae .......................................... ................................. 64
B-1 Effects of beneficial bacterial (BeA) consortium on progression offoliar infection
of Xad-Lux on four cultivars of Anthurium andreanum ..... ........................... 68
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LIST OF ABBREVIATIONS
BCAs - Biological control agents
5MB - Standard mineral base
Xad - Xanthomonas axonopodis pv. dieffenbachiae
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ANTHURIUM
Chapter 1
Literature Review
Anthuriums are mainly perennial, herbaceous, epiphytes with a creeping climbing
habit. Native areas of origin include Columbia, Peru, Central and South America, and
Venezuela (Agrolink, 2003). Anthurium is a genus in the fiunily Areacae, which
comprises over 100 genera and encompasses over 1,500 species (Higaki et a1., 1983).
Spathes are heart shaped and often referred to as the floral portion. The long structure
above the spath is known as the spadix and bears the male and female flowers. Leaves
also are heart shaped with one main midvein and lateral veins, and are attached to long
petioles (Agrolink, 2003).
The genus Anthurium comprises over 600 species from Tropical America
(Kamemoto, 1988a). The best known member within the genus is Anthurium
andreanum, which was discovered in Columbia and Ecuador by Eduard Andre in 1870
(van Uffelen, 1996). In the natural state A. andreanum is epiphytic and can be found in
mountain forests at elevations of2400 ft. In its natural habitat, the spathes of this species
are orange-red and blistered. Many of the anthuriums cultivated today areA. andreanum
hybrids but differ in appearance from the species. The spathes may be smooth or
blistered to varying degrees, and are available in a wide range of colors (Kamemoto,
1988a).
Introduction of A. andreanum to Hawaii from London in 1889 was by S.M.
Damon, and was described as having a spathe with shell-pink color (Neal, 1965). Plants
were grown on the Damon estates on Moanalua from where it was slowly distributed to
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other collectors via vegetative propagation. In the late 1930s and 1940s growers in
Hawaii learned how to propagate anthurium by seed, leading not only to increased
cultivation, but also to increased variation (Kamemoto, 1981). The assortment available
today is a result of crosses between A. andreanum cultivars, as well as crosses between A.
andreanum and other species (van Uffelen, 1996).
ECONONUCnwPORTANCE
Anthuriums became part of the inventory of flower shops in Hawaii during the
1940s. A cut flower industry developed initially from hobbyists to small backyard
growers, to the large scale business operations of today (Kamemoto, 1981). Commercial
operations for anthurium production in 1959 included 266 farms on Hawaii, 88 on Oahu,
7 on Kauai, and 4 on Maui (Hiloweb, 2003). Increased worldwide demand for anthurium
cut flowers in the 1970s boosted sales and increased the production area from 40 acres to
400 acres in 1979. The industry reached its peak in 1980, supplying local, national, and
international markets with up to 232,000 dozen flowers per month (Hiloweb, 2003).
Although yield was at 2.5 million dozen flowers in 1980, supply was insufficient to meet
demand.
The top four producers of anthurium cut flowers worldwide are Holland, Hawaii,
Mauritius, and Jamaica, followed by smaller producers in tropical regions such as the
Philippines, Brazil, Malaysia, Martinique, and Thailand (Deardorff; 1991; Shehata,1992).
The anthurium research program initiated in Hawaii in 1950 by Dr. Haruyuki
Kamemoto led to the development of a breeding program for the commercial
development and release of anthuriums to growers (Kamemoto and Kuehnle, 1996).
Subsequent development of anthuriums for the cut flower industry by breeders in the
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Netherlands has led to the availability ofan assortment of varieties, with red and orange
having the most importance, followed by other colors such as salmon, cherry, and pink
(van Uffelen, 1996).
Initially, anthuriums were grown commercially for the cut flower industry, but
now production has expanded into the potted plant industry. A. scherzerianum cultivars
are the main potted anthuriums in Europe, while in Hawai~ A. andreanum-type cultivars
are more popular (Kuehnle et al., 1996).
Hawaii's floriculture industry was valued at $100 million in 2006 (Hawaii
Agriculture Statistics Service, 2006). Cut flowers sales were valued at $14.1 million, with
anthuriums ranking as the top seller at $5.5 million.
PRODUCTION PRACTICES
Anthuriums grow best between 18°C and 27°C, and require shaded conditions of
50 to 90 percent depending on cultivar, age of plant, and climate (Higaki et al., 1994). A
pathogen-free medium is essential for anthurium culture. A medium that is well aerated
and provides optimal moisture and nutrient retention is required for desirable growth of
anthurium (Higaki et al., 1994; van Os, 2002). Substrates used in the medium will vary
from country to country, depending on availability and cost. In Hawaii, sugar cane
bagasse was considered to be a desirable medium for anthurium culture. but bagasse has
since become unavailable with the decline of sugarcane production. Higaki and Imamura
(1985) found that black cinder, a much cheaper alternative, could be used successfully in
place of bagasse for anthurium flower production.
Plant nutrition is an important cultural factor in commercial production of any
crop. Nutritional treatment of anthurium is a factor that growers can control to optimize
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plant health and production, and prevent susceptibility to diseases and pests (Imamura
and Higaki, 1989). In 1988, Higaki and Imamura (1988) reported that most Hawaiian
growers used one or more of the foIlowing fertilizer treatments:
1.) Osmocote slow-release fertilizer (14-14-14) at 200-350 pounds NI A/yr (225-397
kg NIHa/yr) divided into three equal portions and applied three times per year.
2.) Inorganic chemical fertilizers (non-slow-release fertilizer) at 200-350 pounds
NI A/yr (225-397 kg NlHa/yr) applied 12 times per year.
3.) Solid fertilizer program (lor 2 above) supplemented with foliar fertilizer
applications.
Imamura and Higaki (1989) recommended nitrogen at 300 Ibs/A/yr (340 kg/Ha/yr),
phosphorous at 400 Ibs/ A/yr (454 kglHa/yr), and potassium at 300 Ibs kg! A/yr (340
kglHa/yr) for anthurium production.
Although recommended fertilizer rates provide a general guideline for nutrient
requirements for anthurium, MiIls (1989) and Imamura and Higaki (1989) recommended
leaf tissue analysis at regular intervals for efficient use of nutrients and optimal growth
and flower production. The most recently mature leaf; subtending a 3/4 matured flower
is used for tissue analysis, since it is more representative of the nutrient supply available
for developing leaves and flowers (Mills 1989, Mills and Scoggins 1998). The nutrient
sufficiency ranges for anthurium leafanaIysis is 1.6%-3.0".10 for nitrogen, 0.2%-0.7".10 for
phosphorous, 1.00/0-3.5% for potassium, 1.2%-2.0% for calcium, 0.5%-1.0% for
magnesium, and 0.16%-0.75% for sulfur (Mills, 1989; Jones et aI., 1991).
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Special Practices for Greenhouse-grown Anthuriums
Optimal temperatures for young greenhouse grown anthurium plants are 23°C
during the day, and 22°C at night. For general growing of anthuriums, 18°C to 28°C is
best. A good medium should consist approximately of25% air, 25% water, and 50%
solids. The ratio of coarse to fine material in the media will vary, depending on pot size,
length of cultivation, cultivation system, and available materials. (van Os, 2002) The pH
of the medium should be around 5.5-6.0. Planting of cuttings is recommended in the
spring or summer for fast initial growth. Plants should be planted as deep as possible,
without covering the growing point, allowing the aerial roots near the growing point to
grow into the substrate and provide better anchorage for future growth (Hummelen,
2000). After planting, flower buds should be removed to allow for better root growth.
Leaves should not be removed from cuttings for the first three months to allow
development of thicker growing points and larger flowers.
Fertilization of anthuriums in Holland is based on electrical conductivity or EC,
which is a standard for the total concentration of (mineral) salts in water. The EC is
measured with a meter in milli-Siemens per cm (mS/cm), mhoS/cm, or in 1I0hm (mho).
According to van Spingelen (1998), the target EC values for anthurium are as follows:
1.) During cold/wet periods-l.3 mS/cm and 0.9 mS/cm in water and soil samples,
respectively.
2.) During warm/dry periods - 0.9 mS/cm and 0.7 mS/cm in water and soil samples,
respectively.
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MAJOR DISEASES
Anthuriums were not seriously affected by insects or disease in early years of
production in Hawaii. Disease problems eventually developed as cultivation increased
from small plots to large operations. Significant diseases which affected anthuriums
included anthracnose. root rot, and most important - bacterial blight.
Fungal Diseases
Anthracnose, caused by Colletotrichum gloeosporioides, is a fungal disease of
anthurium as well as other crops grown in tropical and subtropical conditions (Nishijima,
1994). Symptoms of infection usually include an isolated small circular black spot that
eventually grows into an angular shape (Nishijima, 1994). In the 1960s, the disease was
known as spadix rot or "black nose disease" (Kamemoto, 1988a).
A root rot complex of anthurium involves a number of pathogens, including
Pythium splendens, Calonectria crotalariae, Rhizoctonia sp., Phythophthora sp., Pythium
spp., and Fusarium sp. (Nishijima, 1994, Guo and Ko 1991). Symptoms of root rot
include tissue discoloration, decaying odor emanating from roots, overall decrease in
plant vigor, reduction in flower and leaf size, and decreased plant height (Nishijima,
1994).
Bacterial Diseases
Bacterial wilt is a disease caused by Ralstonia solanacearum, also known as
Burkholderia solanacearom, or Pseudomonas solanacearom. The disease is of economic
importance in Mauritius, infecting tomato, eggplant, potato, and anthurium
(Banymandhub-Munbodh, 1998). Symptoms of bacterial wilt include chlorosis, necrosis,
and wilt. Norman and Yuen (1999) reported that distinctions in the symptomology of
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plants systemically infected with Xanthomonas sp. or Ra/stonia solanaceannn could not
be made.
Pseudomonas leaf spot is a disease caused by Pseudomonas cichorii, a bacterium
that infects foliage and flowering ornamentals as well as vegetables. The bacterium
infects plants through hydathodes and wounds. Early symptoms of the disease on foliage
plants include water-soaked lesions anywhere on the leafbut usually on the margins.
Lesions quickly enlarge and become necrotic, forming light and dark zones sometimes
surrounded by a yellow halo. The disease is spread through splashing water, infected
tools, and handling of infected plants (Nishijima and Fujiyama, 1985).
Anthurium blight is a bacterial disease caused by Xanthomonas axonopodis pv.
dieffenbachiae (Xad). Early foliar symptoms involve water soaked spots mainly on the
underside of the leat: near the margin (Nishijima and Fujiyama, 1985; Nishijima, 1994).
Tissues surrounding the infected areas turn yellow and become necrotic. Xad can also
infect anthuriums systemically, preventing the translocation of nutrients and water,
leading eventually to death of the plant (Nishijima, 1988; Nishijima, 1994). Bacterial
blight was first seen in 1971 on Kauai, but it did not have a negative impact on the
industry in Hawaii until 1980 (Nishijima, 1988). The disease is now widespread and can
be found in Australia, California, Florida, Guadalupe, Guam, Jamaica, Martinique, the
Philippines, Puerto Rico, Tahiti, and Venezuela (Lipp et aI., 1992). Bacterial blight can
be spread by a number of ways including contaminated cutting tools, infected plant
material, splashing rain or irrigation water, and aerosols (Nishijima, 1994; Alvarez, et aI.,
1991; 1992; Alvarez and Norman 1993).
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PREVIOUS STUDIES ON ANTHURIUM BUGHT
Monoclonal antibodies have been developed for the identification and
characterization ofXad and other xanthomonads, and have been used to trace the
movement of the bacteria from plant to plant in shadehouses (Alvarez et al., 1988;
Norman and Alvarez, 1989; Lipp et al., 1992; Alvarez and Norman, 1993; Norman and
Alvarez, 1994b). Propagation materials which do not show visual signs of blight still
serve as potential sources of the disease due to latent infections, as seen in tissue cultured
plants (Norman et al., 1993; Norman and Alvarez, 1994a). Latent infection is difficult to
examine without indicators to allow visualization of the bacteria. The creation of a
genetically engineered strain ofXad containing the "lux" gene enabled detection and
observation of movement of the bacteria by exposure to sensitive X-ray film (Alvarez et
al., 1993). This bioluminescent strain ofXad was used to study the infection process,
cultivar susceptibility to the pathogen, and temperature effects on leaf colonization
(Fukui et al., 1996; 1998; 1999c).
DISEASE CONTROL OF BUGHT
Cultural Practiees for Control of Blight
There are various aspects for management of anthurium blight. Sanitation is
important and involves removal of early leaf infections and elimination of systemically
infected plants (Nishijima, 1988; Nishijima, 1994). Disinfection of cutting tools is
important to prevent the spread of blight, since plant materials which show no symptoms
have the potential for latent infection. Growing plants under plastic or glass houses
coupled with drip irrigation rather than overhead or sprinkler irrigation can help reduce
the spread of the bacteria through aerosol and water splash (Alvarez and Norman, 1993).
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Kamemoto and Kuehnle (1989) reported that the change from overhead irrigation to drip
irrigation significantly reduced the incidence of blight in anthurium seedling culture.
Growing anthuriums under cool and shaded conditions slows the progression of the
disease. Inoculated plants exposed to temperatures greater than 27°C were more
susceptible to disease than inoculated plants exposed to lower temperatures (Alvarez, et
al., 1990).
Chemical control of blight is difficult due to the lack of effective bactericides
(Alvarez et al., 1989; Chase 1988). Nishijima and Chun (1991) and Alvarez et al. (1991)
found that the fungicide fosetyl-A1 (A1iette) has the ability to reduce the rate and severity
ofinfection by Xad on anthurium if applied before bacterial infection.
Chemicals have the potential to alter epiphytic and soil microbial populations,
promoting the incidence of disease. Chemicals can stimulate a pathogen, and affect soil
and leaf microorganisms. Diuron (Karmex), a chemical used to control weeds, can be
used as an energy source by XanthomontlS (Mills, 1989).
Nutrition is important in plant susceptibility to diseases. Chase (1989) suggested
that lower fertilizer rates for potted anthuriums could result in fewer leaves susceptible to
Xad and greater flower production. Sakai (1990) reported that higher levels of
ammonium fertilizer led to higher amounts of amino compounds in guttation fluid when
compared to nitrate fertilizers. Increased amounts of amino compounds were associated
with greater plant susceptibility to disease. The use of sufficient amounts of nitrate
fertilizers for plant growth reduced the amount of amino compounds in guttation fluid,
and was expected to reduce the incidence of blight (Sakai, 1991; Sakai, 1992). Higaki et
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al. (1990) reported no differences in blight incidence or susceptibility when comparisons
were made between organic and inorganic fertilizer treatments.
Breeding for Resistance to Anthurium Blight
Many of the early cultivars developed for Hawaii's anthurium industry before
1980 were bred mainly for resistance to anthracnose, while incorporating other desirable
horticultural traits such as color, shape, and yield (Kamemoto, 1988b). Most of the
cultivars were susceptible to blight, but in varying degrees. The introduction of A.
antioquense in crosses with A. andreanum resulted in blight tolerant offspring. Resistant
anthuriums such as the species A. antioquense become infected with the pathogen, but
rarely develop systemic infection (Kamemoto and Kuehnle, 1989).
Genetic Engineering for Resistance to Anthurium Blight
Although some anthuriums are tolerant to Xad, natural genetic resistance to
bacterial blight is not present in anthuriums (Kamemoto and Kuehnle, 1996). Breeding
plants for tolerance to Xad through traditional means is time consuming. Genetic
engineering serves as a means ofintroducing resistance genes from non-plant origins into
anthurium plants.
Agrobacterium-mediated gene transfer has been used to successfully transform
anthuriums (Kuehnle et aI., 1991). Genes that code the antibacterial peptides attacin and
cecropin have been isolated from the cecropia moth (Hya/ophora cecropia) and
genetically engineered into anthuriums (Kuehnle et aI., 1992; 1993; 2003). Transgenic
anthurium plants expressing attacin were less susceptible to Xad, and had fewer bacteria
present when compared to non-transgenic plants (Kamemoto and Kuehnle, 1996).
Kuehnle et. ai, (2003) reported two results for cultivars transformed to express the Shiva-
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1 lytic peptide (a synthetic analog of cecropin B). The cultivar 'Paradise Pink' had
increased tolerance, while the cultivar 'Tropic Flame' had increased susceptibility.
Tissue Cultured Anthuriums
The best means for blight-free production requires the use of plant material that is
guaranteed to be pathogen free. Tissue cultured plants, although highly regarded and
recommended to growers, have the potential for latent infection with Xad (Norman and
Alvarez, 1993; 1994a). Triple indexing is a system which insures that tissue cultured
plants do not serve as sources of inoculum. (Tanabe et aI., 1992).
Biological Control of Anthurium Blight
Biological control is the use of beneficial organisms, natural or modified, to
control the effects of undesirable organisms (Cook, 1988). Cultures of microorganisms
isolated from intemal petiole tissues of anthuriums were examined as a means for
biological control of bacterial blight (Fernandez et aI., 1989). Foliar applications of
microorganisms antagonistic to Xad resulted in inconsistent or insignificant control of the
disease (Fernandez et aI., 1990; 1991).
In later studies, Fukui et aI. (1999a) isolated bacteria from the guttation fluids of
susceptible anthurium cultivars (Marian Seefurth and URI 060) that did not succumb to
infection by Xad. Individually, these beneficial bacteria (BCAs) were not effective in the
suppression ofXad in guttation fluids, but were effective in combination (Fukui et aI,
1999a; 1999b). Foliar applications of the bacterial community were effective in
preventing infection of anthurium leaves by Xad, as well as preventing pathogenic
invasion through wounds (Fukui et aI., 1999b). The beneficial bacteria were later
identified as Sphingomonas ch/oropheno/ica, Microbacterium testaceum,
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Brevimundimonas vesicularis, and Herbaspirilum rubrisubalbicans (Alvarez and
Mizumoto, 2001; 2002).
Fujii (2002) and Fujii et aI., (2002) demonstrated that biological control could be
used simultaneously with genetic modification of anthurium cultivars. The cultivars
'Paradise Pink' and 'Mauna Kea' were engineered to express the Shiva-l lytic peptide,
and did not inhibit growth of the four species of beneficial bacteria.
Biostimulation was observed as an unexpected but beneficial outcome in studies
involving anthurium treatment with BCAs (Alvarez and Mizumoto, 2001). Stage 4
tissue culture plants treated with BCAs had better root systems and greater survival rates
than non-treated plants. Treated plants were more vigorous, flowered sooner, and were
larger in plant height, leaf area, leaf number, shoot and root dry weights.
Feedback inhibition of amino acid patbways
Amino acids have been tested for inhibition of various organisms. Glycine,
cysteine, and serine were tested for control of bacteria that cause foodborne illnesses
(Castellani, 1953; Castellani et aI., 1955). Inhibition of plant growth by lysine, arginine,
tyrosine, proline, threonine, methionine, leucine, and valine, was demonstrated by Miflin
(1969). Sands and Zucker (1976) reported the use of amino acids for control ofseveraI
phytopathogenic pseudo monads. Studies on amino acid biosynthetic pathways led to the
elucidation of specific enzymes that were targets of feedback inhibition. Acetohydroxy
acid synthase (AHAS), the first enzyme in common to valine and isoleucine synthesis,
was the target of valine inhibition in Ecoli (Umbarger and Brown, 1958). Threonine
dehydratase, an enzyme further down in the pathway from AHAS in isoleucine synthesis
of E. coli was inhibited by isoleucine (Umbarger and Brown, 1957).
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LITERATURE CITED
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• K.M. Delate and E.R. Yoshimura, eds. Hawaii Inst. Trop. Agric. Human Res., University of Hawaii, Honolulu.
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Alvarez, A, Lipp, R., Norman, D. 1988. Detection and serological studies. Pages 11-15 in: Proc. Anthurium Blight Conf., 1st. A.M. Alvarez, ed. Hawaii Inst. Trop. Agric. Human Res., University of Hawaii, Honolulu.
Alvarez, A, McElhaney, R., Fuku~ R. 1993. Studies of the infection process in anthurium blight using a bioluminescent strain of Xanthomonas campestris pv. dieffenbachiae. Pages 31-37 in Proc. Hawaii Anthurium Ind. Conf. 6th
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Chase, AR. 1988. Chemical and nutritional aspects ofcontrollingXanthomonas diseases on Florida ornamentals. Pages 32-34 in: Proc. Anthurium Blight Conf., 1st. AM. Alvarez, ed. Hawaii Inst. Trop. Agric. Human Res., University of Hawaii, Honolulu.
Chase, AR. 1989. Effect offertilizer rate on growth of Anthurium andreanum and susceptibility to Xanthomonas campestris pv. dieffenbachiae. Pages 248-49 in: Proc. Anthurium Blight Cont:, 2nd. JA Fernandez, W.T. Nishijima, eds. Hawaii Inst. Trap. Agric. Human Res., University of Hawaii, Honolulu.
Cook, J.R. 1988. Biological control: some concepts, and the potential for application to bacterial blight of anthurium. Pages 35-36 in: Proc. Anthurium Blight Cont:, 1st. AM. Alvarez, ed. Hawaii Inst. Trap. Agric. Human Res., University of Hawaii, Honolulu.
Deardoff, D.C. 1991. Plant bacterial diseases and their control. Pages 66-69 in: Proc. Anthurium Blight Conf., 4th. AM. Alvarez, D.C. Deardorff, K.B. Wadsworth eds. Hawaii Inst. Trop. Agric. Human Res., University of Hawaii, Honolulu.
Fernandez, JA, Tanabe, M.J., Moriyasu, P., Duffy, B. 1989. Biological control. Pages 27-29 in: Prac.Anthurium Blight Conf., 2nd. JA Fernandez, W. T. Nishijima, eds. Hawaii Inst. Trop. Agric. Human Res., University of Hawaii, Honolulu.
Fernandez, JA, Tanabe, M.J., Moriyasu, P., Wolff, W.l 1990. Pages 41-43 in: Prac .. Anthurium Blight Conf., 3rd. A.M. Alvarez, ed. Hawaii Inst. Trop. Agric. Human Res., University of Hawaii, Honolulu.
Fernandez, JA, Tanabe, M.J., Wolff, W.J., Moriyasu, P. 1991. Biological control. Pages 28-30 in: Proc. Anthurium Blight Conf., 4th. AM. Alvarez, D.C. Deardorff, K.B. Wadsworth eds. Hawaii Inst. Trap. Agric. Human Res., University of Hawaii, Honolulu.
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Fujii, T.M. 2002. Evaluation of transgenic anthuriums expressing the Shiva-l gene encoding a synthetic antimicrobial peptide. M. S. Thesis. University of Hawaii at Manoa
Fujii, T.M., Alvarez, A, Fukui, R., Obsuwan, K., Kuehnle, AR. 2002. Effect of transgenic anthuriums producing the Shiva-l lytic peptide on beneficial bacteria. Phytopathology. 92:S27
Fukui, H., Alvarez, AM., Fukui, R. 1998. Differential susceptibility of anthurium cultivars to bacterial blight in foliar and systemic infection phases. Plant Dis. 82:800-806.
Fukui, R., Fukui, H., Alvarez, AM. 1999a. Comparisons of single versus multiple bacterial species on biological control of anthurium blight. Phytopathology. 89:366-373.
Fukui, R., Fukui, R., Alvarez, AM. 1999b. Suppression of bacterial blight by a community isolated from the guttation fluids of anthuriums. Appl. Environ. Microbiol. 65:1020-1028.
Fukui, R., Fukui, H., Alvarez, AM. 1999c. Effect of temperature on the incubation period and leaf colonization in bacterial blight ofanthurium. Phytopathology. 89:1007-1014.
Fukui, R., McElhaney, R., Nelson, S.C., Alvarez, AM. 1996. Relationship between symptom development and actual sites of infection in leaves ofanthurium inoculated with a bioluminescent strain of Xanthomonas compestris pv. dieffenbachiae. Appl. Environ. Microbiol. 62: 1 021-1 027.
Guo, L.Y., Ko, W.H. 1991. Effect of pesticides on root rot ofanthurium. Pages 62-65 in: Proc. Anthurium Blight Conf., 4th. AM. Alvarez, D.C. Deardorft; K.B. Wadsworth eds. Hawaii Inst. Trop. Agric. Human Res., University of Hawaii, Honolulu.
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Higaki, T., Imamura, 1.S. 1985. Volcanic black cinder as a medium for growing anthuriums. HortScience. 20:298-300.
Higaki, T., Imamura, ].S. 1988. Nutritional studies. Pages 19-20 in: Proc. Anthurium Blight Conf., 1st. AM. Alvarez, ed. Hawaii Inst. Trop. Agric. Human Res., University of Hawaii, Honolulu.
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Higaki, T., Watson, D.P., Leonhardt, K.W. 1983. Anthurium culture in Hawaii. Cooperative Extension Service, College of Tropical Agriculture and Human Resources, University of Hawaii at Manoa, Honolulu. Circular 420.
Higaki, T., Watson, D.P., Leonhardt, K.W. 1994. Media. Page 10 in: Anthurium culture in Hawaii. Higaki, T., Lichty, I.S., Moniz, D., eds. Hawaii Inst. Trop. Agric. Human Res., College of Tropical Agriculture and Human Resources, University of Hawaii at Manoa, Honolulu. Research Extension Series 152.
Higaki, T., Imamura I., Tanabe, M., Nishijima, W., Hara, A, Deardon: D., Sewake, K. 1990. Nutritional and cultural effects on anthurium bacterial blight. Pages 7-11 in: Proc. Anthurium Blight Conf., 3rd. AM. Alvarez, ed. Hawaii Inst. Trop. Agric. Human Res., University of Hawaii, Honolulu.
Hiloweb. 2003. Briefhistory of the anthurium tropical flower industry on the big island of Hawaii. http://hiloweb.comlwebmanlhis.html
Hummelen, Harmen. 2000. The start of cultivation. in: Anthur info. 8(1). Anthura B. V., Bleiswijk, Netherlands.
Imamura, I.S., Higaki, T. 1989. Nutrition in anthurium culture. Pages 35-36 in: Proc. Anthurium Blight Conf., 2nd. J.A Fernandez, W. T. Nishijima, eds. Hawaii Inst. Trop. Agric. Human Res., University of Hawaii, Honolulu.
lones, B.I., Wolt: B. Jr., Mills, HA 1991. Plant Analysis Handbook. p.32. MicroMacro Publishing, Georgia,
Kamemoto, H. 1981. Anthurium breeding in Hawaii. Aroideana. 4:77-86.
Kamemoto, H. 1988a. History and development of anthuriums in Hawaii. Pages 4-5 in: Proc. Anthurium Blight Conf., 1st. AM. Alvarez, ed. Hawaii Inst. Trop. Agric. Human Res., University of Hawaii, Honolulu.
Kamemoto, H. 1988b. Breeding for resistance to bacterial blight of anthuriums. Pages 17-18 in: Proc. Anthurium Blight Conf., 1st. AM. Alvarez, ed. Hawaii Inst. Trop. Agric. Human Res., University of Hawaii, Honolulu.
Kamemoto, H., Kuehnle, A 1989. Breeding for blight resistance: a progress report. Pagel0 in: Proc. Anthurium Blight Conf., 2nd. IA Fernandez, W.T. Nishijima, eds. Hawaii Inst. Trop. Agric. Human Res., University of Hawaii, Honolulu.
Kamemoto, H., Kuehnle, A 1996. Breeding antlmriums in Hawaii. University of Hawaii Press, Honolulu.
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Kuehnle, AR., Chen, F.C., Iaynes, 1M. 1993. Status of genetically enfneered anthuriums. Pages 7-8 in: Proc. Hawaii Anthurium Ind. com. 6 . KM. Delate and E.R. Yoshimura, eds. Hawaii Inst. Trop. Agric. Human Res., University of Hawaii, Honolulu.
Kuehnle, AR., Chen, F.C., Iaynes, I.M., Norman, D., Alvarez, A 1992. Engineering blight resistant anthurium: a progress report. p.17-18. Proc. Anthurium Blight Com., 5th KM. Delate, C.H.M. TOme eds. Hawaii Inst. Trop. Agric. Human Res., University of Hawaii, Honolulu.
Kuehnle, AR., Chen, F.C., Sugii, N., Iaynes, I.M. 1991. Engineering of blight resistance in anthurium. Pages 42-43 in: Proc. Anthurium Blight Conf., 4th. AM. Alvarez, D.C. Deardorn: KB. Wadsworth eds. Hawaii Inst. Trop. Agric. Human Res., University of Hawaii, Honolulu.
Kuehnle, A, Kamemoto, H., Rauch, F., Lichty, I., Amore, T., Sugi~ N. 1996. Anthurium cultivars for container production. pp 1-4 in: Horticulture Digest #108. Hawaii Cooperative Extension Service. Last edited 8-24-2001. ht1p:Ilwww2. ctahr.hawaii. edultpss/digest/hd 1 08lhd 1 08 2.html
Kuehnle, A R., Fujii, R., Chen, F. C., Alvarez, A, Sugii, N., Fukui, R., and Aragon, S. L. 2004. Peptide biocides for engineering bacterial blight tolerance and susceptibility in cut flower anthurium. HortScience 39:1327-1331.
Lipp, R.L., Alvarez, AM., Benedict, AA, and Berestecky, I. 1992. Use of monoclonal antibodies and pathogenicity tests to characterize strains of Xanthomonas campestris pv. dieffenbachiae from aroids. Phytopathology. 82:677-682.
Miflin, B.I. 1969. The inhibitory effects ofvarlous amino acids on the growth of barley seedlings. I. Exp. Bot. 20: 810-819
Mills, H.A 1989. Cultural practices and anthurium nutrition. Pages 40-42 in: Proc. Anthurium Blight Com., 2nd. JA Fernandez, W.T. Nishijima, eds. Hawaii Inst. Trop. Agric. Human Res., University of Hawaii, Honolulu.
Mills, H.A, Scoggins, H.L. 1998. Nutritional levels for anthurium: young versus mature Leaves. J. Plant Nutr. 21: 199-203.
Neal, M.C. 1965. In Gardens of Hawaii. Bishop Museum Press, Honolulu. 924 pp.
Nishijima, W.T. 1988. Anthurim blight: an overview. Pages 6-8 in: Proc. Anthurium Blight Conf., 1st. AM. Alvarez, ed. Hawaii lnst. Trop. Agric. Human Res., University of Hawaii, Honolulu.
Nishijima, W.T., 1994. Diseases. Pages 13-18 in: Anthurium culture in Hawaii. Higaki, T., Lichty, J.S., Moniz, D., eds. Hawaii lnst. Trop. Agric. Human Res.,
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College of Tropical Agriculture and Human Resources, University of Hawaii, Honolulu. Research Extension Series 152.
Nishijima, W., Chun, M. 1991. Chemical control ofanthurium blight. Pages 21-23 in: Proc. Anthurium Blight Conf., 4th. A.M. Alvarez, D.C. Deardorft; KB. Wadsworth eds. Hawaii Inst. Trop. Agric. Human Res., University of Hawaii, Honolulu.
Nishijima, W.T., Fujiyama, D.K. 1985. Bacterial blight ofanthurium. Hawaii Cooperative Extension Service. Hawaii Inst. Trop. Agric. Human Res., University of Hawaii Manoa, Honolulu. Commodity Fact Sheet AN-4 (A) Flower.
Norman, D., and Alvarez, A. 1989. A rapid method for presumptive identification of Xanthomonas campestris pv. dieffenbachiae and other Xanthomonads. Plant Dis. 73:654-658.
Norman, D.1., and Alvarez, A.M. 1994a. Latent infections of in vitro anthurium caused by Xanthomonas campestris pv. dieffenbachiae. Plant Cell Tissue Organ Cult. 39:55-61.
Norman, D.l., and Alvarez, A.M. 1994b. Rapid detection ofXanthomonascampestris pv. dieffenbachiae in anthurium plants with a mini plate enrichment I EUSA system. Plant Dis. 78:954-958.
Norman, D.l., Yuen, I.M.F. 1999. First report of Ralstonia (pseudomonas) solanaceannn infecting pot anthurium production in Florida. Plant Dis. 83 (3): 300.
Norman, D., Alvarez, A., Lipp, R. 1993. Latent infections ofXanthomonas campestris pv. dieffenbachiae in tissue-cultured anthurium. Pages 12-16 in: in: Proc. Hawaii Anthurium Ind. Conf. 61ll. KM. Delate and E.R. Yoshimura, eds. Hawaii Inst. Trop. Agric. Human Res., University ofHawai~ Honolulu.
Sakai D.S. 1990. The effect of nitrate and ammonium fertilizer on the contents of anthurium guttation fluid. Pages 18-19 in: Proc. Anthurium Blight Conf., 3rd. A.M. Alvarez, ed. Hawaii Inst. Trop. Agric. Human Res., University of Hawaii, Honolulu.
Sakai D.S. 1991. The effect of nitrogen fertilizer levels on amino compounds in guttation fluid of anthurium and incidence of bacterial blight. Pages 51-52 in Proc. Anthurium Blight Conf., 41ll. A.M. Alvarez, D.C. Deardorft; KB. Wadsworth eds. Hawaii Inst. Trop. Agric. Human Res., University of Hawaii, Honolulu.
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Sakai, W.S., Okimura, S., Hanohano, T., Furutani, C., Sakai, D.S. 1992. A detailed study of nitrogen fertilization, glutamine production, and systemic blight on anthurium cultivars Ellison Onizuka and Calypso. Pages 47-48 in: Proc. Anthurium Blight Conf., 5th.. K.M. Delate, C.H.M .. Tome eds. Hawaii Inst. Trop. Agric. Human Res., University of Hawaii, Honolulu.
Sands, D.C., and Zucker, M., 1976. Amino acid inhibition of pseudo monads and its reversal by biosynthetically related amino acids. Physio!. Plant. Patho!. 9:523-524.
Shehata, S. 1992. Supply-demand and market analysis of the cut-flower industry: a focus on the Hawaiian anthurium industry. Pages 35-38 in: Proc. Anthurium Blight Conf., 5th
• KM. Delate, C.H.M .. Tome eds. Hawaii Inst. Trop. Agric. Human Res., University of Hawaii, Honolulu.
Tanabe, M., Fernandez, J., Moriyasu, P., Crane, S., Wolff; W., Liu, R-W. 1992. Anthurium In Vitro triple indexing. Pages 8-11 in: Proc. Anthurium Blight Conf., 5thKM. Delate, C.H.M .. Tome eds. Hawaii Inst. Trop. Agric. Human Res., University of Hawaii, Honolulu.
Umbarger, H.E., and Brown, B. 1957. Threonine deamination in Escherichia coli II: Evidence for two I-threonine deaminases 1. J. Bacterio!. 73:105-112.
Umbarger, H.E., and Brown, B. 1958. Isoleucine and valine metabolism in Escherichia coli. VIII. The formation of acetol act ate. J. BioI. Chern. 233:1156-1160.
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van Spingelen, 1. 1998. EC in anthurium. in: Anthur info. 6(1). Anthura B.V., Bleiswijk, Netherlands.
van Uffelen, A 1996. Creative Flower A"ang;ng with Anthurium. Kosmos-Z&K Uitgevers B. V., Utrecht
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Chapter 2
Use of Selected Amino Acids to Enhance Effectiveness of Beneficial Bacteria in Biocontrol of Anthurium Blight
ABSTRACT
Beneficial bacteria (Sphingomonas chlorophenolica, Microbacterium testaceum,
Brevundimonas vesicularis, and Herbaspirillum robrisubalbicans) survive in low
populations on anthurium leaves and can be used as biological control agents (BCAs) for
anthurium blight caused by Xanthomonas axonopodis pv. dieffenbachiae (Xad). Studies
were undertaken to improve effectiveness ofBCA treatments by addition of selected
carbon sources to inoculum. Metabolic profiles were evaluated for all strains using
Microlog TM and liquid culture. Among all the carbon sources evaluated, valine and
isoleucine were selected for further studies because each inhibited growth ofXad on a
glucose standard mineral base (SMB) medium when applied individually. When both
amino acids were present, Xad resumed growth. Glutamine and glutamic acid also
reversed inhibition by valine or isoleucine, but log phase growth was delayed for 60
hours. When valine or isoleucine were added to BCA inoculum separately and sprayed
onto anthurlum leaves, disease severity, measured by Xad colonization ofleaftissue was
reduced by 21% to 39% and 26% to 30% respectively, compared to the untreated control.
When applied in the field, disease incidence on anthurium plants treated with valine
combined with BCAs was reduced by 20% to 32% compared to the untreated control,
and 14% to 21% compared to the BCA treatment without amino acids. Use of selected
amino acids to inhibit pathogen growth is a novel and inexpensive approach for
augmenting a biocontrol strategy.
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INTRODUCTION
Anthurium (Anthurium andreanum) is the leading cut flower commodity in the
Hawaiian floriculture industry with an annual value of$ 5.5 million (Hawaii Agriculture
Statistics Service, 2006). Anthurium blight caused by Xanthomonas axonopodis pv.
dieffenbachiae (Xad; syn: X campestris pv. dieffenbachiae) is a devastating disease that
has caused serious losses in anthurium production in Hawaii as well as in other regions of
the world. The disease was reported on Kauai in 1971 (Hayward, 1972), but an outbreak
did not occur in the major production area on the island of Hawaii until the early 1980s
(Nishijima, 1988). Since then the disease has been reported in California (Cooksey,
1985), the Caribbean (Rott and Prior, 1987), the Netherlands (Sathyanarayana et aI.,
1997), Jamaica (Young, 1990), Tahiti (Mu, 1990), the Philippines (Natural, 1990),
Florida (Hoogasian, 1990), Reunion Island (Soustrade et aI., 2000), and Turkey (Aysan
and Sahln, 2003).
Xad enters the hydathodes of anthurium plants causing water soaking at leaf
margins, followed by yellowing and necrosis of plant tissue. The bacteria rapidly invade
the vascular tissue causing systemic infection which kills the plant. Use of a
bioluminescent strain ofXad has allowed accurate visualization of foliar and systemic
infection through light emission from diseased plants (Fukui et aI., 1996).
An integrated approach to disease management includes use ofaxenically
propagated anthurium, planting into pathogen-free cinder, modification of nutritional and
cultural practices, and strict sanitation. Anthurium plants have been bred for blight
resistance, and the bioluminescent strain ofXad was used to evaluate the susceptibility of
various anthurium cultivars to bacterial blight (Fukui et aI .• 1996; 1998). Commercial
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anthurium cultivars also have been genetically modified for enhanced disease resistance
with cercopins (Kuehnle et aI., 2004). Nonetheless, susceptible varieties are still
predominant in anthurium production because of desirable floral characteristics.
Beneficial bacteria indigenous to anthurium have been isolated and investigated
as biological control agents (BCAs). The BCAs significantly reduced infection by Xad
when applied to leaves prior to inoculation and showed promising results for disease
suppression (Fukui et aI., 1999a; 1999b). When used in conjunction with genetically
engineered resistance, the BCAs were still effective, indicating that the combined control
measures are compatible (Fujii et aI., 2002). Nevertheless, methods for increasing their
survival on leaves, hence efficacy, have not been fully explored.
Fukui et aI., (1999a) suggested that competition for organic nutrients was the
underlying mechanism for the inhibition ofXad by the BCAs. If so, then the
effectiveness ofBCA treatments could be enhanced by adding selected carbon sources to
the BCA solutions used for treatment. Carbon sources metabolized only by the BCAs
and not Xad would be most effective. Alternatively, growth ofXad might be reduced by
amino acid products ofbiosynthetic pathways through a mechanism offeedback
inhibition or gene repression.
Amino acids in low concentrations inhibited growth of several microorganisms
including Escherichia coli (Leavitt and Umbarger, 1962), Pseudomonas aeruginosa
(Varga and Horvath, 1966), Bacillus polymyxa (paulus and Gray, 1967; Kuramitsu,
1970), and Rhodospirillwn tenue (Robert-Gero et aI., 1972). Vurro et aI. (2006)
demonstrated that amino acids applied exogenously inhibited growth of a parasitic weed.
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The use of naturally occurring compounds to control pests and diseases is desirable from
an environmental standpoint and thus became the focus of further research.
The objective of this study was to determine whether the effectiveness of the
BCAs could be enhanced by an exogenous supply of organic nutrients. We searched for
carbon sources that selectively favored growth of the BCAs but not Xad, and we explored
the use of amino acids that inhibited growth of the pathogen but not the BCAs.
MATERIALS AND METHODS
Bacterial strains and inoculum preparation. The bioluminescent strain
VI08LRUHl of Xanthomonas axonopodis pv. dieffenbachiae (Xad-Lux) produced by
Rosemary McElhaney through transposon mutagenesis was used to quantifY colonization
of plant tissues in greenhouse experiments and was used for in vitro experiments. This
strain has been stable for more than 15 years and has been used to visualize the infection
process, evaluate resistance of anthurium cultivars, and assess the effect of beneficial
bacteria used for biological control (Fukui et a1., 1999a; 1999b; 1999c). Strain 0150 of
Xad is the original strain without the transposon for bioluminescence, and was used as
inoculum for field experiments. The beneficial strains Sphingomonas chlorophenolica,
Microbacterium testaceum, Brevundimonas vesicu!aris, and Herbaspirillum
rubrisubalbicans (referred to as GUT 3,4, 5, and 6 respectively) were isolated from
guttation fluids of anthurium leaves (Fukui et a1., 1999a). Strains were stored at -80°C
and revived by streaking onto yeast extract dextrose calcium carbonate (YDC) medium.
After 48 hours, inoculum was prepared by suspending the cells in a standard mineral base
(SMB), which contained per liter: 50 ml ofNazHP04 + KHZP04 buffer (1M; pH 6.8); 20
-23 -
ml ofHutner's vitamin-free mineral base (Cohen-Bazire, Sistrom and Stanier, 1957); and
1 g of~)2S04 (Stanier et al., 1966).
Bacterial growth on various carbon sources. Compounds that potentially serve
as sole carbon sources for microbial growth were screened using the Microlog™
metabolic profiling system (Biolog, Inc., Hayward, CAl. Separate microplates were
inoculated with individual bacteria to obtain a profile of carbon sources oxidized by each
strain. Carbon sources that were exclusively used by the beneficial bacteria and not the
pathogen were selected from the carbon utilization profile of each strain and examined
further in liquid culture. Additional amino acids from the aromatic, pyruvate, aspartate,
and glutamate biosynthetic pathways not represented on Microlog plates, were also tested
in liquid culture.
Growth in liquid culture was evaluated in lOX 100 mm test tubes each containing
5 mI total ofSMB and the specific carbon source. Mono- and disaccharides, polyols, and
organic acids were added at a final concentration of2 mglml. Amino acids were added at
a final concentration of 1 mglml except for tyrosine which was added at a final
concentration of O. OS mglml. Each tube was inoculated with an individual strain at 108
colony forming units (CFU)/m1 (OD6OO = 0.1) and incubated at 28°C to 30°C on a shaker
at 200 rpm. Maximum growth was measured at the stationary phase (7 to 10 days) with a
Klett Summerson photoelectric colorimeter (Klett Mfg. Co., Inc., N.Y., USA).
Bacterial growth on potentially inhibitory amino acids was initially determined in
liquid culture using glucose (10 mglml) in 5MB to determine baseline growth. The effect
of adding amino acids at 1 mglml was determined by measuring turbidity with the Klett
Summerson colorimeter, and comparing to the turbidity of the positive control (SMB +
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glucose). Growth of each strain was expressed as a percentage of turbidity produced by
the positive contro\: 0 = no growth; + = 8 - 19%; ++ = 20 - 30%; +++ = 31 - 69"/0; ++++
= 70 -100%.
Growth rates were determined in liquid culture in individual Klett flasks
containing 5MB, glucose (lOmg I ml), and selected amino acids (Img ImI) in a total
volume of30 mi. Flasks were inoculated with 108 CFU/ml (OD6QO = 0.1) and incubated
at 28°C to 30°C on a rotary shaker at 200 rpm. Growth was evaluated approximately
every hour during the lag phase and every half hour during the log phase. The turbidity
of the suspension was recorded as Klett units and was compared to viable cell counts
determined by plating aliquots of appropriate dilutions onto a modified TZC medium
(Table 2-1) (Norman and Alvarez, 1989).
Greenhouse studies. Two Anthurium andreanum cultivars, 'Tropic Mist' (UH
780) and 'Ozaki' were used in this experiment. Anthurium plants (height, 30-50cm)
were potted in cinder and fertilized with NutricoteR (13-13-13 plus microelements;
Chisso Asahi Co., Tokyo, Japan) at a rate of 1.33 g per pot. All plants were placed in a
glasshouse shaded with two layers of saran (30% light transmission) and watered daily.
The effects of 0.1% valine and 0.1% isoleucine on the ability ofXad-Lux to infect
anthurium leaves following spray inoculation were examined in the first greenhouse
experiment. Additional nutrients consisted of 1 % glucose and 0.02% yeast extract. The
experiment consisted of the following ten treatments (4 plants per treatment, two leaves
per plant): BCAs + valine; BCAs + valine + nutrients; BCAs + isoleucine; BCAs +
isoleucine + nutrients; BCAs alone; valine alone; valine + nutrients; isoleucine alone;
isoleucine + nutrients; and a control not treated with BCAs, amino acids, or nutrients.
- 25-
The effect of 0.1% valine and 0.1% isoleucine on inhibition of infection by Xad
Lux was examined in the second greenhouse experiment. No additional nutrients were
added to the solutions in this experiment. The experiment consisted of the following four
treatments (4 plants per treatment, two leaves per plant): BCAs + valine; BCAs +
isoleucine; BCAs alone; and a control not treated with BCAs or amino acids.
The effect of valine at 1.0% or 0.1% on the ability ofXad-Lux to infect anthurium
leaves was examined in the third experiment. The experiment consisted of the following
6 treatments (4 plants per treatment, two leaves per plant): BCAs + valine (1.0%); BCAs
+ valine (0.1 %); BCAs alone; valine alone (1.0%); valine alone (0.1 %); and a control not
treated with BCA or amino acids.
For all greenhouse experiments Gut 3, Gut 4, Gut 5, and Gut 6 were grown on
YDC and cells of each strain were suspended in sterile 5MB and adjusted to 109 CFU/mI.
Equal volumes of each bacterial suspension were mixed and the appropriate amount of
amino acid was added to make the respective solutions. Plants were placed inside clean
plastic bags and the leaves were sprayed uniformly with the respective solutions until
runoff occurred. The bags were closed and the plants were allowed to incubate overnight
at room temperature (22 ± 1°C). The following day, the leaves of each plant were spray
inoculated with a cell suspension ofXad-Lux (1.0 x 107 CFU/ml) and allowed to incubate
overnight in the bags at room temperature (22 ± 1°C). The next day, plants were
removed from the bags and placed in the glasshouse in a randomized complete block
design (RCBO) with four blocks (ten treatments per block, four treatments per block, and
six treatments per block for the first, second, and third greenhouse experiments,
respectively).
-26 -
Severity of leaf infection was detennined by autophotography in which X-ray
film was used to record light emission ofXad-Lux in infected leaves. The percentage of
leaf area infected was used as severity indices as described previously (Fukui et al.,
1996). Assessment of leaf infection was performed four times in each experiment: 21 to
23, 34 to 37, 48 to 51, and 63 to 72 days after inoculation. For both experiments. the two
youngest fully opened leaves of each plant (8 observations for each treatment) were used
for assessment ofleafinfection. Missing data for leaves that fell off was imputed using
linear regression on measurements from the previous assessment dates.
To determine disease severity, the percentage ofleaftissue colonized by Xad was
independently assessed by three examiners and the average scores were transformed by
the arcsine square root transformation and analyzed by analysis of variance. Assessment
day was considered the rep~ed measure, and the means were separated by the Fisher's
least significant difference test.
Field Studies. 'Ozaki' plants were potted in cinder and peat moss and fertilized
with NutricoteR (13-13-13 plus microelements; Chisso Asahi Co., Tokyo, Japan) at a rate
of2 oz (57 g) per pot. All plants were placed in a shade house with 20% light
transmission. Experiments were conducted in Keaau, Hawaii.
The effect of 0.1% valine and 0.1% isoleucine on the ability ofXad strain D150
to infect anthurium leaves was examined in the first field experiment. The experiment
consisted of the following four treatments (1 replication per treatment and 6 plants per
replication): BCAs + valine; BCAs + isoleucine; BCAs alone; and a control not treated
with BCAs or amino acids. Plants were arranged in a completely randomized design
(CRD).
-27 -
The effect of 0.1% valine with the addition of glucose at 1.0% on the ability of
Xad strain DI50 to infect anthurium leaves was examined in the second field experiment.
The experiment consisted of the following 4 treatments (4 replications per treatment and
6 plants per replication): BCAs + valine (0.1%) + glucose (1.0%); BCAs + valine (0.1%);
BCA alone; and a control not treated with BCAs, glucose, or amino acids. Plants were
arranged in a randomized complete block design (RCBD) with four blocks (four
treatments per block).
For both field experiments, The BCAs and solutions were prepared as for the
greenhouse experiments. All leaves of each plant were sprayed uniformly with the
respective solutions until runoff occurred. Two hours later, all plants were spray
inoculated with a cell suspension ofXad (1.0 x 107 CFU/ml). Leaves were inspected
weekly for evidence offoliar infection up to eleven weeks after inoculation. Leaves
showing water-soaking or marginal necrosis and chlorosis were removed weekly. Disease
incidence (number of infected leaves I total leaves) was cumulatively recorded and
averaged. New leaves that emerged during the experiments were added into the total
leaves. The standard error was calculated for each data point.
RESULTS
Bacterial growth on various carbon sources. As a group, the beneficial
bacteria oxidized 23 carbon sources that were not oxidized by Xad (Table 2-2). The three
most promising carbon sources L-arabinose, L-asparagine, and J3-hydroxybutyric were
oxidized by all the beneficial strains but not by Xad. However, when tested in liquid
culture these compounds did not support growth of Gut 4 and 5. Thus, we shifted from
- 28-
screening carbon sources oxidized on Biolog plates to screening amino acids in liquid
culture.
Growth of bacterial strains on amino acids as sole carbon sources is shown in
Table 2-3. Only two of the four beneficial strains used amino acids as sole carbon
sources. Gut 3 grew on alanine and asparagine while Gut 6 grew on eight amino acids
synthesized by four different pathways. Alanine was the only amino acid that sustained
growth ofXad-Lux as a sole carbon source. No single amino acid was suitable for
growth of all the beneficial bacteria but not Xad. Gut 4 and 5 did not show significant
growth on any of the carbon sources tested unless yeast extract at 0.01% was provided as
a growth factor. Therefore, these two strains were not tested further. Subsequent
experiments conducted with glucose as the principle carbon source and amino acids as
inhibitors ofXad growth showed more promise for biological control.
Growth of bacterial strains on 5MB glucose and products of amino acid synthetic
pathways is shown in Table 2-4. Valine, isoleucine, and phenylalanine strongly inhibited
Xad whereas none of the other amino acids tested showed complete suppression of
growth. Valine also inhibited growth of Gut 3, but Gut 6 was not inhibited by any of the
amino acids tested. Valine and isoleucine were selected for further experiments because
water solubility was greater than phenylalanine.
The inhibitory effects of valine and isoleucine were reduced when combined in
equal amounts with non-inhibitory amino acids leucine, glutamine, and glutamic acid
(Table 2-5). When added to the g1ucose-SMB medium, valine inhibited growth of Gut 3
and Xad, and isoleucine inhibited Xad confrrming previous results. The addition of
leucine to the 5MB-glucose-valine combination improved growth ofXad and Gut 3, but
- 29-
did not restore growth to their original levels on 5MB glucose (Table 2-5). On the other
hand, glutamine and glutamic acid reversed inhibition of Gut 3 and Xad by valine.
Leucine, glutamine, and glutamic acid individually permitted growth ofXad in the
presence of isoleucine, but growth was not as heavy as the glucose-SMB control. Valine
combined with isoleucine also reversed inhibitory effects observed when either amino
acid was applied individually.
Gut 6 grew significantly faster than Xad-Lux, reaching its peak in 24 hours,
whereas Xad required approximately 60 hours to enter log phase and then grew at a
slower rate (Fig. 2-lA). Addition of valine to 5MB+glucose doubled growth of Gut 6,
but inhibited growth ofXad.
Valine and isoleucine inhibited growth ofXad-Lux on glucose-SMB when used
separately, confirming previous tube studies (Fig. 2-lB). When valine and isoleucine
were both present Xad-Lux grew on glucose-SMB although log phase growth was
delayed by 20 hours. Nevertheless, growth ofXad was delayed compared to the
5MB+glucose control (Fig. 2-lB).
Glutamic acid enhanced the growth ofXad-Lux compared to the 5MB+glucose
control (Fig. 2-IC). Valine again inhibited growth ofXad-Lux when used alone, but
when glutamic acid was included with valine, Xad-Lux overcame valine inhibition after
approximately 100 h. Glutamine also enhanced growth ofXad on glucose-SMB and
allowed Xad to grow in the presence of isoleucine (Fig. 2-lD). The ability of glutamine
to reverse inhibition by valine was similar to that of glutamic acid. Similar growth
patterns were observed when isoleucine was used instead of valine (Figs. 2-lE and 2-lF).
- 30-
Greenhouse studies. The effects ofbiocontrol treatments on anthurium 'Ozaki'
were first observed three weeks after inoculation as measured by the percentage ofleaf
tissue colonized by Xad (Fig 2-2). The treatments with valine or valine-glucose-yeast
extract increased colonization ofXad when these carbon sources were added alone, but
when added together with the BCAs, the amount of tissue colonized was 12-25% lower
than the control (Xad-SMB) at week 9. Although the BCA-isoleucine treatment appeared
to show less tissue colonization than the BCA treatment alone, differences were not
significant at the 5% confidence level at week 9.
The area ofleaftissue colonized was 30% lower in plants treated with BCAs as
compared to the control plants for all assessment dates (Fig. 2-3). Although plants treated
with BCAs + valine had the lowest disease severity, the difference was not significant than
the other BCA treatments. Nevertheless, the trend warranted further experiments on
additional plants.
Treatments with BCAs and 0.1 % valine resulted in about 30% reduction in the area
ofleaf tissue colonized by Xad on anthurium cultivar 'Tropic Mist' (UH 780) when results
were compared 9 weeks after inoculation (Fig. 2-4). The treatment with valine at 1.1)"/0 did
not significantly improve the BCA treatment, so the 0.1% valine-BCA treatment was
evaluated further in the field.
Field studies. Plants treated with BCAs + 0.1% isoleucine or valine had 30-35%
lower disease incidence than control plants (Fig. 2-5). Significant differences between
treatments and controls were observed 35 -78 days after inoculation with Xad, but the
valine treatment did not differ from the isoleucine treatment.
- 31-
In the second field experiment, plants treated with BCAs + 0.1 % valine showed the
lowest disease incidence, averaging 20% less disease than the control plants on evaluations
between 35 and 78 days after inoculation (Fig. 2-6). Addition of glucose to the BCA
valine treatment did not improve control of Xad infection.
DISCUSSION
Current studies confirmed the effectiveness of the BCAs in reducing disease
severity of anthurium plants inoculated with Xad as previously shown by Fukui et al.
(1999a). In their work, suppression of pathogen growth was attributed to competition for
organic nutrients. Other studies by Fukui et al. (1999b) demonstrated that all four species
of the biocontrol mixture were essential for maximum disease suppression, and that Gut 6
was a key strain for effectively suppressing wound invasion and subsequent leaf infection
by Xad. In current in vitro experiments, Gut 6 grew rapidly entering the log phase 35
hours before Xad started log phase growth. Vowell (2008) showed that populations of
Gut 6 dropped from 109 CFU/ml to approximately 106 CFU/mI one week after spraying
onto anthurium microplants and from 109 CFU/ml to approximately 103 CFU/ml one
week after spraying onto macroplants (Vowell, 2008).
The effort to prolong the survival of the BCAs on the phylloplane by adding
selected carbon sources was not entirely successful, because two of the beneficial species
(Guts 4 and 5) required growth factors to metabolize the principal carbon source, and
addition of 0.0 1 % yeast extract to provide growth factors also increased pathogen growth.
In liquid assays, Gut 3 and 6 were more versatile than Gut 4 and 5, and were stimulated
by selected carbon sources that did not support growth ofXad. However, no single
carbon source enhanced the growth of all four BCAs while not also promoting growth of
- 32-
the pathogen. In contrast, addition of valine to g1ucose-SMB not only enhanced growth
of Gut 6, but also suppressed growth ofXad in culture. Therefore, it was expected that
applications ofvaIine to leaf surfaces would enhance growth of Gut 6 as well as reduce
the ability ofXad to infect anthurium through hydathodes, allowing for a promising
approach to biocontrol.
Feedback inhibition of amino acid biosynthetic pathways has been well described
for E. coli and other bacteria (Castellani, 1953; Castellani et aI., 1955; Leavitt and
Umbarger, 1962; Varga and Horvath, 1966; Paulus and Gray, 1967; Kuramitsu, 1970;
Robert-Gero et aI., 1972), but little information is available on the biosynthetic pathways
of plant pathogens. Among the few, Sands and Zucker (1976) reported amino acid
inhibition of pseudomonads and reversal of inhibition by biosynthetically related amino
acids. In my study, valine and isoleucine were inhibitory to Xad when used individually
in vitro, but inhibition was alleviated when both amino acids were present
simultaneously. Leucine, glutamine, and glutamic acid also alleviated inhibition by
either valine or isoleucine. Similarly, Kajikawa et aI., (2007) observed the mitigation of
isoleucine inhibition on mixed ruminal microbes in the presence ofleucine and valine.
Glutamine and glutamic acid are the major compounds of nitrogen-transport in
plants, and were previously associated with increased susceptibility of anthurium plants
to bacterial blight (Sakai, 1991; Sakai et aI., 1992). Presence of glutamine and glutamic
acid in guttation fluid (Sakai et aI., 1992) may explain the observation that valine or
isoleucine treatments (without BCAs) were not effective in reducing infection by Xad. In
contrast, when BCAs were added to amino acid sprays (valine or isoleucine at 0.1 %),
growth of Gut 6 and other beneficial bacteria may have been stimulated, increasing their
- 33-
ability to compete with Xad in the guttation fluid, resulting in lower infection levels
observed in both greenhouse and field experiments.
Valine in combination with the BCAs improved the efficacy of biological control
treatments, reducing disease by 20% to 39%, which was comparable to disease control
reported for biological control of bacterial spot on tomato, bacterial blight of rice, and
Phytophthora blight of pepper (Flaherty et aI., 2000; Gnanamanickam and Immanuel,
2006; Kim et aI., 2008). The use of amino acids as inhibitors of a bacterial pathogen is a
novel approach to enhancing biological control of anthurium blight.
- 34-
Table 2-1. Relationship between viable ceIl counts and turbidity (measured in Klett units) for bacterial strains used in growth curve studies
Klett Units 50 100 150 200 250 300 350 400
Viable ceIl count (log CFU/ml) Z
Gut 6 Xad
8.7 8.9 9.1 9.0 9.3 9.5 9.4 9.6 9.7 9.6 9.8 9.6 10.1 9.7 10.1 NO
Z Strain designation: Gut 6 = Herbaspirillwn rubrisubalbicans; Xad = Xanthomonas axonopodis pv. dieffenbachiae
- 35-
Table 2-2. Oxidation and growth of beneficial bacteria on carbon sources not utilized by Xanthomonas axonopodis pv. dieffinbachiae.
Carbon Sourcew
L-arabinose
L-asparagine ~-hydroxybutyric acid
D-galacturonic acid glycyl-L-aspartic acid glucuronamide a-ketovaleric acid propionic acid L-rhamnose ~-methyl-D-glucoside
thymidine a-cyclodextrin a-hydroxybutyric acid L-leucine formic acid quinic acid xylitol D-mannitol d-sorbitol
Oxidation of carbon compounds x
ABCDY
ABCD ABCD
ABD AbD aBD ACD ACD AB aB aB AC AD AD aD aD aD BD BD
D-gluconic acid B D p-hydroxyphenylacetic acid B D phenylethylamine B d putrecine B d
Growth in liquid culture AD ad Z
D
A
A
d
D D
WOther carbon sources were oxidized by only one beneficial bacterial strain: a-Dlactose and hydroxy-L-proline were oxidized only by Gut 3; m-inositol was oxidized only by Gut 4; r-aminobutyric acid, r-hydroxybutyric acid, 2-aminoethanol adonitol, D-arabitol, citric acid, D-galactonic acid lactone, D-glucosaminic acid, D-glucuronic acid, i-erythritol, L-phenylalanine, L-pyroglutamic acid, D-serine, and D-saccharic acid were oxidized only by Gut 6.
x Oxidation of carbon compounds evaluated on microplates for gram-negative bacteria (Microlog™ system, Biolog, Inc., Hayward, CA)
Y Strain designation: A = Gut 3 (Sphingomonas chlorophenolica), B = Gut 4 (Microbacterium testaceum), C = Gut 5 (Brevundimonas vesicularis), D = Gut 6 (Herbaspirillum rubrisubalbicans)
• Lower case letters indicate weak oxidation or borderline growth.
- 36-
Table 2-3. Growth of beneficial bacteria and Xanthomonas axonopodis pv. dieffenbachiae on amino acids su~~lied as sole carbon sources in a standard mineraI base.
Amino acid Pathway Strain Y
Gut 3 Gut 4 GutS Gut 6 Xad
valine Pyruvate OZ 0 0 + 0 alanine + 0 0 + + leucine 0 0 0 + 0
isoleucine Aspartate 0 0 0 + 0 lysine 0 0 0 0 0 methionine 0 0 0 0 0 asparagine + 0 0 + 0
glutamine Glutamate 0 0 0 + 0 glutamic acid 0 0 0 + 0
phenylalanine Aromatic 0 0 0 + 0 tryptophan 0 0 0 0 0
tyrosine 0 0 0 0 0 Y Strain designation: Gut 3 (Sphingomonas chlorophenolico), Gut 4 (Microbacterium
testaceum), Gut S (Brevundimonas vesicularis), Gut 6 (Herbaspirillum rubrisubalbicans), Xad (Xanthomonas axonopodis pv. dieffenbachiae)
Z 0 = no growth; + = growth
- 37-
Table 2-4. Growth of beneficial bacteria and Xanthomonas axonopodis pv. die.ffenbachiae on a standard mineral base plus glucose (10 mglml) supplemented with amino acids (1 mgfml) or other products of amino acid synthetic ~athwa~s {AASP}. Amino acid or Pathway Strain Y AASP product
Gut 3 Gut 6 Xad alanine Pyruvate ++++z ++++ ++++ pantothenate +++ ++++ ++ valine 0 ++++ 0 leucine ++++ ++++ ++++
lysine Aspartate ++++ ++++ ++++ isoleucine ++++ ++++ 0
glutamine Glutamate ++++ ++++ ++++ glutamic acid ++++ ++++ ++++
phenylalanine Aromatic ++++ ++++ 0 tyrosine ++++ ++++ +++ tryptophan ++++ ++++ ++
glucose (control) ++++ ++++ ++++ Y Strain designation: Gut 3 (Sphingomonas chlorophenolica), Gut 6
(Herbaspirillum rubrisuba/bicans), Xad (Xanthomonas axonopodis pv. die.ffenbachiae)
Z 0 = No growth; ++ = 20 to 30"10 of the glucose control; +++ = 31 to 69%; ++++ = 70 to 100%
- 38-
Table 2-5. Growth ofbeneficiaI bacteria and Xanthomonas axonopodis pv. dieffenbachiae on a standard mineraI base plus glucose (10 mglml) and combinations of selected amino acids (1 mg/m\).
Amino acids Strain Y
Gut 3 Gut 6 Xad valine O· ++++ 0 valine + leucine + ++++ ++ valine + glutamine +++ ++++ +++ valine + glutamic acid +++ ++++ +++
valine + isoleucine +++ ++++ +++
isoleucine ++++ ++++ 0 isoleucine + leucine ++++ ++++ +++ isoleucine + glutamine ++++ ++++ +++ isoleucine + glutamic acid ++++ ++++ +++
glucose (control) ++++ ++++ ++++ Y Strain designation: Gut 3 (Sphingomonas chlorophenolica), Gut 6
(Herbaspirillum rubrisubalbicans), Xad (Xanthomonas axonopodis pv. dieffenbachiae)
• 0 = No growth; + = turbidity was 8% of the glucose control; ++ = 24%; +++ = 31 to 69%; ++++ = 70 to 100%
- 39-
A 360
xadlSMBlGIc
o 20 40 60 so 100 120 141) 180 180 2DO 221) 241)
Tbno (hours)
300 c
20Q
'00
'" ISMB!GIcIVaVGIu . -I-to_
o 2D 4tI 6l) 80 100 120 140 180 180 20D 22D 240
Tbne (HourS)
E
o 20 40 60 eo tOO 120 140 160 180 20D 220 240
Tbno (Hours)
8
Xsdl 5MBI GIcIIIe! Val
200
'00
.. • .J--II....,_
300
2" 200
'00
..
o 20 40 GO 80 too 120 140 160 180 ZID 220 240
Tbno(hours)
o
XsdISMBlGJc
D~"._~~~L. ______ _ o 20 40 60 80 100 120 140 160 180 20D 22D 240
TJma (hours)
F XsdISMB/GIcIGIn
o 2D 4D 60 80 tao 120 140 160 1SO 2«1 220 240
Tbna (Hours)
Fig. 2-1 Growth of Gut 6 (Herbasprillum rubrisubalbicans) and Xanthomonas axonopodis pv. dieffenbachiae (Xad) in standard mineral base (SMB)containing 1% glucose (Glc), and various combinations of valine (Val), isoleucine (He), glutamic acid (Glu), and glutamine (Gin). The final concentration of each amino acid was 0.1%. The following combinations produced no growth: A) 0, Xad! 5MB/ GlcI Val; B) A, XadI 5MB/ GlcI lie; 0, XadI 5MB/ GlcI Val; C) 0, Xadl 5MB/ GlcI Val; D) 0, Xadl 5MB/ Glcl Val; E) A, Xadl 5MB/ Glcl Ile; F) ll, Xadl 5MB/ GlcI IIe. No growth occurred on the negative controls Gut6/ 5MB and XadlSMB.
- 40-
7D eYal
;60 AAAAA A • Val, GIe, YE
BB BB BBBB BlU" :!50 Gila. 0», YE m A AAAA -40 11 BBBBBBBBB DCorboI
j3Q BlBOA, YaI AAAAA
.5 BB BB B BB Ia BOA, Val, GIc, YE ';20 A AAAA C CCCCCCC mBOA, IJa .,. B B BB B
10 C C CCCCC III BOA, IJa, GIc, YE
0 BlIICA
Week 3 Week 5 Week 7 Week 9
Week 01 evaluation
Fig. 2-2 Effects of 0.1% valine and 0.1% isoleucine treatments on the severity offoliar infection ofanthurium 'Ozaki' by Xad-Lux under greenhouse conditions. Bars within the same week of evaluation with the same letters are not significantly different according to the least significant difference test at the 0.05 level of confidence. Control plants were sprayed with standard mineral base. All plants were sprayed with Xad-Lux at 1.0 x 10' CFU/ml.
50
40 m -os j 30
I 20
'0 ll'I
10 A B B B
0 Week 3
A B B B
A B B B
Week 5 Week 7
Week 01 evaluation
A B B B
Week 9
o Control mBCA, Val lID BCA, De
.BCA
Fig. 2-3 Effects of 0.1% valine and 0.1% isoleucine treatments on the severity offoliar infection of anthurium 'Ozaki' by Xad-Lux under greenhouse conditions. Bars within the same week of evaluation with the same letters are not significantly different according to the least significant difference test at the 0.05 level of confidence. Control plants were sprayed with standard mineral base. All plants were sprayed with Xad-Lux at 1.0 x 10' CFU/ml. This figure is the courtesy ofTomie Vowell, who assisted with the greenhouse studies (Vowell, 2008).
- 41-
50
A A A A A
A A A A A B B B El 1.0% Val
1110.1% Val
o Corrtrol
A A A A B B B B
CC C
B B B iii BCA, 1.0% Val
Ii!IBCA, O.l%VaI
.BCA A AA AAA
Week 3 WeekS Week 7 Week 10
Week of evaluation
Fig. 2-4 Effects of valine treatments on severity offoliar infection ofanthurium 'UH780' by Xad-Lux under greenhouse conditions. Bars within the same week of evaluation with the same letters are not significantly different according to the least significant difference test at the 0.05 level of confidence. Control plants were sprayed with standard mineral base. All plants were sprayed with Xad-Lux at 1.0 x 107 CPU/mi.
90
... 80 .. --S--BCA -+-BCA, Ie
><70 -.-. BCA, Val
180 c-
___ Conlrol
j ~ 80
,5"40
:.t i 30
120 -c 0::. 10
0 0 10 20 30 40 50 60 70 80
Days after Inoculation
Fig. 2-5 Effects of 0.1% valine and 0.1% isoleucine treatments on the progression of foliar infection of anthurium 'Ozaki' by Xad under field conditions. Control plants were sprayed with standard mineral base. All plants were sprayed with Xad-Lux at 1.0 x 107
CPU/ml.
- 42-
90
o
-a-BCA ..... BCA. Gic. Val -.-BCA. Val --Control
10 20 30 40 50 60 70 80
Days after inoculation
Fig. 2-6 Effects of 0.1 % valine treatments on the progression of foliar infection of anthurium 'Ozaki' by Xad under field conditions. Control plants were sprayed with standard mineral base. All plants were sprayed with Xad at 1.0 x 107 CFU/mI.
- 43-
so
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Kuehnle, A R., Fujii, R., Chen, F. C., Alvarez, A, Sugii, N., Fuku~ R., and Aragon, S. L. 2004. Peptide biocides for engineering bacterial blight tolerance and susceptibility in cut flower anthurium. HortScience 39: 1327-1331.
Kuramitsu, H.K. 1970. Concerted feedback inhibition of aspartokinase from Bacillus stearothermophilus. 1. Catalytic and regulatory properties. J BioI Chern. 245:2991-2997.
Leavitt, R.I. and Umbarger, liE. 1962. Isoleucine and valine metabolism in Escherichia coli. XI. Valine inhibition of the growth of Escherichia coli strain K-12. J. Bacteriol. 83 :624-30.
Mu, L. 1990. Anthurium culture and blight in Tahiti. Page 37 in: Proc Anthurium Blight Com., 3rd. A M. Alvarez, ed. Hawaii Inst. Trop. Agric. Human Res., University ofHawai~ Honolulu.
Nishijima, W.T. 1988. Anthurim blight: an overview. Pages 6-8 in: Proc. Anthurium Blight Com., 1st. AM. Alvarez, ed. Hawaii Inst. Trop. Agric. Human Res., University of Hawaii Manoa, Honolulu.
Natural, M. P. 1990. Anthurium blight in the Philippines. Page 38 in: Proc Anthurium Blight Com., 3rd. A M. Alvarez, ed. Hawaii Inst. Trop. Agric. Human Res., University of Hawaii, Honolulu.
-45 -
Nonnan, D. & Alvarez, A. 1989. A rapid method for presumptive identification of Xanthomonas campestris pv. dieflenbachiae and other xanthomonads. Plant Dis. 73:654-658.
Paulus, H., Gray, E. 1967. Multivalent feedback inhibition of aspartokinase in Bacillus polymyxa. 1. Kinetic studies. J. BioI. Chern. 242:4980-4986.
Robert-Gero, M., Le Borgne, L., Cohen, G.N. 1972. Concerted feedback inhibition of the aspartokinase of Rhodospirillum tenue by threonine and methionine: a novel pattern. 1. Bacteriol. 112:251-258.
Rott, P., and Prior, P. 1987. Un deperissement bacterien de I'anthurium provoque par Xanthomonas campestris pv. dieflenbachiae aux Antilles fran~aises. Agron. Trop. 42:61-68.
Saka~ D. S. 1991. The effect of nitrogen fertilizer levels on amino compounds in guttation fluid of anthurium and incidence of bacterial blight. Pages 51-52 in: Proc. Anthurium Blight Conf., 4th. A. M. Alvarez, D. C. Deardorfl; and K. B. Wadsworth, eds. Hawaii Inst. Trop. Agric. Human Res., University of Hawaii, Honolulu.
S~ W. S., Okimura, S., Hanohano, T., Fu~ S. C., S~ D. S. 1992. A detailed study of nitrogen fertilization, glutamine production, and systemic blight on anthurium cultivars Ellison Onizuka and Calypso. Pages 47-48 in: Proc. Anthurium Blight Conf., 5th. K. M. Delate and C. H. M. Tome eds. Hawaii Inst. Trop. Agric. Human Res., University of Hawaii, Honolulu.
Sands, D.C., and Zucker, M., 1976. Amino acid inhibition of pseudo monads and its reversal by biosynthetically related amino acids. Physiol. Plant. Pathol. 9:523-524.
Sathyanarayana, N., Reddy, O. R, Latha, S., and Rajak, R L. 1998. Interception of Xanthomonas campestris pv. dieflenbachiae on Anthurium plants from the Netherlands. Plant Dis. 82:262.
Soustrade, 1, Gagnevin, L., Roumagnac, P., Gambin, 0., Guillaumin, D. , and Jeuffrault, E. 2000. First report of anthurium blight caused by Xanthomonas axonopodis pv. dieflenbachiae in Reunion Island. Plant Dis. 84: 1343.
Stanier, R.Y., Palleroni, N.J., and Doudoroff, M. 1966. The aerobic Pseudomonas: a taxonomic study. J. Gen. Microbiol. 43:159-271.
Varga, J.M., Horvath, I. 1966. Growth inhibition of Pseudomonas aeroginosa by valine. J. Bacteriol. 92(5):1569.
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Vowell, T. 2008. Reducing bacterial blight ofanthurium caused by Xanthomonas axonopodis pv. dieffenbachiae by improving survival of beneficial bacteria used for biological control. M.S. Thesis. University of Hawaii, Honolulu, Ill.
Vurro, M., Boari, A, Pilgeram, A L., and Sands, D. C. 2006. Exogenous amino acids inhibit seed germination and tubercle formation by Orobanche ramosa (Broomrape): Potential application for management of parasitic weeds. BioI. Control 36:258-265.
Young, F.Y. 1990. Anthurium blight in Jamaica. Page 37 in: Proc. Anthurium Blight Conference., 3rd. A M. Alvarez, ed. Hawaii Inst. Trop. Agric. Human Res. University of Hawaii, Honolulu.
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Chapter 3
Optimizing Growth of Anthurium Microplants with Mineral Nutrients and Beneficial Bacteria
ABSTRACT
Plantlets derived from tissue culture are vulnerable upon deflasking and transplanting
into community pots. Establishment of vigorous microplants is desirable for healthy
productive plants. Studies were undertaken to optimize growth of microplants with
inorganic nutrients and anthurium biological control agents (BCAs). Applications of
NutricoteR at 1.33 g per pot stimulated growth without desiccation of microplants. Plants
treated with BCAs and 1.33 g ofNutricoteR grew better than plants that received only
BCAs or only NutricoteR• A standard mineral base (SMB) provided the best growth for
the BCAs when compared to growth on v.. strength MiracIe-GeoR or v.. strength modified
Hoagland solution. The 5MB also provided better growth stimulation of anthurium
plants when compared to Miracle-GeoR or modified Hoagland solution. Biostimulation
by the BCAs was observed on all varieties of anthurium in this study.
INTRODUCTION
Anthurium plants are vital to the floriculture industry of Hawaii with cut flowers
valued at $ 5.5 million (Hawaii Agriculture Statistics Service, 2006). The use of tissue
cultured plants to replenish stocks and increase production acreage is important for
grower operations. Optimal growth of microplants transplanted from tissue culture
allows for better establishment of healthier plants.
Free living bacteria that colonize a plant and enhance its growth are referred to as
plant growth promoting bacteria (pGPB). Some PGPBs are associated with the roots of
- 48-
plants (Han et aI., 2005), while some are found on the phyIIosphere (Bashan and Bashan,
2002). Some PGPBs are capable of occupying both niches (Cabailero-MeIIado et aI.,
2004). Beneficial bacteria used as biological control agents (BCAs) were also found to
stimulate root and shoot growth ofanthurium plants (Alvarez and Mizumoto, 2001). The
biostimulation was observed on two cuItivars of Anthurium ClTIlireanum and one cultivar
of syngonium.
The objective ofthis study was to optimize growth ofanthurium microplants with
inorganic nutrients in conjunction with BCAs, and to determine ifbiostimulation by the
BCAs was widespread among cultivars.
MATERIALS AND METHODS
Plant materials and growth conditions. Four cu1tivars were obtained from Dr.
Adelheid Kuehnle in the Department of Tropical Plant and Soil Sciences, University of
Hawaii at Manoa: UH780 ('Tropic Mist'), UH965 ('Rudolph'), UHl3ll, UHl469, and
UH1651. Anthurium plants were deflasked and transplanted into medium that consisted
of two parts redwood soil conditioner (Kellogg Garden Products, Carson, CA) to one part
grade 2 perlite in community pots (15 cm azalea pots, 20 plants per pot). Community
pots were placed into plastic boxes with a thin layer of water in the bottom of the box to
maintain high humidity, and placed under fluorescent lights. Pots were propped on top of
Petri plates to prevent contact with the water on the bottom of the box, and the medium
was moistened every other day. Average temperature was 27.9°C.
Bacterial strains and inoculum preparation. Bioluminescent strain
VI08LRUHl of Xanthomonas axonopodis pv. dieffenbachiae (Xad-Lux) and the
biological control agents (BCAs) Sphingomonas chlorophenolica, Microbacterium
- 49-
testaceum, Brevundimonas vesicularis, and Herbaspirillum rubrisubalbicans (referred to
as GUT 3, 4, 5, and 6 respectively) were used in this study. Strains were stored at -80°C
and revived by streaking onto yeast extract dextrose calcium carbonate (YDC) medium.
After 48 hours, inoculum was prepared by suspending the cells in a phosphate buffer
(O.OIM, pH 6.9).
Effects of fertilizer treatments and beneficial bacteria on antburium
microplants. Microplants ofanthurium varieties UH780, UH 965, and UH 1469 were
transplanted as above. In the first and second experiment, the effect ofNutricoteR (13-
13-13 plus microelements in a 70 day release formulation; Chisso Asahi Co., Ltd.,
Tokyo, Japan) and Miracle-GroR (Scotts Miracle-Gro products, Inc., Maysville. OR.
USA) on growth of anthurium microplants was examined. The treatments consisted of
NutricoteR (17 g per pot), NutricoteR (17 g per pot) with a daily spray application of~
strength Miracle-GroR, and a control (sprayed with phosphate buffer). Percentage of
plant death by desiccation was recorded.
The second experiment was conducted only on UH780 with a reduction in the
amount ofNutricoteR from 17 g to 1.33 g per pot and a reduction in the frequency of~
strength Miracle-GroR foliar application to once a week instead of daily. The effect of
weekly applications ofBCAs (applied at 109 colony forming units (CFU)/mI) combined
with fertilizer treatments on anthurium plants were also examined in this experiment.
The treatments consisted ofBCA with NutricoteR, BCAs with NutricoteR and ~ strength
Miracle-GroR, BCAs with no fertilizer, NutricoteR
, NutricoteR with ~ strength Miracle
GroR and a control (sprayed with phosphate buffer). Microplants that received BCA
treatments were soaked in a BCA solution 30 minutes before transplant into community
- 50-
pots and plants that did not receive BCAs were soaked in phosphate buffer. Percentage of
plant death by desiccation was recorded. Growth parameters such as plant height, canopy
width, leaf length, and leaf width were collected approximately 15 weeks after transplant
into community pots. Leaflength and width measurements were taken on the three
youngest mature leaves per plant. The experiment consisted of20 plants per treatment.
Each plant was considered a replicate. Parameter means of the different BCA treatments
combined with fertilizer were compared to their corresponding treatments without the
BCAs using the Student's t-test (p = 0.05).
The third experiment was conducted to examine the effect ofBCAs on UR780,
UR1311, UR1469, and UR1651. All community pots (20 plants per pot) were fertilized
with NutricoteR at 1.33 g per pot. Microplants that received BCA treatments were soaked
in a BCA solution 30 minutes before transplant into community pots and plants that did
not receive BCAs were soaked in phosphate buffer. Subsequently, treated plants received
weekly foliar applications ofBCAs at 109 CFU/ml, while control plants received weekly
foliar applications of phosphate buffer. Growth measurements such as plant height,
canopy width, leaf length, and leaf width were collected approximately IS weeks after
transplant into community pots. Leaf length and width measurements were taken on the
three youngest mature leaves per plant. The experiment consisted of 60 BCA treated
plants and 60 control plants of each anthurium variety. Each plant was considered a
replicate. Means ofBCA treated and control plants of each variety of anthurium were
compared using the Student's t-test (p = 0.05).
Bacterial growth on various mineral solutions. Three nutrient solutions were
used in this study: A standard mineral base (SMB), which contained per liter: 50 ml of
- 51-
Na2HP04 + KH2P04 buffer (1M; pH 6.8); 20 ml ofHutner's vitamin-free mineral base
(Cohen-Bazire, Sistrom and Stanier, 1957); and 1 g of<NHIhS04 (Stanier et al., 1966),
Miracle-GroR, and a modified Hoagland solution (Taiz and Zeiger, 2002). The Miracle
GroR and modified Hoagland solutions were diluted to 'A strength to reflect a reduced
fertilizer concentration recommended for anthurium micropiants. 5MB is a mineral base
used for bacterial growth and not for plant growth and was not diluted.
Bacterial growth in liquid culture was evaluated in 10 X 100 mm test tubes each
containing 5 mI total of 5MB, 'A strength Miracle-GroR, or 'A strength modified Hoagland
solution and glucose added at a final concentration of2 mglml. Each tube was inoculated
with an individual strain at 108 CFU/mi (00600 = 0.1) and incubated at 28 to 30° C on a
shaker at 200 rpm. Growth was measured at 5 days with a Klett Summerson
photoelectric colorimeter (Klett Mfg. Co., Inc., N.Y., USA).
Effects of mineral solutions on anthurium microplants. The effects of 5MB
(17,090 !1M. 49,660 !1M. and 16,660 }.lM N, P, K respectively), Miracle-GroR, and a
modified Hoagland solution (16,000 !1M. 2,000 IJM, and 6,000 }.lM N, P, K respectively)
were examined on microplants of anthurium cultivar UH780. All mineral solutions were
diluted to 'A strength and sprayed weekly onto microplants. Plant height, canopy width,
leaf length, and leaf width, were collected approximately 15 weeks after transplant into
community pots. Each treatment consisted often plants, and each plant was considered a
replicate. Data was analyzed by analysis of variance and means were separated by the
Fisher's least significant difference test.
- 52-
RESULTS
Effects offertilizer treatments and beneficial bacteria on anthurium
microplants. Applications ofNutricoteR at 17g per pot resulted in death of anthurium
plants from the three cultivars in this study (Table 3-1). At 30 and 50 days after
transplant, UH1469 had fewer desiccated plants compared to the other cultivars, but at 80
days, percent of desiccated plants was close to that ofUH780. UH965 had the greatest
percentage of desiccated plants when treated with NutricoteR, but also had the highest
number of dead control plants. Applications ofNutricoteR at 17 g per pot and daily
applications of '.4 strength Miracle-GroRwere most detrimental to survival of all cultivars
in this study (Table 3-1). UH1469 had the least amount of desiccated plants at 30 and 50
days after transplant. UH780 and UH965 had up to 80% plant death after 30 days and a
5-10% increase in plant death by 50 days after transplant. All cultivars had almost total
plant desiccation by 80 days after transplant. Reduction in the amount ofNutricoteR to
1.33 g per pot and reduction in frequency of '.4 strength Miracle-GroR sprays to once a
week considerably increased plant survival of cultivar UH 780 (Table 3-2).
Treatments with BeAs significantly enhanced the growth of cultivar UH780
(Table 3-3). Moreover, the combination of beneficial bacteria and inorganic fertilizer
further enhanced plant growth. Beneficial bacteria in combination with NutricoteR
produced the greatest growth stimulation.
BCA treatments stimulated growth ofUH131l, UH1651, UH1469, and UH780
(Fig. 3-1). All cultivars showed increased growth over the untreated control in the
parameters of plant height, width of plant canopy, leaf length, and leaf width. Overa1~
UHI651 was most growth-stimulated, followed by UH1311, UH780, and UH1469.
- 53-
Bacterial growth on various mineral solutions. Growth of beneficial bacteria
and Xad were tested on nutrient solutions designed for plants, and compared to 5MB
which was designed for bacterial growth (Fig. 3-2). None of the strains grew in the
mineral solutions alone. Beneficial strains Gut 3 and 6 grew better in 5MB + glucose
when compared to either 14 strength modified Hoagland solution + glucose or 14 strength
Miracle-GroR + glucose. Gut 4 and 5 are fastidious strains and would not grow without
the addition of a growth factor.
Effects of mineral solutions on anthurium microplants. 5MB was best suited
for growth of beneficial strains, and was tested for effects on growth ofUH 780 (Fig. 3-
3). Plant height, width of plant canopy, leaflength, and leaf width of plants treated with
14 strength 5MB were significantly greater than control plants and plants treated with 14
strength modified Hogland solution.
DISCUSSION
Beneficial bacteria have been isolated and used as a consortium of biological
control agents (BeAs) for the reduction of disease caused by Xanthomonas axonopodis pv.
dieifenbachiae (Fukui et. aI., 1998; 1999a; 1999b). Biostimulation was an unexpected
effect observed on plants treated with BeAs. Alvarez and Mizumoto (2001) reported
stimulation of shoot and root development of tissue cultured anthurium and syngonium
cultivars treated with BeAs after removal from sterile conditions. This study further
confirmed the stimulatory properties of the BeAs by the increased growth observed on four
additional cultivars of Anthurium andreanum. The mechanism of how this biostimulation
operates is not known.
- 54-
Anthurium microplants were susceptible upon defiasking. Stimulating the plant
growth after transplant was critical to healthy plant development. In this study, the
inorganic nutrients as well as the BCAs stimulated growth of the microplants. The initial
rates ofNutricoteR and Miracle-GroR that was tested caused desiccation of all the
anthurium varieties. The reduction in the amount of NutricoteR to 1.33 g and Miracle
GroR foliar applications to once a week was more conducive to plant establishment and
survival. The use ofBCAs in conjunction with the inorganic fertilizers was synergistic,
and enhanced growth of plants more than if either was used separately.
Mirac1e-GroR and a modified Hoagland solution designed for plant nutrition were
compared to a standard mineral base (SMB) designed for bacterial growth. The purpose
was to substitute Miracle-GroR or modified Hoagland solution for the bacterial culture
solution and to apply to plants if either formulation supported growth of beneficial bacteria
as well as 5MB. All solutions were tested at 'A strength on plants to represent the common
application in plant nutritional studies and provide a weak solution that would not desiccate
sensitive microplants. The 5MB supported th~ best growth of the beneficial bacteria.
When sprayed onto microplants, 'A strength 5MB provided better growth for the anthurium
microplants when compared to 'A strength modified Hoagland solution. This was probably
due to the higher levels of phosphorous and potassium in 5MB attributable to the
sodium/potassium phosphate buffer.
The use of anthurium beneficial bacteria and different sources of plant nutrition
were examined for optimal growth of anthurium microplants. Biostimulation by the BCAs
was observed on all anthurium varieties tested. An initial soak of microplants in a BCA
solution before transplant, the application of 1.33g ofNutricoteR per 15 cm community pot,
- SS-
and weekly applications ofBCAs in 5MB are recommended for optimal growth of
microplants.
- S6-
Table 3-1. Effect offertilizer treatments on growth of three anthurium cultivars. Days after % Desiccated plants (death) for each
Treatment" transplant variety UH UH UH UH UH 780 780 965 1469 1469
Control 30 0 0 30 0 0 Nutricote 50 35 65 5 15 Nutricote + Miracle Gro 80 80 80 40 5
Control 50 0 0 30 0 0 Nutricote 65 65 80 40 25 Nutricote + Miracle Gro 90 90 85 75 50
Control 80 0 0 30 0 0 Nutricote 65 65 90 70 55 Nutricote + Miracle Gro 100 95 95 95 75
'Control plants were sprayed daily with phosphate buffer (0.01M, pH 6.9), Nutricote K was applied at 17 g per pot, and V4 strength Miracle-GroR was sprayed daily.
Table 3-2. Survival ofUH 780 treated with reduced nutrient applications 30, 50, and 80 days after transplant.
Control Nutricote
Treatment"
Nutricote + Miracle Gro
Control Nutricote Nutricote + Miracle Gro
Control Nutricote Nutricote + Miracle Gro
Days after transplant
30
50
80
% Survival of 00 780
100 100 100
100 100 100
100 100 100
'Control plants were sprayed weekly with phosphate buffer (0.01M, pH 6.9) , Nutricote R was applied at 1.33 g per pot, and V4 strength Miracle-GroR was sprayed weekly.
- 57-
Table 3-3. Mean parameter measurements for UH 780 plants treated with biological control agents (BCAs) and inorganic fertilizer.
Mean Parameter Measurements" Plant Leaf Leaf
Height Canopy Length Width Leaf TreatmentY {cm) Width {cm} {cm} {em} number
BCA 4.4* 4.9* 1.6* 1.1 6.5 Control 3.8 4.0 1.4 1.1 6.1
BCA+Nutircote 8.4* 9.2* 3.6* 2.1* 8.1 Nutricote 5.8 6.2 2.2 1.5 7.4
BCA+Nutricote+Miracle-Gro 7.9* 8.2 3.4* 2.1* 7.7* Nutricote+Miracle-Gro 7.0 7.5 2.7 1.8 6.3 'Control plants were sprayed weekly with phosphate buffer (0.01M, pH 6.9), Nutricote R
was applied at 1.33 g per pot, 'A strength Miracle-GroR was sprayed weekly, and BCAs were suspended in phosphate buffer at 109 colony forming units I m1 at sprayed weekly.
Z Parameter means of the different BCA treatments combined with fertilizer were compared to their corresponding treatments without the BCAs using the Student's t-test. Values with asterisks are significantly different from the corresponding values without BCAs (P = 0.05)
- 58-
• 1~=reatOOl A
7
56 iP :!4
i 3
2 0
UH 1311 UH 1651 UH 1469 UH7B11
11
I~~u:reatedl B i 10 _ e i!: 8
I 7 a
~ 6 .. 4 'I;
I " 2 1 0
UH 1311 UH 1651 UH 1469 UH760
4
I~=remedl • C
_3 .[
12 E
0 UH1311 UH 1651 UH 1469 UH7B11
4
I~:,~reatedl D
." • ... • 12 E
0 lni 1311 UH 1651 UH 1469 UH760
Figure 3-1. Effect of beneficial bacteria (BCAs) on growth parameters offour varieties of Anthurium andreanum. Mean parameter measurements ofBCA treated and control plants of each variety of anthurium were compared using the Student's t-test. Bars with asterisks are significantly different (p = 0.05) from the corresponding values for control plants. A Comparison of average plant height. B. Comparison of average canopy width C. Comparison of average leaf length. D. Comparison of average leaf width.
- 59-
400 mGut3 350 III Gut 4 300 It! Gut 5 CD
.3 250 IIII Gut 6 II > 200 .Xad ~ .l!! 150 lII:
100
50
0
5MB Miracle Gro Hoagland
D-Glucose No Carbon Source
Figure 3-2. Growth of anthurium beneficial bacteria and Xanthomonas axonopodis pv. dieffenbachiae in selected mineraI solutions (Standard mineral base, Miracle-Gro, and modified Hoagland solution) and D-glucose.
ABB
PIBnI LOBI 1 LeBf2
• 1148MB
EJ1/4 HoBglarul o Control
LeBf3
Figure 3-3. Comparison of plant height, canopy width, and leaf length and width of anthurium UH 780 treated with modified 14 strength Hoagland solution and 14 strength Standard Mineral Base (SMB). Bars with the same letter are not significantly different according to the Fisher's least significant difference (P = O.OS).
- 60-
UTERATURE CITED
Alvarez, A., and Mizumoto, C. 2001. Bioprotection and stimulation of aroids with phylloplane bacteria. Phytopathology. 91:S3.
Bashan, Y., and de-Bashan, L.E .. 2002. Protection of tomato seedlings against infection by Pseudomonas syringae pv. tomato by using the plant growth-promoting bacterium Azo.!pirillum brasilense. Appl. Environ. Microbiol. 68:2637-2643.
Caballero-Mellado, J., Martinez-Aguilar, L., Paredes-Valdez, G., and Estrada-de los Santos, P. 2004. Burkhokkria unamae sp. nov., an N2-fixing rhizospheric and endophytic species. Int. J. Syst. Evol. Microbiol. 54:1165-1172.
Fukui, H., Alvarez, A. M., and Fukui, R 1998. Differential susceptibility ofanthurium cultivars to bacterial blight in foliar and systemic infection phases. Plant Dis. 82:800-806.
Fukui, R, Fukui, H., and Alvarez, A. M. 1999a. Suppression of bacterial blight by a community isolated from the guttstion fluids of anthuriums. Appl. Environ. Microbiol. 65: 1020-1028.
Fukui, R, Fukui, H., and Alvarez, A. M. 1999b. Comparisons of single versus multiple bacterial species on biological control of anthurium blight. Phytopathology. 89:366-373.
Han, J., Sun, L., Dong, X., Cai, Z., Sun, X., Yang, H., Wang, Y., Song, W. 2005. Characterization of a novel plant growth-promoting bacteria strain Delftia tsurohatensis HR.4 both as a diazotroph and a potential biocontrol agent against various plant pathogens. Syst. Appl. Microbiol. 28(1):66-76.
Hawaii Agricultural Statistics Service. 2006. United States Department of Agriculture. Released 9-10-07. http://www.nass.usda.gov/hi/flower/flower.pdf
- 61 -
Appendix A
Susceptibility of Anthurium antioquense 'Cotton Candy' to Anthurium Blight and Bioprotection by Beneficial Bacteria
MATERIALS AND METHODS
Plant materials and growth conditions. Anthurium antioquense cultivar
'Cotton Candy' was transplanted form cinder into 6 in azalea pots with medium that
consisted of two parts redwood soil conditioner (Kellogg Garden Products, Carson, CA)
to one part perlite. Plants were placed in the greenhouse under saran (30% light
transmission). Average minimum and maximum temperatures were 23.3°C and 32.4°C,
respectively.
Bacterial strains and inoculum preparation. The bioluminescent strain
VI08LRUHl of Xanthomonas axonopodis pv. dieffenbachiae (Xad-Lux) and the
biological control agents (BCAs) Sphingomonas chlorophenolica, Microbacterium
testaceum, Brevundimonas vesicularis, and Herbaspirillum rubrisubalbicans were used
in this study. Strains were stored at _80°C and streaked onto yeast extract dextrose
calcium carbonate (YDC) medium. After 48 hours, inoculum was prepared by
suspending the cells in a phosphate buffer (0.01M, pH 6.9).
Plant inoculation. Each of the BCA strains were suspended in sterile phosphate
buffer and adjusted to 1 rf colony forming units (CFU)/ml. Equal volumes of each
bacterial suspension were mixed. Plants were placed inside clean plastic bags and the
leaves were sprayed uniformly with the BCA consortium until runoff occurred. Control
plants were sprayed with phosphate buffer until runoff. The bags were closed and the
plants were allowed to incubate overnight at room temperature (22:1: 1°C). The
following day, plants were spray inoculated with a cell suspension ofXad-Lux at 1.0 x
- 62-
108 CFU/ml and allowed to incubate overnight at room temperature (22 :I: 1 QC). The next
day, plants were removed from the bags and returned to the glasshouse. The experiment
consisted of eight BCA treated plants and eight non-treated controls.
Data collection. Leaves were inspected weekly for evidence offoliar infection
up to 23 weeks after inoculation. Leaves showing water-soaking were considered to be
infected by Xad. Disease incidence (number of infected leaves I tota1leaves) was
recorded and averaged. The standard error was calculated for each data point.
RESULTS
A. antioquense cultivar 'Cotton Candy' showed minor symptoms mainly at the
leaf margins but water soaking did not develop into necrotic tissue. Disease incidence
progressively increased throughout the experiment, however, BCA treated plants had at
least 30% lower disease incidence compared to the untreated control for the duration of
the experiment (Fig. A-I).
CONCLUSION
In this experiment, disease incidence (evidenced by water soaking) was 85%.
However, since the water-soaked areas did not develop into fully necrotic tissues, the
plants appeared to be resistant. This phenomenon could be examined further with X-ray
film exposed by the bioluminescent strain of the pathogen to provide a more accurate
determination of leaf infection as well as colonization ofleaftissue by Xad.
- 63-
g 100 EI SCA ... >< 90 0 Non-treated -j 80
co !! 70 to J!! j 60 ~
fl - 50
~ ~ 40 to
~ J!! 30 0
j 20 ~:: ~~ ~:
10 r; t. :::. - :~:
.;-: M ~~~ 0 ::::
:::: ~:; .:~
f:~ ~: .::
:::: ~: ~;
~~ ~~ , :' :::! ~ , ~!~ :~~
~i :::: :;::: l~ ill; .:~ :~:
1':: ;~: -::;: U
::.: i~~ l~j
:::: ~:: :j~i ~l: :* ~~ ;;:: -{
t l ~:: ;:! ::::
t g l~ ~~: ::,:
ll~ ;.;. ::~
:1 :;~: ~: I ~:;
.:%
I ~! :::: .::~ ~ l:~ "',,! :::" ::;;
52 59 75 81 89 96 103 112 121 132 139 147 153 163 Daysaftermocu~on
Fig. A-I Effects of beneficial bacterial treatments on the progression of disease incidence of Anthurtum antioquense cultivar 'Cotton Candy' by Xanthomonas axonopodis pv. dieffenbachiae.
- 64-
AppendixB
Bioprotection of Five Cultivars of Anthurium Microplants
MATERIALS AND METHODS
Plant materials and growth conditions. Microplants of anthurium cultivar
UH780 ('Tropic MisC), UH13II, UH1469, and UHI651 were transplanted from
community pots into 5.1 cm2 pots with medium that consisted of two parts redwood soil
conditioner (Kellogg Garden Products, Carson, CA) to one part perlite. Plants were
placed in the greenhouse under saran (30% light transmission). Average minimum and
maximum temperatures were 19.5°C and 31. 7°C, respectively.
Bacterial strains and inoculum preparation. Bioluminescent strain
VI08LRUHI of Xanthomonas axonopodis pv. dieffenbachiae (Xad-Lux) and the
biological control agents (BCAs) Sphingomonas chlorophenolica, Microbacterium
lestaceum, Brevundimonas vesicularis, and Herbaspirillum rubrisubalbicans were used
in this study. Strains were stored at -80°C and streaked onto yeast extract dextrose
calcium carbonate (YDC) medium. After 48 hours. inoculum was prepared by
suspending the cells in a phosphate buffer (O.OIM, pH 6.9).
Plant inoculation. Each of the BCA strains were suspended in sterile phosphate
buffer and adjusted to I rf colony forming units (CFU)/ml. Equal volumes of each
bacterial suspension were mixed. Plants were placed inside clean plastic bags and the
leaves were sprayed uniformly with the BCA consortium until runoff occurred. Control
plants were sprayed with phosphate buffer until runoff. The bags were closed and the
plants were allowed to incubate ~vernight at room temperature (22:!: 1°C). The
following day, plants were spray inoculated with a cell suspension ofXad-Lux at 1.0 x
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107 CFU/ml and allowed to incubate overnight at room temperature (22 ± 1°C). The next
day, plants were removed from the bags and returned to the glasshouse.
Data collection. The experiment was conducted twice. Incidence and severity
data were collected 4 weeks after inoculation with Xad-Lux. Leaves were destructively
removed from plants and taped onto construction paper. X-ray film was placed over the
leaves and used to record light emission ofXad-Lux in infected leaves. Incidence data
was calculated as number of infected leaves divided by total leaves X 100. Means of
BCA treated and control plants of each variety of anthurium were compared using the
Student's t-test (P = 0.05). A disease severity index (Table B-1) was used to rate the
overall severity of disease per treatment based on Xad colonization ofleaftissue as
evidenced by X-ray film. The severity index was calculated with the following formula:
RESULTS
SI = 1: (No. of leaves in each severity class x class No.) x 100 (Total No. leaves) x (Total classes-I)
Pretreatment of anthurium leaves with the BCA consortium reduced infection by
Xad-Lux for the anthurium cultivars tested (Table B-1). The more susceptible varieties
ofanthurium were UH1311, UH1469, and UH965, while UH780 and UHI65 I were less
susceptible to Xad. Reduction of disease incidence was significant for UHI311 and
UHI469 for both experiments I and IT (Fig. B-1).
CONCLUSION
Effects ofbiocontrol are most evident with the more susceptible cultivars of
anthurium.
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Table B-1. Disease severity of Anthurium andreanum microplants treated with beneficial bacteria and inoculated with Xanthomonas axonopodis pv. diq
100 A 8 8D
flllBCA o Control
~ 8D
51 70
8D
H 50
#1 40 *
I 30
20 ... 10
* 0 Uti 1311 Uti 1651 Uti 1489 Uti 7110
100 B 90 flllBCA
8 o Control ~ 80
iiI 7D
60
H 50
#) 40
I 30
20 ... 10
0 Uti 1311 UH 1651 UHI489 UH 7110
Fig. B-1. Effects of beneficial bacterial (BCA) consortium on foliar disease incidence by Xad-Lux on four cultivars of Anthurium andreanum. Mean disease incidence ofBCA treated and control plants of each variety of anthurium were compared using the Student's t-test. Bars with asterisks are significantly different (p = 0.05) from the corresponding values for control plants. A Experiment I; B. Experiment n
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AppendixC
Comparison of Anthurium Microplants Treated With Beneficial Bacteria and Two Inoculum Levels of Xanthomonas axonopotlis pv. dieffenbachiae
MATERIALS AND MEmODS
Plant materials and growth conditions. Microplants of anthurium variety
UH780 ('Tropic Mist') were transplanted from community pots into 5.1 cm2 pots with
medium that consisted of two parts redwood soil conditioner (Kellogg Garden Products,
Carson, CA) to one part perlite. Plants were placed in the greenhouse under saran (30%
light transmission).
Bacterial strains and inoculum preparation. Bioluminescent strain
VI08LRUHI of Xanthomonas axonopodis pv. dieffenbachiae (Xad-Lux) and the
biological control agents (BCAs) Sphingomonas chlorophenolica, Microbacterium
testaceum, Brevundimonas vesicularis, and Herbaspirillum rubrisubalbicans were used
in this study. Strains were stored at -80DC and streaked onto yeast extract dextrose
calcium carbonate (YDC) medium. After 48 hours, inoculum was prepared by
suspending the cells in a phosphate buffer (0.01M, pH 6.9).
Plant inoculation. Each of the BCA strains were suspended in sterile phosphate
buffer and adjusted to 1 rf colony forming units (CFU)/ml. Equal volumes of each
bacterial suspension were mixed. A total of twenty-four plants was used in this study.
Twelve plants were placed inside clean plastic bags and the leaves were sprayed
uniformly with the BCA consortium until runoff occurred. Twelve plants were sprayed
with phosphate buffer until runoff. The bags were closed and the plants were allowed to
incubate overnight at room temperature (22 ± I DC). The following day, one set of plants
(6 BCA treated and 6 buffer treated) was spray inoculated with a cell suspension ofXad-
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Lux at 1.0 x 108 CFU/ml and the second set (6 BCA treated and 6 buffer treated) was
sprayed with Xad-Lux at 1.0 x 109 CFU/ml. The bags were sealed and the plants were
allowed to incubate overnight at room temperature (22 :!: 1°C). The next day, plants were
removed from the bags and returned to the glasshouse.
Data coUection. Incidence and severity data were collected 3 weeks after
inoculation with Xad-Lux. Leaves were destructively removed from plants and taped
onto construction paper. X-ray film was placed over the leaves and used to record light
emission ofXad-Lux in infected leaves. Incidence data was reported as number of
infected leaves divided by total leaves X 100. A disease severity index (Table C-l and
C-2) was used to rate the overall severity of disease per treatment based on Xad
colonization ofleaftissue as evidenced by X-ray film. The severity index was calculated
with the following formula:
51 = ~ (No. of leaves in each severity class x class No.) x 100 (Total No. leaves) x (Total classes-I)
RESULTS
Control plants that were inoculated with Xad at 1.0 x 108 CFU/ml had 21% more
disease incidence than the BCA treated plants (Table C-1). The control plants also had a
higher severity ofinfection.
Although the disease incidence and severity was greater for plants inoculated with
Xad at 1.0 x 109 CFU/ml, the BCAs still suppressed disease as was seen with the lower
Xad inoculation (Table C-2). BCA treated plants had a disease incidence of 12.5%, in
contrast to a disease incidence of 61.5% in the untreated control. The untreated control
also had a higher disease severity than the BCA treated plants.
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Plants inoculated with Xad at 1.0 X 109 CFU/mI were more infected than plants
inoculated with Xad at 1.0 X 108 CFU/mI.
CONCLUSION
The BCAs were effective at suppressing disease incidence and severity of
anthurium plants inoculated with high levels ofXad.
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Table C-l. Disease incidence and severity of anthurium plants challenged with Xanthomonas axonopodis pv. dieJ[enbachlae at 108 CFU/ml
No. Leaves Total No. % Leaves Severity Treatment Plant # infected Leaves infected Rating
Control
BCA Treated
1 2 3 4 5 6
Total Severity
1 2 3 4 5 6
Total Severity
1 5 20 1 1 4 25 1 1 5 20 1 1 5 20 1 2 5 40 1,1 1 5 20 1 7 29 24.14
2.68"
0 3 0 0 0 7 0 0 0 5 0 0 1 6 16.67 1 0 5 0 0 0 4 0 0 1 30 3.33
0.37
" 1: <No. ofleaves in each severity class x class No.) x 100 (Total No. leaves) x (Total classes-I)
Severity Class based on percentage of leaf area colonized as evidenced by X-ray film:
o = No infection 1 = 2.5 10 10 2 = 11 to 20 3 =211030 4 =311040 5 =411050
6 =511060 7 =611070 8=711080 9 =8110 90 10=9110100
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Table C-2. Disease incidence and severity ofanthurium plants challenged with Xanthomonas axonopodis pv. dieffonbachiae at 1 r:f CFU/ml
No. Leaves Total No. % Leaves Severity Treatment Plant # infected Leaves infected Rating
Control
BCA Treated
1 2 3 4 5 6
Total Severity
1 2 3 4 5 6
Total Severity
3 3 100 1, 1, 1 3 5 60 1, 1, 1 3 5 60 1, 1, 1 2 4 50 1,2 3 4 75 2, 1, 1 2 5 40 1,1 16 26 61.54
7.69"
1 7 14.29 1 0 4 0 0 0 4 0 0 1 4 25.00 1 1 7 14.29 1 1 6 16.67 1 4 32 12.50
1.39
z 1: (No. ofleaves in each severity class x class No.) x 100 (Total No. leaves) x (Total classes-I)
Severity Class based on percentage of leaf area colonized as evidenced by X-ray film:
o = No infection 1 = 2.5 10 10 2=111020 3 =211030 4 =311040 5 =411050
6=511060 7 =611070 8 =711080 9 =811090 10=9110100
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Appendix D
Feedback lnhibition of Valine, Leucine and Isoleucine Biosynthesis
Isopropylmalate synthase
isopropylmalate
i 2-oxoisocaporate
1 leucine
Pyruvate ---+
1 Pyruvate
acetolactate
2-oxoisovelerate
1 Valine
Hydroxyethylthiam ine -pp
Acetolactate synthase
Threonine
1 2-oxobutyrate
Threonine dehydratase
acetohydroxybutyrate
j 2-oxo-3-methylveletate
1 Isoleucine ----~
Adapted from: Miflin , BJ . 1977 . Modification controls in lime and space. In (H. Smith ed.) Regulation or enzyme synthesis and activiLY in higher plants. Academic Press New York, pp 23-40
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AppendixE
Overall Conclusions
Growers have been struggling with anthurium blight since the outbreak of this
disease in the 1980s. A. andreanum cultivars are the mainstay of Hawaiian anthurium
production because of the large showy flowers used in floral arrangements. These
cultivars however, are not resistant to blight. In my studies, the A. antioquense cultivar
'Cotton Candy' was resistant to infection by Xad, but lacked the desirable floral
characteristics of A. andreanum cultivars.
The use of biological control may add value in managing disease caused by Xad.
The use ofBCAs if applied in the industry can help keep susceptible cultivars with
desirable horticultural attributes in production by protecting them and adding some level
of tolerance to blight. In these studies, the BCAs were effective at suppressing disease
incidence and severity of anthurium plants inoculated with high levels ofXad, and the
effects ofbiocontrol were most evident with the more susceptible cultivars of anthurium.
Furthermore, the addition of valine to BCA treatments increased bioprotection under both
greenhouse and field conditions.
An added benefit to the use ofBCAs is growth enhancement, which was observed
on four cultivars of anthurlum microplants. Accelerated growth may speed up the
process from tissue cultured plants to mature flowering plants.
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