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. QL b. I",,J..
ii To my grandmothers, Josefina G. Toves and Rosario Villamor
iii 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.
iv 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
v 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
vi 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
vii 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 ofAnthurium 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 ofXanthomonas axonopodis pv. dieffenbachiae ...... 69
D. Feedback inhibition of valine, leucine, and isoleucine biosynthesis ...... 74
E. Overall conclusions ...... 75
viii 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 ofAnthurium andreanum microplants treated with beneficial
bacteria and inoculated with Xanthomonas axonopodis pv. dieJlenbachiae ...... 67
ix 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
x 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 ofvaline (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
xi 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
ofAnthurium 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
xii LIST OF ABBREVIATIONS
BCAs - Biological control agents
5MB - Standard mineral base
Xad - Xanthomonas axonopodis pv. dieffenbachiae
xiii Chapter 1
Literature Review
ANTHURIUM
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 ofA. 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
- 1 - 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
-2- 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
-3- 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 NIA/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
NIA/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).
-4- 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.
-5- 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
-6- 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).
-7- 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 ofthe 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).
-8- 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 ofthe 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
-9- 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 ofA. 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-
- 10- 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 ofthe 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,
-11- 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 ofbacteria 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 ofE. coli was inhibited by isoleucine (Umbarger and Brown, 1957).
- 12- LITERATURE CITED
Agrolink. 2003. Anthurium. http://agrolink.moa.my/doalbdclgenera1anthurium.html
Alvarez, AM., Mizumoto, C.Y. 2001. Bioprotection and stimulation ofaroids with phylloplane bacteria. Phytopathology. 91:S3.
Alvarez, AM., Mizumoto, C.Y. 2002. Beneficial bacteria protect microaroids from bacterial blight. Phytopathology. 92:S4. Publication no. P-2002-0021-AMA
Alvarez, A, Norman, D. 1993. Alternatives for control of anthurium blight using information gained from epidemiological studies. Pages 17-21 in: Proc. Hawaii th Anthurium Ind. Conf. 6 • K.M. Delate and E.R. Yoshimura, eds. Hawaii Inst. Trop. Agric. Human Res., University of Hawaii, Honolulu.
Alvarez, A, Lipp, R., Bushe, B. 1989. Resistance of bacteria to antibiotics used for control ofanthurium blight. Pages 11-12 in: Proc Anthurium Blight Conf., 2nd. JA Fernandez, W.T. Nishijima, eds. Hawaii Inst. Trop. Agric. Human Res., University of Hawaii, Honolulu.
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.
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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
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- 16- 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.
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- 19- 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.
- 20- 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
- 21- 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.
- 22- 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 ofXanthomonas 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 +
- 24- 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 ofthe 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 Viable ceIl count (log CFU/ml) Z Gut 6 Xad Klett Units 50 8.7 8.9 100 9.1 9.0 150 9.3 9.5 200 9.4 9.6 250 9.7 9.6 300 9.8 9.6 350 10.1 9.7 400 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. Oxidation of Growth in Carbon Sourcew carbon compounds x liquid culture L-arabinose ABCDY AD L-asparagine ABCD ad Z ~-hydroxybutyric acid ABCD D D-galacturonic acid ABD glycyl-L-aspartic acid AbD glucuronamide aBD a-ketovaleric acid ACD propionic acid ACD L-rhamnose AB A ~-methyl-D-glucoside aB thymidine aB a-cyclodextrin AC A a-hydroxybutyric acid AD L-leucine AD d formic acid aD quinic acid aD xylitol aD D-mannitol BD D d-sorbitol BD D D-gluconic acid B D p-hydroxyphenylacetic acid B D phenylethylamine B d putrecine B d WOther carbon sources were oxidized by only one beneficial bacterial strain: a-D lactose 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) YStrain 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 8 360
Xsdl 5MBI GIcIIIe! Val
200
xadlSMBlGIc '00.. • .J--II....,_ o 20 40 60 so 100 120 141) 180 180 2DO 221) 241) o 20 40 GO 80 too 120 140 160 180 ZID 220 240 Tbno (hours) Tbno(hours)
300 c 300 o 2"
20Q 200
XsdISMBlGJc
'00 '00 '" .. .-I-to_ ISMB!GIcIVaVGIu D~"._~~~L. ______o 2D 4tI 6l) 80 100 120 140 180 180 20D 22D 240 o 20 40 60 80 100 120 140 160 180 20D 22D 240 Tbne (HourS) TJma (hours) F E XsdISMB/GIcIGIn
2D 4D 60 80 tao 120 140 160 1SO 2«1 220 240 o 20 40 60 eo tOO 120 140 160 180 20D 220 240 o Tbno (Hours) 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 AAAAA A • Val, GIe, YE ;60 BB BB BBBB BlU" :!50 Gila. 0», YE m A AAAA -40 11 BBBBBBBBB DCorboI BlBOA, YaI j3Q 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 A B B B 40 m A B B B -os j 30 o Control mBCA, Val A B B B lID BCA, De I 20 .BCA '0 ll'I 10 A B B B
0 Week 3 Week 5 Week 7 Week 9 Week 01 evaluation
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 B B B El 1.0% Val 1110.1% Val o Corrtrol A A A A A B B B iii BCA, 1.0% Val A A A A Ii!IBCA, O.l%VaI B B B B .BCA A AA AAA CC C
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 -S--BCA ... 80 .. -+-BCA, Ie -><70 -.-. BCA, Val ___ Conlrol 80 c-1 j ~ 80 ,5"40 :.t i 30 120 c -0::. 10
0 0 10 20 30 40 50 60 70 80 90 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- -a-BCA ..... BCA. Gic. Val -.-BCA. Val --Control
o 10 20 30 40 50 60 70 80 so 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- LITERATURE CITED
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Castellani, A.G. 1953. Inhibiting effects of amino acids and related compounds upon the growth of enterotoxigenic micrococci in cream pastry. Appl Microbio!. 1: 195- 199.
Castellani, A. G., Makowski, R., and Bradley, W. B. 1955. The inhibiting effect of serine upon the growth of the indigenous flora of cream filling. Appl Microbio!. 3: 132- 135.
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Cooksey, DA 1985. Xanthomonas blight ofanthurium andreanum in California. Plant Dis. 69:727.
Flaherty, J.E., Jones, 1.B., Harbaugh, B.K., Somodi, G.C., and Jackson, L.E. 2000. Control of bacterial spot on tomato in the greenhouse and field with h-mutant bacteriophages. RortScience 35:882-884.
Fukui, R., Fukui, H., McElhaney, R., Nelson, S. C., and Alvarez, A M. 1996. Relationship between symptom development and actual sites of infection in leaves of anthurium inoculated with a bioluminescent strain ofXanthomonas campestrispv. dieffenbachiae. Appl. Environ. Microbiol. 62:1021-1028
Fuku~ R., Alvarez, A M., and Fukui, R. 1998. Differential susceptibility of anthurium cultivars to bacterial blight in foliar and systemic infection phases. Plant Dis. 82:800-806.
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Fuku~ 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.
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Hoogasian, C. 1990. Anthurium blight threatens state's floral economy. Florist 24: 55-57.
Kajikawa, H., Tajima, K., Mitsumori, M., and Takenaka, A 2007. Inhibitory effects of isoleucine and antagonism of the other branched-chain amino acids on fermentation parameters by mixed ruminal microbes in batch cultures and rumen simulating fermenters (Rusitec). Animal Sci. J. 78:266-274.
Kim, H.S., Sang, M.K., Jeun, Y.C., Hwang, B.K., and Kim, K.F. 2008. Sequential selection and efficacy of antagonistic rhizobacteria for controlling Phythophthora blight of pepper. Crop Prot. 27:436-443. .
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.
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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.
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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.
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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.
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-46 - 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.
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- 47- 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
R BCAs or only Nutricote • 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 ofAnthurium 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 ofXanthomonas 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~
R strength Miracle-Gro , 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.
R R The treatments consisted ofBCA with Nutricote , BCAs with Nutricote and ~ strength
R R R Miracle-Gro , BCAs with no fertilizer, Nutricote , Nutricote 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 R Miracle-Gro , 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 R containing 5 mI total of 5MB, 'A strength Miracle-Gro , 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 R (17,090 !1M. 49,660 !1M. and 16,660 }.lM N, P, K respectively), Miracle-Gro , 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 R plant desiccation by 80 days after transplant. Reduction in the amount ofNutricote to R 1.33 g per pot and reduction in frequency of '.4 strength Miracle-Gro 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 ofAnthurium 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 ofthe 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 ofbeneficial 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 ofthe 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. Days after % Survival Treatment" transplant of 00 780 Control 30 100 Nutricote 100 Nutricote + Miracle Gro 100 Control 50 100 Nutricote 100 Nutricote + Miracle Gro 100 Control 80 100 Nutricote 100 Nutricote + Miracle Gro 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- • A 7 1~=reatOOl 56 iP :!4 i 23 0 UH 1311 UH 1651 UH 1469 UH7B11 11 B i_ 10e I~~u:reatedl i!: 8 7 I a ~ 6 .. 4 'I; "2 I 1 0 UH 1311 UH 1651 UH 1469 UH760 4 C I~=remedl • _3 .[ 12 E 0 UH1311 UH 1651 UH 1469 UH7B11 4 D I~:,~reatedl ... • • ."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 CD It! Gut 5 .3 250 II IIII Gut 6 > 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 • 1148MB EJ1/4 HoBglarul o Control PIBnI LOBI 1 LeBf2 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 1rf 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: 1QC). 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 - 80 co j 70 !!to J!! 60 :::: ~:; .:~ ~ j .:: 50 f:~ ~: fl :::: ~; - ~~ ~: , :' ~ 40 , :::! ~ ~ to :~~ ~ J!! ~~ :::: 0 30 :;::: ~!~ :~: ill; .:~ ~i ::.: 1':: l~ :::: ;~: -::;: 20 ~~ ~:: U ~l: l~j ;;:: -{ ~:: i~~ ~~ :::: ~:: ~: :j~i :* ;:! ;.;. t. t l l~ ::,: ::~ 10 r; :::. t :;~: g .:% ~~: ~: j ~:; :::: .::~ .;-: ~ :::" :~: :::: M ~~~ :1 l:~ "',,! ~! ll~ ::;; - 0 I I 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 ofAnthurtum 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 ofXanthomonas 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 - 65- 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: SI = 1: (No. of leaves in each severity class x class No.) x 100 (Total No. leaves) x (Total classes-I) RESULTS 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. - 66- Table B-1. Disease severity ofAnthurium andreanum microplants treated with beneficial bacteria and inoculated with Xanthomonas axonopodis pv. diq![enbachiae Severity Index x Cultivar SCA Y Control Z Experiment I UH 1311 3.8 11.0 UH 1651 0.7 1.9 UH 1469 0.0 6.8 UH780 0.0 0.3 UH965 1.7 3.6 Experiment II UH 1311 2.0 7.3 UH 1651 0.6 0.9 UH 1469 2.0 4.9 UH780 1.2 1.9 UH 965 2.9 6.4 x l: (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 6=51to60 1 =2.5to 10 7 =61 to 70 2 = 11 to 20 8 =71to80 3 =2lt030 9 =81 to 90 4 =31t040 lO=91 to 100 5 =41to50 YTreated with biological control agents (BCAs) Sphingomonas cnlorophenolica, Microbacterium testaceum, Brevundimonas vesicularis, and Herbaspirillum rubrisubalbicans followed by inoculation with X axonopodis pv. die.ffenbachiae Z Treated with phosphate buffer (0.01M, pH 6.9) followed by inoculation with X axonopodis pv. die.ffenbachiae - 67- 100 A flllBCA 8 8D o Control ~ 8D 70 51 8D H 50 #1 40 * 30 I... 20 10 0 * Uti 1311 Uti 1651 Uti 1489 Uti 7110 100 B 90 flllBCA 8 o Control ~ 80 7D iiI 60 H 50 #) 40 30 I... 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 ofAnthurium 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 - 68- 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 1rf 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- - 69- 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. -70 - 9 Plants inoculated with Xad at 1.0 X 10 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. -71- 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 1 1 5 20 1 2 1 4 25 1 3 1 5 20 1 4 1 5 20 1 5 2 5 40 1,1 6 1 5 20 1 Total 7 29 24.14 Severity 2.68" BCA Treated 1 0 3 0 0 2 0 7 0 0 3 0 5 0 0 4 1 6 16.67 1 5 0 5 0 0 6 0 4 0 0 Total 1 30 3.33 Severity 0.37 " 1: -72 - Table C-2. Disease incidence and severity ofanthurium plants challenged with Xanthomonas axonopodis pv. dieffonbachiae at 1r:f CFU/ml No. Leaves Total No. % Leaves Severity Treatment Plant # infected Leaves infected Rating Control 1 3 3 100 1, 1, 1 2 3 5 60 1, 1, 1 3 3 5 60 1, 1, 1 4 2 4 50 1,2 5 3 4 75 2, 1, 1 6 2 5 40 1,1 Total 16 26 61.54 Severity 7.69" BCA Treated 1 1 7 14.29 1 2 0 4 0 0 3 0 4 0 0 4 1 4 25.00 1 5 1 7 14.29 1 6 1 6 16.67 1 Total 4 32 12.50 Severity 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 6=511060 1 = 2.5 10 10 7 =611070 2=111020 8 =711080 3 =211030 9 =811090 4 =311040 10=9110100 5 =411050 -73 - Appendix D Feedback lnhibition of Valine, Leucine and Isoleucine Biosynthesis Pyruvate ---+ Hydroxyethyl Threonine thiamine -pp Threonine 1 1 dehydratase Isopropylmalate Pyruvate Acetolactate 2-oxobutyrate synthase synthase acetolactate acetohydroxybutyrate isopropylmalate i j 2-oxoisocaporate 2-oxoisovelerate 2-oxo-3-methylveletate 1 1 1 leucine Valine Isoleucine ----~ Adapted from: Miflin, BJ. 1977. Modification controls in lime and space. In (H. Smith ed.) Reg ulation or enzyme synthesis and activiLY in higher plants. Academic Press New York, pp 23-40 - 74- 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 ofA. 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. -75 -