Bioprotection of Potted Ornamental Aroids Against Bacterial Blight Progress Report — June 1999
Progress Report for Research Supported by the American Floral Endowment May 25, 1999
Project title:
“Changing Production Technology to Protect Ornamental Aroids from Bacterial Blight”
Institution:
University of Hawaii
AFE Grant Amount:
$20,000
Grant Period:
7/1/98 to 6/30/99
Project Completion Date (as on proposal application):
May 30, 1902
Project Leader:
Anne M. Alvarez
Title:
Plant Pathologist and Professor
Address:
Department of Plant Pathology
3190 Maile Way
University of Hawaii
Honolulu, HI 96822
Telephone: (808) 956-7764
Fax:(808) 956-2832
e-mail: alvarez@hawaii.edu
Additional researchers:
Carla Mizumoto: Research Associate
Tomie Shiraishi: Student Research Assistant
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Review of industry needs addressed and project objectives
Industry needs:
This project addresses two questions of general interest to the floriculture industry.
- Current governmental pressures to restrict pesticide use in agriculture have increased the urgency of understanding how biological factors can be adjusted and/or manipulated to protect plants against disease. In this project we seek to understand the mode of action of four selected biological control agents (BCAs), their survival on leaf surfaces and/or within plants, and their roles in preventing infection by a widely-dispersed bacterial pathogen.
- Production of pathogen-free plants in vitro stipulates that essentially no other organisms are cultured with the desired plant (i.e. axenic culture is required). However, when such plants are first exposed to the natural environment they are susceptible to colonization by numerous unidentified microorganisms. In this project we seek to establish a beneficial mixed population of bacteria on and within microplants soon after they are deflasked from axenic culture.
The hypothesis:
Establishment of beneficial endophytic bacteria will protect plants from subsequent invasion by harmful bacteria.
The model plant-pathosystem for these studies is bacterial blight of anthurium caused by Xanthomonas campestris pv. dieffenbachiae (Xcd). This pathogen causes a serious bacterial blight of ornamental and food crops in the family Araceae, including spathiphyllum, aglaonema, and xanthosoma. Plants can be protected from disease by particular mixtures of antagonistic bacteria. We have selected and identified four bacteria from different species that act as antagonists of Xcd and thereby prevent infection through openings at leaf margins (hydathodes), stomates wounds, and roots. We currently seek to establish these species in deflasked plantlets during their first three months of growth in community pots.
Future anticipated benefits:
Growers could fill an important niche by producing disease-free, tissue-cultured and bio-protected ornamental aroids for market. Based on promising results of previous experiments, BCAs should protect young plants from disease. Understanding biological using mixtures of bacterial antagonists will help establish basic principles of biological control that can be extended to control bacterial diseases in other potted ornamentals.
Project Objectives:
- To determine the optimal conditions for applying biological control agents (BCAs) to protect tissue-cultured, potted anthurium plants from bacterial blight.
- To determine the earliest possible point at which the protective population of BCAs can be introduced to plants in vitro.
- To determine the level and duration of bio-protection when potted plants are challenged with different levels of Xcd inoculum.
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Summary of work conducted since inception
Work plan:
The project is on schedule with the research timetable as outlined for the project for the first year. Formulations for enhancing growth of BCAs on plants have been evaluated and tested on plants. In vitro-grown anthurium plants (four cultivars, 100 plants of each cultivar) were shipped from Twyford, Inc. at monthly intervals. Two subsets (20 plants each) were immersed in a mixture of BCAs for thirty minutes immediately on arrival. Treated and non-treated plants were planted into community pots, grown for three months to establish roots, then transplanted into trays and placed on greenhouse benches. BCA formulations and numbers of treatments were compared in ten separate monthly trials with respect to the survival of individual BCA species in and on plants. The effect of BCA treatment on disease development following a challenge inoculation with the pathogen in the greenhouse was then analyzed using a bioluminescent reporter strain of Xcd to quantitatively measure the extent of pathogen invasion in plant tissues. Survival of BCAs in and on the tissue-cultured plants in community pots and greenhouse trays was evaluated using semiselective culture media containing selected antibiotics.
Results obtained to date:
- Shelf-life of the BCA-solution used as inoculum was examined by testing the viability of all four BCAs in water or buffer suspensions at various temperatures. BCA mixtures were stable at room temperature for 22 days in phosphate buffer (pH 6.9). Populations declined gradually after three weeks. Further experiments to evaluate BCA inoculum preparation are underway.
- BCAs survived within and on plantlets and were detectable as long as 67 days after the initial soak and spray treatment. Populations declined gradually from the initial levels (2.5 to 5.5 log units which are equivalent to 300 to 300,000 colony-forming units per plant) (Fig. 1). One component of the mixture (strain Gut6) declined more rapidly than the other three strains.
- Growth enhancement of tissue-cultured microplants was an unexpected effect of BCA-treatments during the first three months as they were being established in community pots. Roots became established more rapidly and there was a greater percentage of rooted plantlets in community pots. Plants were taller, more vigorous, and leaves generally were larger at an earlier date. Growth enhancement was observed for all cultivars, but the effect was most pronounced on the cultivar Marian Seefurth (Fig 1A and 1B).
- Bioprotection was obtained when plants were challenge inoculated with Xcd in the greenhouse following repeated sprays of BCAs as plants were established in community pots and later on the greenhouse bench (Fig 2C). Bioprotection was observed for all five cultivars (Fig 2D), but differences between treated and non-treated plants were most pronounced with Marian Seefurth (Fig 2E – 2H).
- Cultivars Marian Seefurth, Pink Frost, Tropic Fire, Cotton Candy, and Bubble Gum were compared for susceptibility to Xcd following BCA treatment. The four latter cultivars (genetic background: antioquense) are significantly more resistant to bacterial blight than Marian Seefurth (genetic background: andreanum). Foliar symptoms were not pronounced on resistant cultivars. However, once systemically infected, the plantlets collapsed and died rapidly. Pink Frost appeared to be most resistant in two trials, but resistance trials need further repetition.
Conclusions:
BCAs survive in equilibrium in buffered suspensions for at least 21 days without a significant decline in population. Relatively stable populations of BCAs are established within and on plantlets during the first month of plant growth in community pots. The BCAs protect against plant death during the first three months, resulting in better establishment of plantlets prior to transplanting to greenhouse trays. Foliage is more luxurient and plants are larger. BCAs protect plants from infection by Xcd provided that the BCA populations are high at the time of pathogen inoculation.
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Future research
Next Steps:
- Current results are promising, but experiments will be repeated to confirm the above findings under various conditions. Growth enhancement resulting from BCA-treatment of plantlets in community pots will be quantified. Measurable parameters (height, total fresh weight, root weight, and percentage of plants established) will be statistically compared.
- Experiments will be undertaken to find methods of extending the shelf-life of BCA inoculum.
- Experiments will be undertaken to compare methods of enhancing survival of BCAs within and on plants. Timing and frequency of sprays will be examined in detail.
- Cultivars will be evaluated for differential resistance to bacterial blight as well as the effectiveness of BCAs in protection from bacterial blight.
Anticipated industry benefits:
If both growth enhancement and bio-protection is repeatedly observed, these findings could have a definite impact on plant propagation and production methods used commercially in industry. Growth enhancement of plantlets in community pots was a new and unexpected finding that adds value to the bio-protection provided through treatment with the mixture of BCAs.
Recent publications:
The following publications report the results of this work as well as results from a closely related USDA-funded project on non-target pesticide effects on the effectiveness of biological control of anthurium blight.
- Fukui, R., Fukui, H., and Alvarez, A.M. 1999. Comparisons of single versus multiple bacterial species on biological control of anthurium blight. Phytopathology 89:366-373.
- Fukui, R., Fukui, H. and Alvarez, A.M. 1999. Suppression of bacterial blight by a bacterial community isolated from the guttation fluids of anthuriums. Appl. and Environ. Microbiol. 65:1020-1028.
- Alvarez, A.M., Fukui, R., Fukui, H., and McElhaney, R. 1999. Bioprotecting anthurium against bacterial blight. Grower Talks, 2-99:74-78.
Captions for Figure 2
Growth enhancement and bio-protection of anthurium by biological control agents (BCAs).
(A) Marian Seefurth plants treated with BCAs (two community pots at top) are larger and more vigorous than non-treated plants (bottom). Growth of treated plants was more uniform and a greater percentage of the microplants became established in community pots. The photograph was taken 9 weeks after planting.
(B) Growth enhancement of Marian Seefurth plants 9 weeks after removal from agar, immersion in a mixture of BCAs, and planting into community pots. Treated plants (right) are taller than non-treated plants (left).
(C) Ten-month-old anthurium plants 10 weeks after inoculation with Xanthomonas campestris pv. dieffenbachiae (Xcd). Nine plants at left were treated with BCAs. Non-treated plants were in nine pots (right). Seven plants are dead and only two plants (one leaf per plant) survived after inoculation with Xcd.
(D) Five-month-old anthurium plants in greenhouse trays. After three months growth in community pots, plants were again soaked in BCAs and transplanted into trays. Treated plants (right tray) were larger and more vigorous than non-treated plants (left tray). Empty wells in trays at left are the result of missing plants (approximately 25% of each non-treated cultivar) that failed to establish roots in community pots and were unavailable for planting into trays.
(E). Non-protected Marian Seefurth plants following inoculation with Xcd (inoculum concentration = 103 colony-forming units/ml). Four of six inoculated plants died from systemic blight 13 weeks after inoculation, and the two remaining plants showed severe foliar infection. Note necrotic margins of infected leaves.
(F) BCA-treated plants inoculated with Xcd . Two infected leaves remained 13 weeks after inoculation. One of the six plants died. Several others previously had infected leaves, but the leaves fell off and plants recovered.
(G-H) Eight BCA-treated plants from an earlier experiment (top and side-views). Three plants had leaves that showed symptoms 4 weeks after inoculation; however, the infected leaves eventually fell off and plants became symptomless. No evidence of internal infection was revealed in symptomless plants by the bioluminescence assay. In three repeated experiments 75 to 90% of BCA-treated plants recovered and produced flowers, whereas only 10% of the non-treated plants were still alive 14 weeks after inoculation. Photographs of BCA-treated plants were taken 28 weeks after inoculation.
