Changing Production Technology To Protect Ornamental Aroids From Bacterial Blight Progress Report - May 2000
Progress Report for Research Supported by the American Floral Endowment
May 28, 2000
Project title:
“Changing Production Technology to Protect Ornamental Aroids from Bacterial Blight”
Institution:
University of Hawaii
AFE Grant Amount:
- $20,000, 1998-1999
- $20,000, 1999-2000
Grant Period:
7/1/98 to 12/31/00
Project Completion Date:
December 31, 2001 (requested)
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 Assistant
Tomie Shiriashi, Alan Asano: Student helpers
David Warganich: Graduate student admitted to the program for Fall, 2000
I. 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 anthunum caused by Xanthomonas campestris pv. die ffenbachiae (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.
II. Summary of work conducted:
This report will summarize the work completed during the second year of the granting period (for previous results, please see progress report, May 25, 1999). During the second year, we continued work on survival of beneficial bacteria used as biological control agents (BCAs) and expanded the growth enhancement studies to other aroids (syngonium and dieffenbachia). Syngonium cultivars, White Butterfly (WE) and Lemon Lime (LL), are susceptible to bacterial blight caused by Xanthomonas campestris pv. die ffenbachiae (Xcd). Experiments were designed to evaluate the effect of protective spray/dip treatments of microplants. The leaf blot method (described previously) was used to evaluate the extent of infection.
A. Survival of beneficial bacteria:
We have completed survival studies to determine how long the BCAs remain on leaf surfaces in a quiessent or semi-dormant state. Scanning electron microscopy was performed to determine the sites of survival and the host-plant interaction. The photographs will be shown in the next report after a time-series of experiments are completed to determine the relationship between physical presence and viability of cells.
B. 1. Growth enhancement:
Growth enhancement was observed after applying BCAs as beneficial bacteria to leaf surfaces and as drench to roots. The effect was observed for six cultivars of anthunum. Results are shown on Figure 1 for anthurium cultivars Marian Seefurth and Pink Frost. These observations were repeated at monthly intervals for each successive shipment of microplants from Florida.
B. 2. Cultivar susceptibility:
Differences in cultivar susceptibility were reported previously for anthunum cultivars. For syngonium in the first series of experiments (cool environment) Cultivar LL was slightly more susceptible to infection than WB. In a second series of experiments (warmer environment) the difference between the two syngonium cultivars was greater, cultivar LL being definitely more susceptible than WB.
B. 3. Bioprotection:
Anthunum: Experiments with six different cultivars of anthunum have confirmed that plants can be protected from infection by the pathogen Xcd if leaves are sprayed with mixtures of BCAs. The bioprotective effect has been repeated five times starting with tissue-cultured anthurium cultivar, Rudolph, and continuing with plants at different ages up to two years. However, it is dear that bioprotection does not provide immunity to disease. High inoculum levels of Xcd under either warm or cool conditions are sufficient to obtain 100% kill of nonprotected plants; 36% of the protected plants died under warm conditions and 15 to 25% died under cooler conditions. Continued post-inoculation treatment of protected plants resulted in regeneration of new leaves and foliage. Plants remained symptomless and autophotographs confirmed that Xcd no longer survived in the vascular tissue. The symptomless plants eventually developed flowers (two per year) and were as vigorous as plants that had never been inoculated with the pathogen. However, on reinoculation with Xcd, the plants again developed symptoms. Results of experiment 8E are shown in Figure 2.
Syngonium: Treatments with bioprotective bacteria resulted in 14 to 25% fewer infected leaves for cultivars LL and WB, respectively (Figures 3 and 4).
Conclusions:
Bioprotection with mixtures of beneficial bacteria has broader effects than first anticipated. We have repeatedly demonstrated the effect on various cultivars of anthurium and now have evidence that another aroid, syngonium, can be protected with the same mixture of bacteria. Growth effects, though not as striking, definitely occur on syngonium.
III. Future research:
At the request of AFE last year, we have expanded the growth-enhancement studies to other ornamentals. We will continue to investigate other aroids and eventually other ornamentals in different plant families. This aspect, however, will not dominate the research, as it is still important that we analyze the plant-microbial interactions sufficiently to determine the survivability of BCAs on plant surfaces, to establish the necessary dosage to achieve a significant effect, and to find the best means of providing BCA inoculum in an appropriate formulation for growers. These aspects will be the focus of the third year of these studies.
A. Next Steps:
With the support of the College of Tropical Agriculture and Human Resources at the University of Hawaii, I have now secured funds to employ a graduate teaching assistant who also will help part-time with this project. This added support will enable me to undertake more detailed studies in the formulation of inoculum and the host-pathogen interactions so that the BCAs can be efficiently utilized. We expect to make significant progress during the third year of this project.
B. Anticipated industry benefits:
Last year I indicated that 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. We can now state that both effects have been repeatedly observed. Thus, during the third year, we will place greater emphasis on producing a microbial product for the industry.
IV. Recent publications:
(including those reported last year)
- Fukui, R., Fukui, H., and Alvarez, A.M. 1999. Effect of temperature on the incubation period and leaf colonization in bacterial blight of anthunum. Phytopathology 89:1007-1014.
- Fukui, R., Fukul, H., and Alvarez, A.M. 1999. Comparisons of single versus multiple bacterial species on biological control of anthunum 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. AppI. Environ. Microbiol. 65:1020-1023.
- Alvarez, A.M., Fukui, R., Fukui, H., and McElhaney, ft 1999. Bioprotecting anthunum against bacterial blight. Grower Talks, 2-99:74-78.
Figure 1A.
Growth enhancement of anthurium cultivar Marian Seefurth treated with beneficial bacteria (left). Microplants were deflasked, shipped from Florida to Hawaii then soaked in a mixture of beneficial bacterial leaf epiphytes prior to planting the microplants into community pots. They were sprayed at regular intervals for three months while roots were established, then uprooted, soaked in a suspension of the beneficial bacteria and transplanted into 1.5-inch pots. Twenty treated plants (left) had abundant foliage and were larger than non-treated plants (right). Only 14 of 20 non-treated plants were alive after three months because six of twenty non-treated plants had an inadequate root system and/or developed root disease. The remaining 14 plants grew slowly, producing fewer and smaller leaves than treated plants. Photographs were taken eleven months after the initial treatment of microplants.
1B.
Growth enhancement of anthurium cultivars Pink Frost and Tropic Fire treated with beneficial bacteria (left). Microplants were treated as described for Marian Seefurth. After three months microplants were soaked in beneficial bacteria and planted into 40-well trays, half a tray for Pink Frost (top) and half for Tropic Fire (bottom). Missing wells indicate plants had not developed sufficient roots for transplanting into trays. Growth enhancement due to beneficial bacteria is revealed both by the increased plant size as well as the number of plants surviving after three months in community pots.
1C.
Growth enhancement of syngonium cultivar White Butterfly. Plants at left were treated at regular intervals and transplanted into 1.5-inch pots 22 days after planting into community pots; plants at right were not treated. Photographs were taken 50 days after initial treatment of microplants.
1D.
Side view of the same 15 syngonium White Butterfly (treated plants, left; non-treated plants, right) showing difference in plant height.
Figure 2.
Bioprotection of Anthurium cultivar “Rudolph” with beneficial bacteria. Treated plants (Fig. 2A) were sprayed with beneficial bacteria at weekly intervals, then inoculated with the bacterial blight pathogen, Xanthomonas campestris pv. dieffenbachiae (Xcd). Treated plants developed lesions on some leaves, but all non-treated plants died. Foliar sprays for treated plants were continued at regular intervals, and the infected leaves eventually fell off, after which each plant developed 2-3 flowers. One year after the first challenge inoculation, treated plants were again sprayed with the pathogen. Internal infection was assessed 19, 33, and 47 days after inoculation using the leaf blots to detect bioluminescence (See Figures 3 and 4). Visual symptoms were recorded photographically 60 days after the second inoculation (Fig. 2B).
Figure 3.
Leaf blots of non-treated plants (Syngonium cultivar Lemon Lime) 20 days following inoculation with the leaf blight pathogen,, Xanthomonas campestris pv. dieffenbachiae (Xcd). Leaves were detached and grouped on blotting paper. Leaves from individual plants were distinguished from each other by the orientation of their leaf tips (Fig. 3A). For example, plant 1 (upper left) had 9 leaves; plant 2 (upper right) had 13 leaves; plant 3 (lower section) had 16 leaves. No blight symptoms were observed. Leaves were exposed to X-ray film in the dark for 8 hours, after which films were developed. Dark areas on the film (Fig. 3B) indicate internal infection by Xcd. When dry, films were placed over the leaves, and leaf margins were traced with a black pen. The leaf areas were determined using an image scanner, and the percentages of infected leaf areas were calculated.
Figure 4.
Leaf blots of treated plants. Syngonium cultivar Lemon Lime was sprayed with a mixture of leaf epiphytes (beneficial bacteria) at regular intervals during establishment of microplants in community pots (for details, see methods). Twenty-four hours following the last spray. plants were sprayed with Xcd. Leaves were detached 20 days following inoculation and processed as for non-treated plants (See caption for Fig. 3). The absence of dark areas indicates that Xcd was not able to infect leaves when protected by the beneficial bacteria.
