Integrated Biocontrol Strategies for Disease Control on Greenhouse Ornamentals Using Poinsettia as a Model System

Industry needs and project objectives

The competitive nature of the floriculture industry in the USA and the stiff competition from foreign firms has forced many growers to become more efficient in production. This means that adequate control measures must be found disease losses due to recurring pathogens like Botrytis, Pythium, and Rhizoctonia. As a part of developing improved disease control strategies, studying of new technologies for the use of biological control (biocontrol) is under way in the USA and elsewhere. Biocontrol takes advantage of environmentally-safe microorganisms, predators, or parasites that are introduced during crop production for disease or pest control. Several microorganisms like Burkholderia cepacia and binucleate Rhizoctonia have been identified as effective biocontrol agents previously. The technology must now be developed to extend their range of effectiveness against multiple pathogens as well as develop formulations that provide suitable shelf life, efficacy, and user-friendliness in the greenhouse environment. Firms that adopt the use of proven microbial biocontrol agents with EPA registration for disease control will enjoy an advantage of short re-entry periods under the Worker Protection Standard (WPS) as well as a low worker exposure hazard. The use of chemical fungicides may result in pathogen resistance over time, but long-term use of bicontrol is not an issue because plant pathogens do not develop resistance to biocontrol agents.

The first objective of this research is to understand the mechanisms associated with biocontrol of several diseases on greenhouse ornamentals (Rhizoctonia stem rot, Pythium dampimg-off, and Botrytis gray mold). Understanding mechanisms involved in biocontrol is essential for developing strategies to control plant diseases as well as assessing possible resistance development of pathogens to biocontrol agents.

The second objective is to assess the integrated use of a nonpathogenic, binucleate Rhizoctonia (BNR) and Burkholderia cepacia to optimize the efficacy of biocontol. In some cases, it is hard to control plant diseases during the entire crop production period by applying single biocontrol agent. Additional application of the same biocontrol agent or integrated use of two or more biocontrol agents needs to be considered. This approach may provide benefits to ornamentals industry by extending crop protection period and controlling wide range of plant diseases.

Summary of work conducted

1. Pyrrolnitrin production and biocontol activity of Burkholderia cepacia

   The mechanisms by which B. cepacia suppress growth of plant pathogens are most likely the production of antibioticslike pyrrolnitrin and phenazine. To investigate the relationship between pyrrolnitrin production by strain 5.5B of B. cepacia and biocontrol activity of Rhizoctonia stem rot of poinsettia, spontaneous mutation of 5.5B has been conducted and optimal cultural conditions for 5.5B have been studied to enhance pyrrolnitrin production by 5.5B.

   Spontaneous mutants of B. cepacia and their biocontrol activity

   The wild type (5.5B) was spread on the rifampicin containing media (100ug/ml) and mutants were evaluated by their colony morphology and disease control ability in the greenhouse. Nine spontaneous mutants were selected and tested in the greenhouse experiment. Each mutant including the wild type strain were incubated in nutrient broth media for 5 days, then adjusted to a cell concentration at 10⁹/ml with distilled water. Disease control varied depending on mutants of 5.5B. Rifampicin-resistant strain 21-2 showed the same level of disease control as the wild type of 5.5B, while rifampicin-resistant strain 13-1 showed no biocontrol activity on stem rot. The other rifampicin-resistant strains varied in their ability to control stem rot. Pyrrolnitrin production of these mutants was assessed by thin layer chromatography. Cultures of each mutant were extracted with ethyl acetate and visualized on TLC plates with Van Urk¬ís reagent. Wild type strain 5.5B and rifampicin-resistant strain 21-2 produced a distinct purple spot (0.9 cm in diameter) on the TLC plate which indicated rifampicin-resistant strain 21-2 still produced pyrrolnitrin at same concentration as the wild type. However, only a faint signal (0.2 cm in diameter) was detected when rifampicin-resistant strain 13-1 was analyzed by using TLC. These results suggest that the production of pyrrolnitrin by B. cepacia may play a major role in controlling Rhizoctonia stem rot of poinsettia.

   The effect of cultural conditions on the production of pyrrolnitrin

   Cultural conditions such as nutrition factors and pH of media can affect growth of microorganisms and production of antibiotics. To enhance pyrrolnitrin production and biocontrol activity by B. cepacia 5.5B, nutrient broth (NB), Luria-Bertani (LB) and a minimum salts media were used at two different pH. Growth rate of B. cepacia in NB was faster than on the other two media during the initial 3 days of incubation. After 5 days of incubation, B. cepacia in NB and minimum salts media reached the same cell concentration, but the cell concentration in LB was lower. B. cepacia incubated in NB and minimum salts media showed the same amount of biocontrol ability in the greenhouse experiment, but the biocontrol activity of B. cepacia incubated in LB was slightly lower than other two media. Change of pH in NB during growth of B. cepacia was greater than in minimum salts media. The pH in NB rose from 5.8 to 7.8 after 7 days of incubation. The pH in minimum salts media only increased from 5.8 to 6.2. No differences in biocontrol ability were observed when B. cepacia was cultured at different pH values and applied in the greenhouse experiment.

2. Induced resistance by BNR isolates

   Non-pathogenic, binucleate Rhizoctonia are known to induce plant resistance to plant pathogens. Systemic resistance of poinsettia against Rhizoctonia stem rot induced by two BNR isolate (BNR621 and P9023) was investigated as a part of understanding mechanisms involved in biocontrol. Poinsettia cuttings acquired from BNR treated stock plants of three cultivars (Angelica White, Freedom Red, and V14 Glory) were challenge-inoculated with Rhizoctonia. A series of time gaps between BNR treatment and challenge inoculation (3 days to 8 weeks) was provided to give BNR time to colonize in the root system and to induce systemic resistance. Colonization of BNR in soilless mix and on root system was also assessed from each treatment by sampling with a multiple pellet soil-sampler and culturing pellets on selective media. Induced resistance by BNR was observed in the treatments with 10 or more days between BNR treatment and challenge inoculation. Disease severities of stem rot on cuttings of Angelica White, Freedom Red, and V14 Glory from these treatments were 15, 20, and 28% lower than the infested control, respectively. The same level of BNR population was detected (23.4 propagules/g soilless mix) from soilless mix regardless of cultivars, while difference in root colonization of BNR among cultivars was observed. Number of BNR propagules colonized on the root system of V14 Glory at 10 days after BNR treatment was higher (106 propagules/g root) than Angelica White (80 propagules/g root) and Freedom Red (84 propagules/g root). Host variation for biocontrol may be due to differences in host genotype or level of inoculum used in this experiment and further study is needed. No induced resistance was observed when only one week was given between BNR treatment and inoculation with Rhizoctonia, indicating that one week is not long enough for BNR to induce resistance.

3. Sequential application of two biocontrol agents

   To extend the protection of a crop from plant pathogens, additional application of the same biocontrol agent, integrated use of chemicals or combined application of two or more biocontrol agents needs to be considered. Two isolates of binucleate Rhizoctonia(P9023, BNR 621) as well as B. cepacia 5.5B have been characterized as effective biocontrol agents for stem rot of poinsettia. These two biocontrol agents, binucleate Rhizoctonia (BNR) and B. cepacia, with different possible mechanisms of biocontrol were applied sequentially to provide crop long protection against Rhizoctonia stem rot in following manner.

   Biocontrol Application	Rooting Cube Stage	At Transplanting
   Strain 5.5B	BNR
   BNR	Strain 5.5B
   Strain 5.5B	Strain 5.5B
   BNR	BNR
   Combination	Strain 5.5B + BNR	Strain 5.5B + BNR

   Treatment of rooting cubes with B. cepacia during the poinsettia root development stage was effective in suppression of Rhizoctonia stem rot. Disease severity of stem rot in B. cepacia-treated rooting cubes was 57% lower than in infested controls. No biocontrol activity was observed in rooting cubes treated with BNR. At transplanting, soilless media were amended with BNR in a pesta formulation or drenched with a B. cepacia suspension. Isolates of BNR were more effective in control of stem rot for up to 8 weeks after transplanting compared to B. cepacia. Disease severity in BNR treated pots was 67% lower than infested controls, but only 34% lower in B. cepacia treated pots. BNR isolates were not pathogenic on poinsettia plants. These data indicate that the effectiveness of biocontrol agents is affected by the timing of application. In the poinsettia-Rhizoctonia stem rot system, applying B. cepacia at the propagation stage and then using BNR at the time of transplanting was the most effective bioconrtol strategy.

   In combination application, both P9023 and strain 5.5B were applied to rooting cubes or soilless media at the same time. At rooting cube stage, combination application of these two biocontrol agent showed some level of disease control (19% lower than infested control), but the effectiveness was significantly lower than in the rooting cubes treated with B. cepacia alone. After transplanting, similar results were observed in combination application. Disease severity in P9023 and strain 5.5B treated pots was only 35% lower than infested control, while 67% lower in the pots treated with P9023 alone. Loss of biocontrol efficacy in the combination application may due to the interaction between two biocontrol agents.

4. Population dynamics of two biocontrol agents

   Establishment of biocontrol agents in the plant root zone and maintenance of high population levels are critical for successful biocontrol. The relationship between rhizosphere (zone of root influence) and non-rhizosphere colonization by strain 5.5B and BNR isolates and their biocontrol ability was studied. Strain 5.5B successfully colonized and maintained its population level (10⁷/g root) up to 5 weeks after the application on the poinsettia root system. However, the population of 5.5B in non-rhizosphere declined rapidly and reached a non-detectable level at 7 weeks after application. Multiple application of 5.5B (1^st application in rooting cubes and 2^nd application at transplanting) showed the highest root colonization, but the biocontrol activity was not significantly improved compared to sequential application. When BNR isolates were introduced to soilless mix at the transplanting, root colonization of BNR was detected 3 days after BNR treatment. The population of BNR in the poinsettia root system reached the highest level (134 propagules/g root) at 7 days after treatment and declined to 52 propagules per gram of root at 8 weeks after treatment. These data suggested that root colonization of these biocontrol agents is critical for controlling Rhizoctonia stem rot.

   Loss of biocontrol activity from combination application of two biocontrol agents (applying strain 5.5B and BNR simultaneously at transplanting) has been previously observed. To evaluate the relationship between population of the biocontrol agents and loss of disease control efficacy, rhizosphere colonization was studied in combination application. Root colonization of strain 5.5B was consistently lower compared to population level of root system treated 5.5B alone. Similar results were observed for BNR colonization in combination application compared to single application of BNR. It can be assumed that root colonization of the biocontrol agents and disease control activity is closely related and there is an interaction between BNR and 5.5B when they are applied at the same time.

5. E. Biocontrol of Botrytis blight on poinsettia

   Numerous experiments have been conducted with these biocontrol agents and other candidates for Botrytis control without success to date.

Outline next steps

Strains of B. cepacia have emerged as new potential pathogenic microorganisms isolated from respiratory system of cystic fibrosis patients. Characterization and sub-classification of strains of B. cepacia with molecular biology techniques have been done in recent studies and it is suggested that there may be ecological and biochemical differences between environmental and clinical strains of B. cepacia. To proceed federal registration of B. cepacia as a commercial biocontrol agent by EPA, the environmental strains need to be characterized and differentiated from the clinical strains of B.cepacia.

Appropriate formulation and delivery methods are critical for efficacy of disease control as well as shelf-life of biocontrol agents. Various formulation methods were suggested for B. cepacia such as using peat moss and freeze-drying. The future research project will include developing appropriate formulations for B. cepacia and cost-effectiveness analysis of formulation methods so that the biocontrol agent could be marketed.

This entry was posted on Thursday, June 1st, 2000 at 2:07 pm and is filed under Disease Management, Progress Reports. You can follow any responses to this entry through the RSS 2.0 feed. You can leave a response, or trackback from your own site.