Mechanisms of Action of Bacterial Biological Control Agents

Abstract

Bacterial biocontrol agents (BCAs) have been widely employed for minimizing the incidence and severity of several economically important crop diseases. Studies on the interactions between the bacterial BCAs and target microbial plant pathogens have revealed the involvement of different mechanisms of their biocontrol activities. Bacterial antagonism may be due to production of toxic metabolites (enzymes, antibiotics and volatile organic compounds), competition for nutrients and space, prevention of pathogen colonization of host tissues and induction of resistance in plants to crop diseases. The bacterial species included in the genera Pseudomonas, Bacillus, Burkholderia, Lysobacter, Serratia and Pantoea have been reported to be effective against several plant pathogens by acting through one or more mechanisms. The rhizobacterial species are potent biocontrol agents and also efficient promoters of plant growth, thus providing double benefits to the treated plants. Factors that influence the efficiency of the biocontrol agents have been studied. These studies are useful to select the suitable bacterial strain(s) that could provide higher level of protection to the plants under a particular set of environmental conditions existing in different agroecosystems.

Appendices

Appendix 5.1: Visualization of Effects of the Metabolite 2,4-diacetylphloroglucinol (2,4-DAPG) of Pseudomonas spp. on Fungal Pathogen Using Confocal Laser Scanning Microscope (CLSM) (Islam and Fukushi 2010)

A.	Preparation of fungal hyphal cells for visualization
i.	Grow the fungal pathogen (Aphanomyces cochlioides) on potato dextrose agar (PDA) at room temperature (25 °C); cut out agar disks (6 mm diameter) from the growing edges; place the disk individually 30 cm apart from the colony of bacterial biocontrol agent (Pseudomonas fluorescens ECO-001) in four replicates: prepare control plates without the BCA and incubate at 25 °C for 5 days in the dark
ii.	Harvest the fungal hyphae, using a sterile corkborer (6 mm diameter) from the colony edges growing toward the BCA colonies for observations under confocal laser scanning microscope (CLSM)
iii.	Prepare different concentrations of DAPG and latrunculin B (0.1, 0.5, 1.0 and 5.0 μg) in acetone; place the solutions on individual sterile paper disks (8 mm diameter × 1.5 mm thickness) (Advantec Toyo, Japan) and dry the disks by evaporating acetone under vacuum
iv.	Place the disks 2 cm apart in petriplates containing PDA inoculated with mycelial plugs (6 mm diameter); cut from the edge actively growing fungal pathogen colony and incubate at 25 °C
B.	Preparation of specimen and observation under CLSM
i.	Remove mycelial plugs using a sterile cork borer (6 mm diameter) from the edge of the actively growing hyphae of the pathogen paired with bacterial colony or DAPG or latrunculin B-treated paper disks and use plugs removed from untreated plates as control.
ii.	Fix the mycelial plugs with 6 % paraformaldehyde in 60 mM 1,4-piperazine- diethanesulfonic acid buffer (Sigma) pH 7.0 with 100 μM MBS (m-maleimidobenzoyl N-hydroxyl succinimide ester, Pierce) for 30 main at room temperature; rinse three times in a buffer solution and transfer to glass slides for sectioning
iii.	Section the upper portion of the agar plug uniformly (0.25 mm thickness) with a sterilized stainless blade
iv.	Stain the sections for 30 min in 0.66 μM RP in 60 mM Pipes buffer (pH 7.0); rinse in buffer and mount in 50 % glycerol with 0.1 % p-phenylene-diamine and observe under CLSM
v.	Use scan time perframe 1.08 s; to obtain a DIC image and perform averaging at four scans per frame
vi	Repeat the experiment three times with three replicates for each experiment

Appendix 5.2: Assessment of Effect of Phenazines on Microsclerotial Germination of Verticillium spp. by Microplate Assay (Debode et al. 2007)

i.	Place nylon mesh filters (pore size 41 μm, diameter 25 mm, Millipore, USA) in the wells of 96-well microplate; embed the microsclerotial preparation (20 μl) in the well (approx. 50 microsclerotia/well); add aliquots of 180 μl bacterial suspension (2 × 107 and 2 × 109 CFU/ml); use sterile physiological solution (180 μl) for controls and incubate for 2 days at 24 °C
ii.	Retrieve the filters from the wells separately; place them on sterile filter paper under sterile conditions and dry the filters
iii.	Place the filters on soil-pectate-tergitol agar (SPTA) plates containing each 50 mg/l of chloramphenicol, tetracycline and streptomycin sulfate and incubate for 10 days
iv.	Examine under the dissecting microscope and record the effect on percentage of microsclerotial germination and formation of secondary microsclerotia in different treatments
v.	Maintain five replications and repeat the experiment once

Appendix 5.3: Assessment Antagonistic Activity of Bacterial Antagonists Against Agrobacterium spp. (Dandurishvili et al. 2010)

A.	Dual-culture ‘sandwich’ assay
i.	Fill a petridish with suitable medium seeded with the test antagonist strain and fill another petridish similarly with medium inoculated with the target pathogen strain (overnight culture) at appropriate dilution (105 cells/ml)
ii.	Join the open plates together and tightly seal with parafilm maintain suitable control (without the antagonistic strain)
iii.	Incubate the plates at 28 °C and examine the samples taken at 24-h interval
B.	Bioassays for tumorigenicity in the greenhouse
i.	Raise the tomato seedling nursery in plastic seed trays for 25–30 days at 25 °C in a growth chamber; transplant the seedlings at 2–3 leaf stage into bigger containers and placed in the greenhouse
ii.	Grow the bacterial pathogen and the BCA strains separately in suitable medium for 48 h at 28 °C in a shaker; centrifuge the suspension at 8,000 rpm for 15 min; resuspend the bacterial cells in tap water and adjust the concentration to 108 cells/ml
iii.	Soak the roots of tomato seedlings in glass vessels containing the BCA cell suspension at 107 cells/ml and transplant the seedlings into containers placed in the greenhouse
iv.	Make wounds by scratching on the stem surface of seedlings with a needle at two sites (second and third internodes); inject into the wound sites with pathogen suspension (10 μl, 2–5 x 108 cells/ml) and treat the control plants similarly only with the pathogen cell suspension
v.	Inoculate tomato seedling in the wound sites first with the antagonist cell suspension; after 7 days inoculate the pathogen in the same wounds by injecting the pathogen suspension (10 μl/wound site)
vi.	Record the tumor formation in tomato plants inoculated by two different methods and express the disease incidence in terms of tumor fresh weight in different treatments

Appendix 5.4: Assessment of Activities of Enzymes in Papaya Fruits Treated with Pseudomonas putida MGY2 (Shi et al. 2011)

A.	Phenylalanine-ammonia lyase (PAL) (EC4.3.1.5) activity
i.	Perform all steps at 4 °C; homogenize pericarp tissue samples (1 g) in 2 ml of extraction buffer (50 mM Tris–HCl buffer, pH 8.8 containing 15 mM B-mercaptoethanol, 5 mM EDTA, 5 mM ascorbic acid, 1 mM PMSF and 0.15 % PVP (w/v)); filter the homogenate through cheesecloth (four layers); centrifuge at 12,000 g for 20 min at 4 °C and use the supernatant as crude enzyme
ii.	Prepare the reaction mixture (3 ml) containing 16 mM L-phenylalanine, 50 mM Tris–HCl buffer (pH 8.8), 3.6 mM NaCl and 0.1 ml of enzyme solution and incubate at 37 °C for 40 min
iii.	Stop the reaction by adding 500 μl of 6 mM HCl; centrifuge at 12,000 g for 10 min to precipitate the denatured protein
iv.	Record the absorbance at 290 nm using a spectrophotometer before [step (ii)] and after incubation
v.	Use cinnamic acid (analytical grade) as standard; one unit of PAL activity is equal to the amount enzyme that produced 1 μmol of cinnamic acid within 1 h
vi.	Express the results as μmol of cinnamic acid/mg protein/h
B.	Catalase (CAT) (EC1.11.1.6) activity
i.	Grind pericarp tissues (5 g) from five papaya fruits in 20 ml of 0.05 M sodium borate buffer (pH 7.0, containing 5 mM mercaptoethanol) with 0.5 g polyvinyl polypyrrolidone (PVPP).
ii.	Add 0.2 ml of enzyme preparation to 2.8 ml of 40 mM H2O2 (dissolved with 50 mM sodium phosphate buffer, pH 7.0) as a substrate.
iii.	Measure the decomposition of H2O2 by the decline in absorbance at 240 nm
iv.	One unit of CAT is equal to the amount of enzyme per microgram protein that decomposed 1 mol of H2O2/min at 30 °C
C.	Peroxidase (POD) (EC 1.11.1.7) activity
i.	Homogenize the pericarp tissues (1 g) in 4 ml of extraction buffer (0.2 M sodium phosphate buffer, pH 6.4 containing 0.2 % PVP (w/v)) at 4 °C; centrifuge at 12,000 g for 20 min and use the supernatant as crude enzyme
ii.	Incubate the mixture containing 0.5 ml enzyme extract and 2 ml of guaiacol substrate (100 mM sodium phosphate, pH 6.4, containing 8 mM guaiacol) for 5 min at 30 °C
iii.	Determine the increase in absorbance at 460 nm after adding 1 ml H2O2 (24 mM)
iv.	One unit of POD activity is equal to the amount of enzyme per microgram protein that increased 0.01 in absorbance at 460 nm in 1 min