w6 biological control of bacterial pathogens
TRANSCRIPT
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Biological Control of Phytopathogenic Bacteria
Ent 547 Fundamentals of Biological Control
Fall 2005
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Phytopathogenic Bacteria Prokaryotic
Covalently closed circular DNA in a nucleoid. May contain plasmids. No organelles 70s ribosomes
Small, 1-10 microns x 0.5 – 1 micron. Reproduction binary fission. Endospores. Entry into plant via wounds (trichome breakage, pruning,
grafting, root tip elongation) or natural openings (stomata, hydathodes, lenticels).
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Phytobacterial Lifestyles Obligate parasites – fastidious bacteria.
Wall-less prokaryotes. Rickettsia. Grass endophytes. Seed-borne.
Facultative saprophytes. Prefers host but can live or survive outside host for short periods
of time (1 week to 4-5 years). Seed-borne
Facultative parasites. Opportunistic pathogens, generally efficient pathogens once
ingress is obtained. Can survive outside of host (soil) for years.
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Importance of Bacteria Used in basic research. Industrial uses. Consumer goods (Xanthan gums, flavor, texture). Medical uses (antibiotics). Agricultural (nitrogen fixation). May be the oldest forms of life. Involved in carbon, nitrogen, and sulfur cycles.Cause disease in animals, plants,
and humans.
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Mo
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Gram Positive Bacterial Cell Wall
From Nancy Perry, University of Manchester. http://www.teaching-biomed.man.ac.uk/student_projects/2001/mnlf8np2/homepage.htm
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Gram Negative Cell Wall
From Nancy Perry, University of Manchester. http://www.teaching-biomed.man.ac.uk/student_projects/2001/mnlf8np2/homepage.htm
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Taxonomy Gram positive
Bacillus Coryneform Clostridium Streptomyces
Gram negative Acidovorax Agrobacterium Burkholderia (Ralstonia) Erwinia Pantoea Pseudomonas Rhizomonas Xanthomonas Xylophilus
Fastidious Phloem-limited bacteria
Cell-wall free bacteria
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Symptoms of a Bacterial Infection in Plants Necrosis – dead, dying tissue margins, leaf streaks,
stripes, cankers, lesions, spots, blights, vascular and pith necrosis.
Chlorosis – yellow with adjacent necrotic tissue or alone. Watersoaking. Wilting – vascular occlusion from cells, gum,
polysaccharide, tyloses. Soft rots – pectolytic enzymes, water release. Hyperplasia – overgrowth, galls, knots.
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Signs of a Bacterial Infection Bacterial ooze or slime, especially under moist
conditions. Bacterial gum, under drier conditions. Bacterial scale, crust, or flake under when dried. Bacterial streaming.
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Bacterial Disease Management Resistant cultivars Limit moisture with management Sanitation Antibiotics Copper based pesticides Bioantagonists
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Microbial Pesticides for Bacterial Disease Control
Organism Product Target Hosts Formulation/application
Agrobacterium radiobacter
Norbac 84-CNogallGalltrol A
Crown gall Fruit and nut trees, caneberries, roses, ornamental nursery stock
Live agar culture/water
Bacillus subtilis
Rhizo-Plus, Rhizo-Plus Konz
Streptomyces scabies
Potato Water dispersible granule/seed treatment, soil drench, dip
Bacillus subtilis QWT713
Serenade Erwinia amylovora (and fungi)
Stone fruits (and other crops)
Wettable powder
Pseudomonas fluorescens A506
BlightBan A506 Erwinia amylovora, frost damage
Almond, apple, apricot, blueberry, cherry, peach, pear, potato, strawberry, tomato
Wettable powder/bloom time spray
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Microbial Pesticides for Bacterial Disease Control
Organism Product Target Hosts Formulation/application
Pseudomonas fluorescens
Conquer Pseudomonas tolaasii
Mushrooms
Burkholderia solanacearum
PSSOL Burkholderia solanacearum
Vegetables
Streptomyces lydicus
Actinovate Soilborne fungal pathogens
Greenhouse and nursery crops, turf
Water-dispersible granule
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Potential Agents Other Bacteria
Wild type Azospirillum brasilense Other Bacillus species Streptomyces praecox Pantoea agglomerans
Mutants Mutants of Burkholderia solanacearum hrp mutants
Other bacteria (mutants) Bacteriophage - bacterial viruses. Bacteriocins – small peptides that inhibit the growth of
various bacteria.
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Antagonism Mechanisms Antibacterial metabolites Siderophores Nutrient deprivation, niche exclusion Induced resistance Plant growth promotion
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Background
Aztecs 1200 A.D. Chinampas Potential Biological
control organisms Trichoderma spp. Pseudomonas spp. Fusarium spp.
Incorporated organic material (manure)
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First Biological Control of Plant Pathogenic Bacteria
Potato scab or common scab of potatoes Streptomyces scabies Streptomyces acidiscabies
Millard and Taylor, 1927 Added green grass cuttings Added Streptomyces praecox
Competition for active sites
Called observation “starving out” Recent work in the 1990’s
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Background
Thus, biological control studies with bacteria has examined for over 70 years
Sources of biological control bacteria Suppressive soils On aerial plant parts (epiphytes, phylloplane) On root surfaces (epiphytes, rhizoplane) Colonizing plant pathogens (hyperparasites) Plant disease causing bacteria (phytopathogens)
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Principles
Baiting Schisler, D. A. and Slininger, P. J. 1994. Selection and
performance of bacterial strains for biologically controlling Fusarium dry rot of potatoes incited by Gibberella pulicaris. Plant Dis. 78:251-255.
Formulation Mechanisms of pathogen suppression
substrate competition and niche exclusion siderophores antibiotics induced resistance (not really biological
control?)
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Examples Products
Agrobacterium radiobacter Bacillus subtilis Pseudomonas fluorescens – Erwinia amylovora, Pseudomonas
syringae pv. syringae
Reports Azospirillum brasilense – root stimulant Burkholderia mutants Erwinia carotovora subsp. betavasculorum hrp- mutants Pantoea agglomerans (Erwinia herbicola) – Erwinia amylovora Bacteriophage and bacteriocins
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Crown Gall Agrobacterium
tumefaciens Crown gall on a wide range
of dicotyledonous plants especially apple, pear, peach, cherry, almond, raspberry and roses
A separate strain, biovar 3 causes crown gall of grapevine
Gram negative, motile rod, related to Rhizobium
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Agrobacterium tumefaciens Fairly ubiquitous in soil and cosmopolitan Can live saprophytically for up to two years Fairly efficient colonizer of the rhizosphere Pathogenic determinants are on the Ti (tumor-inducing)
plasmid (pTi) or the Ti plasmid Are chemotactically attracted to sugars, and other root
components However, A. tumefaciens strains with the Ti plasmid are
more strongly attracted to wound phenolic compounds such as acetosyringone (10-7 M)
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Infection
At greater concentrations (10-5 to 10-4 M), acetosyringone activates vir genes, these lead to the production of permeases for opine uptake, and an endonuclease that excises the T-DNA (transferred DNA)
The T-DNA is released, enters and integrates into plant DNA, T-DNA codes for opines, IAA, and novel plant metabolites (agrocinopines, opines, nopalines)
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Agrobacterium radiobacter: Galltrol-A,
Nogall, Diegall, Norbac 84C Agrobacterium radiobacter strain K84
Controls only nopaline producing A. tumefaciens strains This is the first biological control product for any plant disease Alan Kerr in the 1970’s
Target Pathogen/Disease: crown gall disease caused by Agrobacterium tumefaciens
Crop: fruit, nut, and ornamental nursery stock Formulation: aqueous suspension containing bacterial
cells, methyl cellulose, and phosphate buffer (refrigerate), agar plates, peat substrate
Application: root, stem, cutting dip, or spray
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Agrobacterium radiobacter K84 Similar to A. tumefaciens (same biovar) except
does not have the Ti plasmid Has pAGK84 which codes for agrocin 84 and
pNOC which codes for nopaline uptake and catabolism
Mechanism of action pNOC – competition for nopaline Niche competition – efficient colonizer of roots and
wound sites (chromosomal) Agrocin 84
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Agrocin 84
Agrocin84 is an adenine nucleotide with a 6 glucofuran and a methylated pentamide attached (fraudulent nucleotide)
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Agrocin84
Highly selective for nopaline producing AT strains Ti plasmid of sensitive A. tumefaciens strains has
NOC (nopaline catabolism) and ACC (agrocinopine catabolism) genes and permeases for uptake
agrocinopene permeases imports A84 A84 blocks DNA synthesis
Luckily, the majority of AT strains are nopaline producing strains
A. radiobacter K1026 is Tra- , first genetically engineered microbe released for widespread use
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Bacillus Gram positive, soil borne, motile, endospore
producing (req. oxygen), facultative anaerobe, prokaryote.
Can be found in manure and associated with plants. There are nearly 50 species known of which only B.
anthracis (anthrax) and B. cereus (food poisoning) cause disease in humans.
Known producers of bioactive metabolites act as pheromones, antibiotics, plant growth hormones, etc.
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Bacillus subtilis A13: Epic, Kodiak, Rhizo Plus, Serenade, System 3
Bacillus subtilis A13 Registered on peanut in 1988 Registered on cotton and broad bean in 1990
Background Broadbent et al., 1977 Inhibited fungi (Phytophthora spp., Pythium spp.,
Fusarium spp., Sclerotium spp., Rhizoctonia spp.) Stimulated growth of eggplant, dahlia and cabbage in
steamed soil Seed treatment: Carrots (48%), Oats (33%), Peanuts (37%)
yield increases
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Kodiak
Biocontrol Agent: Bacillus subtilis Target Pathogen/Disease: Rhizoctonia solani,
Fusarium spp., Alternaria spp., and Aspergillus spp. that attack roots
Crop: cotton, legumes Formulation: dry powder; usually applied with
chemical fungicides Application: added to a slurry mix for seed
treatment; hopper box treatment
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Bacillus species Mode of action
Antibiosis Plant growth promotion Induced resistance
Wulff et al. 2002. Biological control of black rot (Xanthomonas campestris pv. campestris) of brassicas with an antagonistic strain of Bacillus subtilis in Zimbabwe. Eur. J. Plant Pathol. 108:317-325.
Wulff et al. 2002. Biochemical and molecular characterization of Bacillus amyloliquefaciens, B. subtilis, and B. pumilus isolates with distinct antagonistic potential against Xanthomonas campestris pv. campestris. Plant Pathol. 51:574-584.
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Pseudomonas fluorescens
BlightBan A506: Fireblight
Conquer, Victus: targets P. tolassii in mushrooms
Weller and Thomashow 2-fluoroglucinol phenazine
Lindow Frostban
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FireBlight Fireblight is caused by Erwinia amylovora Transmitted by bees and insects to flowers Pathogen enters flower nectaries and invades
the vascular system of the plant P. fluorescens is an effective protectant – site
exclusion Pantoea agglomerans (Erwinia herbicola) similar
mechanism.
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Disease cycle of fireblight.
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Burkholderia solanacearum Burkholderia (Pseudomonas, Ralstonia) Kempe, J. and L. Sequeira. 1983. Biological control of
bacterial wilt of potatoes: attempts to induce resistance by treating tubers with bacteria. Plant Dis. 67:499-503. Inoculated avirulent strains of B. solanacearum, virulent but
incompatible strains of B. solanacearum, and saprophytic or pathogenic pseudomonads
Found Incompatible strain 70 (plantain) Avirulent B. solanacearum strain B82 P. fluorescens strain W163
Induced resistance
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Genetic Modification of B. solanacearum
Burkholderia solanacearum and many other bacterial plant pathogens have hypersensitivity and pathogenicity “clusters”
The hypersensitive reaction Rapid, localized plant cell death upon contact with a
pathogen Phytoalexin accumulation Pathogenicity related protein increase Lipoxygenases increase Pathogen sequestering and death
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Hrp- mutants of B. solanacearum Hrp = hypersensitivity pathogenicity gene cluster Mutants
Decreased pathogenicity Decreased vascular spread Populations usually lower than wildtype
In combination with wildtype Mutant populations are increased Wildtype populations are decreased
Mechanisms Competition Bacteriocin mediation?
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Bacteriophage Bacteriophage are obligate intracellular viral
parasites of bacteria and are compose of nucleic acids and protein
Range in size up to 200 nm long. All have a “head” structure Many but not all have a tail Uses
Diagnostic tool Identification and taxonomic tool
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Genetic manipulation Loper et al. Erwinia carotovora subsp.
betavasculorum Bacteriocin (phage) Out minus mutants Nearly 100% suppression of
the soft rot pathogen, E. c. subsp. carotovora in potato tubers
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Bacteriocins Most bacteriocins are proteinaceous compounds
that are active again closely related bacteria There are exceptions (Agrocin 84) Reports
Burkholderia solanacearum inhibited on plants dipped in a non-pathogenic, bacteriocin producing strain of B. solanacearum
Xanthomonas campestris pv. oryzae infection incidence and severity reduced with non-pathogenic, bacteriocin producing strains.
Purified bacteriocin from Pseudomonas syringae pv. ciccaronei (isol. From carob tree) – inhibited P. s. pv. savastanoi in vitro and in planta.
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Summary Bacterial agents
Bacillus Pseudomonas Burkholderia Streptomyces
Other agents Bacteriophage Bacteriocins
Mechanisms Antibiosis Induced resistance