Biological Control of Phytopathogenic Bacteria

Ent 547 Fundamentals of Biological Control

Fall 2005

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).

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.

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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Gram Positive Bacterial Cell Wall

From Nancy Perry, University of Manchester. http://www.teaching-biomed.man.ac.uk/student_projects/2001/mnlf8np2/homepage.htm

Gram Negative Cell Wall

From Nancy Perry, University of Manchester. http://www.teaching-biomed.man.ac.uk/student_projects/2001/mnlf8np2/homepage.htm

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

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.

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.

Bacterial Disease Management Resistant cultivars Limit moisture with management Sanitation Antibiotics Copper based pesticides Bioantagonists

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

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

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.

Antagonism Mechanisms Antibacterial metabolites Siderophores Nutrient deprivation, niche exclusion Induced resistance Plant growth promotion

Background

Aztecs 1200 A.D. Chinampas Potential Biological

control organisms Trichoderma spp. Pseudomonas spp. Fusarium spp.

Incorporated organic material (manure)

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

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)

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?)

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

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

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)

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)

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

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

Agrocin 84

Agrocin84 is an adenine nucleotide with a 6 glucofuran and a methylated pentamide attached (fraudulent nucleotide)

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

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.

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

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

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.

Pseudomonas fluorescens

BlightBan A506: Fireblight

Conquer, Victus: targets P. tolassii in mushrooms

Weller and Thomashow 2-fluoroglucinol phenazine

Lindow Frostban

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.

Disease cycle of fireblight.

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

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

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?

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

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

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.

Summary Bacterial agents

Bacillus Pseudomonas Burkholderia Streptomyces

Other agents Bacteriophage Bacteriocins

Mechanisms Antibiosis Induced resistance