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.
Morphology
Gram Positive Bacterial Cell Wall

From Nancy
Perry,
University of
Manchester.
http://www.tea
ching-
biomed.man.a
c.uk/student_p
rojects/2001/m
nlf8np2/home
page.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  Gram negative
 Bacillus  Acidovorax
 Coryneform  Agrobacterium
 Clostridium  Burkholderia (Ralstonia)
 Streptomyces  Erwinia
 Pantoea
 Pseudomonas
 Fastidious Phloem-limited  Rhizomonas
bacteria  Xanthomonas
 Cell-wall free bacteria  Xylophilus
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 Norbac 84-C Crown gall Fruit and nut Live agar
radiobacter Nogall trees, culture/water
Galltrol A caneberries,
roses, ornamental
nursery stock
Bacillus Rhizo-Plus, Streptomyces Potato Water
subtilis Rhizo-Plus scabies dispersible
Konz granule/seed
treatment, soil
drench, dip
Bacillus Serenade Erwinia Stone fruits (and Wettable
subtilis amylovora (and other crops) powder
QWT713 fungi)

Pseudomonas BlightBan A506 Erwinia Almond, apple, Wettable

fluorescens amylovora, apricot, powder/bloom
A506 frost damage blueberry, cherry, time spray
peach, pear,
potato,
strawberry,
tomato
Microbial Pesticides for Bacterial Disease Control

Organism Product Target Hosts Formulation/

application
Pseudomonas Conquer Pseudomonas Mushrooms
fluorescens tolaasii

Burkholderia PSSOL Burkholderia Vegetables

solanacearum solanacearum

Streptomyces Actinovate Soilborne Greenhouse and Water-

lydicus fungal nursery crops, dispersible
pathogens turf 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