BIOL 640 Charles R. Lovell
MICROBIAL INTERACTIONS
December 4, 2006 With Plants
I. Parasitism
A. Relevant plant surface properties
1. Adhesion to the plant cell surface is a prerequisite for
infection
2. The primary cell wall has substantial strength, but
little rigidity, allowing expansion
a. Major structural components of the primary cell wall
1. -cellulose constitutes the microfibrillar phase
of walls (analogous to glass fibers in fiberglass)
2. Matrix polysaccharides consist of hemicelluloses
and pectins
a. Hemicelluloses include xylans, xyloglucans,
glucomannans, galactoglucomannans, and
noncellulosic mixed -glucans
b. Pectins are a mixture of neutral and acidic
polysaccharides which are water soluble and gel
forming
3. At least three types of hydroxyproline-rich
glycoproteins are also found
a. Insoluble, cellulose-associated
b. Soluble lectins and agglutinins
c. Soluble, high molecular weight arabinogalactan
glycoproteins
4. Noncovalent bonding is thought sufficient for
stable association of polymeric constituents
3. The rigid secondary cell wall provides structural
strength
a. Hydrophobic lignin deposition replaces water,
encrusting microfibrilar and matrix polysaccharides
1. Covalent linkages are formed between lignin and
hemicellulose and proteins
b. Lignin synthesis
1. Lignins are formed by enzymatic oxidation of the
phenolic group and subsequent, random
polymerization of phenoxy radicals of coumaryl,
coniferyl, and sinapyl alcohols
a. Monomers are joined by ether and carbon-carbon
linkages
4. Outer coatings vary with location and with plant tissue
function
a. Exposed surfaces and older root surfaces are covered
with hydrophobic materials composed of a structural
network of long-chain hydroxy fatty acid esters
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waterproofed with wax (long chain aliphatics)
1. Translucent, membranous cuticles are characterized
by cutin (insoluble, high molecular weight, long-
chain hydroxy fatty acids)
2. Periderms (bark) are characterized by the suberin
complex (aliphatic polyester plus
polyphenylpropanoid polymers)
b. Mucilages cover root regions active in water and
nutrient uptake
1. The extent and possibly type of mucilages produced
depend on plant species, soil type, and region of
the root
a. Maize root-cap slime contains 30% (w/w)
protein, with the balance mostly polysaccharide
5. Plant defensive compounds
a. Preformed compounds, including phenylpropanoids,
terpenoids, enzymes, and H2O2 are associated with the
epidermal wall
1. Silicon, lignin, and cyanogenic glycosides or
glucosinolates may also be present
b. Phytoalexins are postformed defensive compounds
1. Many groups of secondary metabolites are included
(phenylpropanoids, flavonoids, stilbenes,
terpenes, polyacetylenes)
2. Increased production of reactive oxygen species
during the first few hours of infection has also
been observed
a. Some species of plant pathogens (such as
Xanthomonas oryzae pv. oryzae) produce high
levels of catalase and superoxide dismutase as
soon as growth begins
d. Cellulose (insoluble glucan) acts as a wound sealant
and can be deposited or removed quickly
B. Opportunistic parasites
1. This category includes only organisms capable of
resisting or overcoming plant defense mechanisms
a. Saprotrophic bacteria can readily grow on dead plant
material external to the plant
1. Some saprophytic bacteria and fungi (epiphytes)
also colonize the plant surface
2. Types of plant parasites
a. Necrotrophic bacteria and fungi can invade plants but
only grow well on dead plant tissue
1. Some necrotrophic fungi attack plants during one
stage of their life cycle, but not during others
2. Necrotrophs gain entry into the plant through
wounds, kill plant cells, and grow
saprotrophically on the dead plant tissue
a. Obligate necrotrophs do not compete well as
true saprotrophs, spending almost all of their
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life cycle as parasites of living plants
b. Biotrophs grow parasitically in a living plant for
most of their life history, most with an independent
saprotrophic stage
1. Obligate biotrophs grow on or in plant tissues at
all times and include the rusts, smuts and powdery
mildews (Claviceps purpurea) among the fungi
a. Biotrophs typically grow either intracellularly
or intercellularly, interacting with living
plant cells
1. Fungi that grow intracellularly penetrate
plant cell walls and develop specialized
hyphae, called haustoria, which seal to the
host cell wall
2. The interaction between the host plant and the
biotrophic fungus is more specific than that
between the host and necrotrophic fungi
a. Fungal specificity for plant hosts may be due
in part to plant signal compounds, such as
isoflavones
1. Motile zoospores of some fungi are released
under conditions of soil flooding and
nutrient deprivation
2. Motile zoospores of plant-pathogenic
oomycetes are actively chemotactic to these
compounds
3. Ectomycorrhizal fungi can also be considered
biotrophic (see below)
3. Environmental factors affecting parasite virulence
a. Temperature should be high enough to support rapid
growth
b. Soil moisture should be high enough to prevent
desiccation of the parasite
c. Soil nutrient levels should be high enough to support
initial pre-penetration growth of the parasite
4. Plant defenses
a. Mechanical barriers
1. These include cuticles, bark, and epidermal cells
a. Plants can often wall off an invasion site with
cork cells or localized thickening of cell
walls (callose deposits)
b. Fungi grow at the tips of their hyphae,
allowing them to push through the barriers
c. Bacteria and viruses typically enter the host
through a wound
1. Some viruses are introduced by the sucking
mouth parts of insect vectors
b. Chemical defenses
1. Pre-formed defensive chemicals include phenolic
compounds such as tannins and catechol, saponins
and lactones
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a. Such toxins are often concentrated in waxes
b. Some pre-formed defensive chemicals are
harmless until activated by fungal enzymes
2. A variety of metabolic changes resulting in
production of toxins occur in infected plants
a. This class of toxins is called phytoalexins
c. Plant defenses do not appear to be specific to
antagonistic microorganisms, mutualists can also be
excluded
C. Specialist parasites
1. A wide variety of plant viruses are found
a. A virus is typically parasitic on one or only a few
plant species
2. Agrobacterium interaction with dicots
a. Crown gall disease is caused by Agrobacterium
tumefaciens
1. Agrobacterium infects the plant through wounds,
usually near the crown (junction between roots
and stem) of the plant
a. Agrobacterium is actively chemotactic and is
attracted to wound exudates
b. Specific receptors located on the surfaces of
the plant cells interact with a polysaccharide
on Agrobacterium cells
2. The bacteria stimulate the plant cells around the
wound to multiply, forming a tumorlike growth, or
gall
a. Generally only wounded plants are infected
because the new cells developing around the
wound are most susceptible to infection
1. Only young, actively dividing plant cells
produce the Agrobacterium receptor compound
2. Agrocins interfere with binding to the plant
cell wall
b. The probability of successful infection and
the extent of the damage to the plant are
largely dependent on the host plant species
c. Phenolics produced by the host plant when
wounded have been shown to activate
Agrobacterium virulence
3. Crown gall disease is an example of genetic
parasitism
b. A. tumefaciens carries one or more large plasmids
1. One of these plasmids is the Ti (tumor inducing)
plasmid
a. The Ti plasmid carries about 100 genes
2. The bacterium attaches to the plant cell wall and
the plasmid enters the plant cell very rapidly
3. Part of the plasmid, the T-DNA (transferred DNA)
integrates into the plant cell chromosome
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a. The T-DNA genes are expressed along with the
normal plant genes, causing the production of
chemicals not normally produced in the plant
and of plant hormones
1. Opines are derived from amino acids and are
produced in infected plant cells
a. Opines serve no purpose in the plant,
but serve as carbon and nitrogen sources
for the agrobacteria
1. Other soil bacteria and fungi can also
utilize opines
2. In some tumors, opine production and
specific utilization by the infecting
Agrobacterium strains defines a highly
specific niche (the opine concept)
a. The advantage of tumor opine
production for the Agrobacterium
strain is not clear for all plant
hosts
b. Opine production is Agrobacterium strain
specific
c. Opines also stimulate plasmid exchange
between Agrobacterium strains and
increase the virulence of Ti plasmids
d. Agrobacterium strains can be classified
on the basis of the opines they utilize
1. Octopine (derived from arginine) is
used by the octopine strains
2. Other opines include lysopine (from
lysine), histopine (from histidine),
octopinic acid and nopalinic acid
(from ornithine), and nopaline (from
arginine)
2. Plant growth hormones (auxin and
cytokinins) are also expressed from the T-
DNA, stimulating growth of the tumor
3. The Ti plasmid also encodes the enzymes
necessary for opine catabolism by the
Agrobacterium, but these genes are not
incorporated into the plant genome or
expressed in the plant
b. Regions of nucleotide sequence homology between
the plant chromosome and the Ti plasmid exist,
probably facilitating integration of the
plasmid into the plant chromosome
c. Conservative DNA sequences in the pathogenic
functions virD2 (endonuclease) and ipt (T-DNA
cytokinin synthesis) have been used to
distinguish phytopathogenic from nonpathogenic
Agrobacterium strains
3. In at least some cases there is a high degree of host
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specificity of specialist parasites, with some evidence
for coevolution of host and parasite
II. Commensalism
A. The phyllosphere
1. The terrestrial phyllosphere
a. Leaf and stem surfaces are colonized by numerous
species of bacteria, yeasts, and filamentous fungi
1. Many organisms grow as aggregates coated with
substantial exopolymeric material
a. These aggregates have been identified as
biofilms by some workers
b. Yeasts, filamentous fungi, and bacteria dominate the
phyllosphere microflora at different times during
development of terrestrial plant leaves
1. Typical microbial succession on leaves starts with
bacteria as pioneers when the bud breaks, followed
by yeasts and filamentous fungi
a. Competition for nutrients seems to be an
important determinant in this succession
b. Antagonism between yeasts and filamentous fungi
is well known
1. Production of antifungal compounds by some
yeasts has been reported
2. Yeasts and yeastlike fungi dominate the leaf
surface during much of the growing season and
their biomass can exceed that of other types of
microorganisms by 50 to 1
a. Some common species, such as Aureobasidium
pullulans, Sporobolomyces spp., Rhodotorula
spp., and Cryptococcus spp. may reach 107
CFU·g-1 (fresh wt) of leaf material on some
plant species
b. Similar species of yeasts occur through the
year and across a range of plant species,
although numbers are variable and influenced by
sampling time
c. Reasons for the dominance of terrestrial phyllosphere
microflora by yeasts are mostly unknown
1. Possible explanations include:
a. Cuticle utilization
b. Adaptation to environmental extremes
c. Tolerance to pollutants and inhibitors such as
phytoalexins
d. Antibiotic production
2. Production of antimicrobial compounds is common in
phyllosphere yeasts, but not in yeasts from other
environments
d. A variety of factors appear to influence species
composition of phyllosphere bacterial communities
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1. Host effects including plant species and leaf age
and position
2. Physical environmental conditions
3. Availability and density of inoculum
a. In an experiment in which Pseudomonas syringae
was inoculated onto leaves at different cell
concentrations (from 105 - 109 ml-1), the
strain exhibited the lowest mortality rates
and lowest proportional decline of the
inoculum at the highest inoculum levels
1. Inoculating a low number of live cells mixed
with a high number of heat-killed cells also
resulted in lower mortality and inoculum
decline
b. Density dependence may result from cooperative
protection factors, such as extracellular
polysaccharides (which can prevent dehydration)
or neighbor effects from other cells (i.e.
physical protection from growth in cell masses)
c. Airborne bacteria successfully colonizing the
phyloplane are often from plant foliage
1. Immigration of airborne bacteria to newly
emerged leaves is partially blocked by
adjacent leaves
a. This is one effect of leaf position on
microbial succession
2. Bacteria from different plant species
growing in close proximity to a given plant
can readily colonize that plant due to
airborne transmission
a. Seasonal increases in epiphytic bacteria
on navel orange leaves is due largely to
immigration from nearby plants having
high bacterial loads
4. Presence and density of competitors
a. Coexistence of epiphytes is inversely
correlated with similarity of carbon source
utilization patterns (ecological similarity)
5. Genetic recombination through conjugative plasmid
transfer and transduction
2. The marine phyllosphere
a. Emergent marine grasses
1. Standing dead stalks of the smooth cordgrass,
Spartina alterniflora, is host to a complex
community of eukaryotic algae, diatoms,
cyanobacteria, and bacteria, though the
composition of this phyllosphere community differs
in estuaries of differing turbidities
a. In estuaries having relatively low turbidity,
the phyllosphere microflora is dominated by
cyanobacteria, which are also responsible for
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most of the epiphytic nitrogen fixation
1. The composition of the cyanobacterial
assemblage changes over an annual cycle,
with heterocystous species (Calothrix spp.
and Nostoc spp.) dominating in the spring
and nonheterocystous species (Lyngbya spp.
and Phormidium spp.) dominating in the fall
2. Nitrogen fixation activity was localized in
the ephiphytic biomass, not in the
underlying stem material
b. In estuaries having high turbidity,
cyanobacteria are less important and
heterotrophic bacteria are the dominant
nitrogen fixing organisms
1. Analysis of recoverable nifH sequences
reflected a heterotrophic nitrogen fixing
assemblage dominated by α-Proteobacteria
b. Submerged seagrasses
1. The seagrass Halophila stipulacea supports a large
and very diverse epiphyte community
a. A clonal library containing 16S rDNA sequences
recovered from epiphytes by PCR amplification
yielded 103 sequences distinguishable by RFLP
methods
1. These clones fell into 58 different clusters
2. Only a few clones were sequenced; these
sequences were affiliated with a plastid-
like sequence recovered from marine snow and
with a marine Hyphomonas strain
B. The rhizosphere
1. The rhizoplane of the salt marsh cordgrass, Spartina
alterniflora, is heavily colonized by sulfate reducing
bacteria
a. These organisms have 16S rDNA sequences similar to
those of Desulfococcus and Desulfosarcina
b. Abundances of these organisms vary seasonally,
apparently in response to differing levels and/or
qualities of organic matter inputs during different
stages of plant ontogeny
c. The sulfate reducers are apparently resistant to or
capable of utilizing oxygen introduced through the
plant arenchyma
2. Roots of several freshwater and marine macrophytes
support dissimilatory iron reduction
a. Iron reduction rates on the roots of freshwater
plants are sufficient to account for substantial
organic matter turnover in the rhizosphere
1. The plants studied to date are from mildly
acidic, peat-rich environments
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b. Iron reduction on the roots of marine plants occurs
at much lower rates and may not be as significant to
organic matter turnover
1. Iron reduction was not inhibited by molybdate,
but this does not completely refute participation
by sulfate reducers since iron reduction by some
of these organisms is not molybdate sensitive
3. Bacterial cells on the rhizoplane are often aggregated,
with microcolonies readily apparent
III. Mutualism
A. Non-specific (non-obligatory) mutualism
1. The phyllosphere
2. The rhizosphere
a. The rhizosphere is defined as the subsurface plant
mass plus all surrounding soil/sediment detectably
affected by the plant
1. Major plant impacts on the rhizosphere microbiota
a. Organic enrichment from mucigel and soluble
exudates
b. Introduction of oxygen by plants with
aerenchymatous root tissue
1. Roots of salt marsh plant species raise
sediment redox potential even when
pronounced oxidation of the rhizosphere is
not visible
2. Oxygen introduction in the rhizosphere can
stimulate nitrification, followed by
denitrification after diffusion of NO3- into
anoxic sediment
3. Methane oxidizing bacteria are associated
with the rhizoplane and endorhizosphere of
rice and the freshwater marsh plants
Pontederia cordata (pickerel weed) and
Sparganium eurycarpum (bur reed)
a. When the root medium was oxic, methane
oxidation accounted for 88 and 63% of
total methane depletion for S. eurycarpum
and P. cardata, respectively, in split
chamber experiments
b. Methane oxidation under suboxic
conditions was not detected for S.
eurycarpum but accounted for 68% of
methane losses for P. cardata
1. During a suboxic incubation, dissolved
oxygen decreased by 19% in S.
eurycarpum chambers, but increased by
232% in P. cardata chambers, due to
the greater capacity of P. cardata to
oxygenate the rhizosphere
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2. Rhizosphere effects are strongly seasonal and vary
with plant type
a. Rice rhizosphere supports lower microbial
biomass during flowering
b. Mycorrhizal interactions
1. A wide variety of fungi form associations with
plant roots
a. The fungi tend to have very broad host ranges
and are fully capable of independent existence
1. Mycorrhizae are associated with about 90% of
all land plants
b. A total of seven types of mycorrhizal
interactions are known
c. Interaction between the fungi and the plant
root is apparently modulated by phenolic
compounds produced by the plant
d. Interactions are established best in soils
having low nutrient contents
e. Detection and identification of mycorrhizal
fungi is commonly performed using PCR
2. The commonest type of mycorrhiza is the vesicular-
arbuscular mycorrhiza (zygomycetes, about 80
species)
a. No obvious morphological changes occur in the
root, making this type of mycorrhiza hard to
recognize
b. The fungus grows between the root cortical
cells and also penetrates these cells
1. Hyphae that penetrate the cells of the
cortex form growths called arbuscules and
vesicles (swellings)
a. Arbuscules pass nutrients from the host
to the fungus until destroyed by host
anti-fungal compounds
2. No outer sheath is formed, but hyphae do
grow out into the soil from the root
a. Signature phospholipid fatty acids,
particularly 16:1ω5, can be used for
estimating fungal biomass in roots and
soils
1. This PLFA can be analyzed for both
structural lipids and storage lipids
to differentiate biomass of mycelium
and storage structures
c. Fungi that have formed vesicular-arbuscular
mycorrhizas become obligate symbiotes
1. They obtain their carbon and energy from the
host
d. Mycorrhizas are important in transfer of
inorganic nutrients (particularly phosphate)
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from the soil to plants and between plants
1. Fungal biomass decreases with increasing
soil phosphorus
a. High phosphorus levels can also inhibit
spore germination, hyphal growth, early
colonization of roots, and growth of the
extraradical mycelium
2. The mycorrhizal interaction also promotes
drought resistance in the host plant
3. Spore germination and hyphal growth during
colonization of new roots by mycorrhizas
appears to be stimulated by rhizosphere
bacteria
3. Ectomycorrhizas form a mantle or sheath around the
root
a. Hyphae grow into the root, surrounding the
cortical cells, but not penetrating them, and
extend out into the surrounding soil
b. The fungus releases growth hormones (fungal
auxins) that produce structural changes in the
root
1. Root hair growth is inhibited and the root
takes on a coral-like appearance
c. Several thousand different basidiomycetes form
ectomycorrhizas
1. Different fungi may form ectomycorrhizas on
different roots of the same tree without
competing with each other
d. Ectomycorrhizas protect the root from other
parasitic organisms and secrete antibiotics to
inhibit the growth of potential parasites
e. Phosphate travels much more quickly thorough
the fungal sheath than through bulk soil,
increasing the phosphate supply to the root
1. Sucrose passes from the tree to the fungus,
which converts it to carbohydrates the tree
cannot use
2. In the presence of high levels of nitrogen,
fungal auxin production is inhibited,
preventing the formation of ectomycorrhizae
f. Ectomycorrhizal geographical distribution
follows that of the host plants, but is also
heavily influenced by abiotic environmental
parameters
b. Non-specific mutualist and commensal rhizosphere
bacteria
1. A great variety of saprotrophic bacteria grow in
root-associated soils and on the rhizoplane
a. Growth of these bacteria is stimulated by root
exudates (and oxygen, in some cases), which
strongly influence the development and species
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composition of the rhizosphere microflora
1. Evidence for bacterial growth stimulation in
the rhizosphere
a. Enterobacter cloacae inoculated into
wheat plant microcosms was metabolically
active in the rhizosphere, but activity
was not detectable in bulk soil
2. Evidence for plant selection for specific
bacterial populations
a. Plant selection for specific populations
of fluorescent pseudomonads was
demonstrated in flax and tomato, and was
stronger in flax
3. Non-plant influences on rhizosphere
microflora
a. Active predation on saprotrophs by soil
nematodes has been demonstrated
b. Soil type exerts a major influence on
rhizosphere fluorescent pseudomonads;
apparently a stronger influence than the
plant itself
1. Rhizosphere assemblages are distinct
from those of uncultivated soil, but
also differ between plants of the same
type grown in different soils
b. Rhizosphere colonization by non-specific
mutualistic and commensalistic bacteria
1. Some rhizosphere bacteria adhere directly to
the root surface through the action of
specific agglutinins
2. Substantially competition for colonization
sites exists
a. E. cloacae introduced into non-sterile
soil wheat mesocosms was only able to
colonize the spermosphere (seed exterior)
1. In the absence of competition (sterile
soil), this organism colonized all
depths of the root
c. Fluorescent pseudomonad populations associated
with roots of flax and tomato are different
from those isolated from surrounding soil
2. Most of these bacteria probably have little impact
on growth of the plants, while others are
detrimental (pathogens) or helpful
a. Nitrogen fixing rhizosphere bacteria do appear
to enhance plant belowground productivity,
often without having much impact on aboveground
productivity
b. Disease suppression
1. Disease suppression depends on a combination
of traits, including at least some of the
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following:
a. Root colonization
b. Induction of resistance in the plant
1. Growth of harmless bacteria in the
rhizosphere can help to induce the
plant "hypersensitive" response,
increasing production of phenolics and
phytoalexins
c. Competition for nutrients, particularly
iron
d. At least some of disease suppressing
organisms produce antimicrobial
compounds, though these compounds do not
appear to confer any decisive advantage
in rhizosphere colonization
2. Some root-colonizing Pseudomonas strains
suppress plant diseases caused by soil
pathogens
3. The rhizospheres of aquatic plants can be sites of
elevated geochemical activity
a. Oxygen introduction into anoxic sediments by
roots of aerenchymatous plants creates an oxic-
anoxic interface in the root zone
b. Methane dynamics
1. Methane transport through aboveground plant
tissues can exceed that by diffusion across
the sediment/water or sediment/air
interfaces
2. Aquatic plant rhizospheres are enriched in
methane oxidizing bacteria and have elevated
levels of methane consumption
a. Type II methylotrophs are most abundant
in rhizospheres of the grass
Calamogrostis canadensis, though Type I
organisms are also found
b. Methane oxidizing bacteria are most
abundant and active in the rhizoplane of
rice (Oryza sativa) and are also found in
the endorhizosphere
c. Nitrogen dynamics
1. Rates of nitrogen fixation are elevated in
the rhizosphere relative to bulk sediment,
due to root exudation and (in anoxic
sediments) oxygen introduction
2. The release of oxygen into the rhizosphere
stimulates nitrification
3. Diffusion of nitrate into anoxic sediments
supports denitrification
B. Specific mutalisms
1. Endophytic bacteria in grasses
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a. A variety of nitrogen fixing bacterial species are
specific mutualists in plants
1. These include Azoarcus sp., Acetobacter
diazotrophicus and Herbaspirillum sp. found in
various tissues of rice, Kallar grass, sugar cane
and sorghum
a. A. diazotrophicus has also been recovered from
sweet potato stems and roots and from coffee
roots and rhizosphere soil
2. Most of these organisms do not survive well in and
cannot be isolated from root-free soil
1. Growth on the plant surface can occur, at least
with some species, and these endophytic
bacteria are relatively easily cultivated in
the laboratory
a. Obligate endophytes would neither grow on
plants or in artificial culture
b. These organisms spread systematically within plant
tissues, but there is no evidence for intracellular
growth in living plant cells
c. The bacteria are physiologically active and stimulate
plant growth, but their exact contribution(s) to the
plants are not completely understood
1. Azoarcus sp. have high levels of nif gene
expression in Kallar grass and rice
2. Rhizobium-legume interaction
a. Under appropriate conditions, Rhizobium can initiate
formation of nodules on the root of leguminous plants
1. This interaction is an example of symbiosis in
which both host and commensal benefit
b. The infection process
1. Different strains of Rhizobium have different host
specificities
a. These specificities are established by
interaction of complementary carbohydrates
b. In some cases, different Rhizobium species can
nodulate the same plant
1. The specific Rhizobium strain present in a
nodule can be determine through PCR-RFLP
approaches (see Molecular Biology notes)
2. The target plant carbohydrate is distributed in a
gradient on the plant root hairs, with the highest
concentrations on the root hair tip
a. Specific surface carbohydrates at one pole of
Rhizobium cells and the host plant root hairs
are immunologically cross-reactive, indicating
structural similarity
1. Cyclic -(1,2)-glucans may be involved in
attachment since mutants unable to produce
them are defective in their ability to
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attach
b. A glycoprotein, trifoliin A, acts as a lectin,
binding specifically to the cross-reactive
carbohydrates, forming a bridge between the
bacterial cell and the root hair surface
c. Ca2+ and Mg2+ are required for specific binding
of R. leguminosarum bv. phaseoli to the host
plant Phaseolus vulgaris and cause tighter
binding of the bacteria to the roots
3. The correct Rhizobium population is essential for
successful nodulation
a. The plant releases soluble nutrients which
allow the Rhizobium population to multiply
b. Rhizobium manifests a chemotactic response to
soluble compounds released by the plant
1. This response brings the cells into close
proximity to the root hair
c. Nodule formation
1. Flavonoids or isoflavonoids secreted by the host
plant induce expression of several nodulation
(nod) genes
a. The nod gene products are enzymes involved in
synthesis of the Nod factors, species-specific
substituted lipooligosaccharides
b. nod genes may be expressed prior to adsorption
of the rhizobia to the root
2. The Nod factors cause a specific deformation of
the root hair, curling, and division of
meristematic cells
a. The root hair curls around the bacterial cell
3. The bound bacterium (or possibly the plant)
produces polygalacturonidase, which allows
penetration of the root hair cell wall
a. Cell wall synthesis by the root hair is
redirected toward the site of infection
b. The new cell wall material is laid down in the
form of an infection thread, growing inwardly
toward the root cortex
c. The nucleus of the root hair doubles in size
and directs this process
d. The infection thread contains Rhizobium cells
lying end to end in a polysaccharide matrix
4. As the infection thread enters the cortex, some
cortex cells are stimulated to divide, forming
the nodules
a. The infection thread penetrates a cortex cell
and deposits Rhizobium cells into the cytoplasm
b. The intracellular bacteria, singly or in groups
are then surrounded by a peribacteroid membrane
d. The cells deposited by the infection thread undergo
radical changes in morphology, becoming bacteroids
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1. Some bacteroids (depending on strain and host) are
as much as 40x the size of the original rods
2. Bacteroids are nonmotile (Rhizobium is motile),
thin walled and pleomorphic
3. Free living Rhizobium is an obligate aerobe,
bacteroids are microaerophiles
4. The bacteroid chromosome is a truncated form of
the Rhizobium chromosome, incapable of supporting
independent cell division outside the plant
5. Bacteroides are very restricted in the carbon
sources they can utilize compared to free-living
Rhizobium
e. A direct product of the symbiosis is leghemoglobin
1. The globin chains are produced by the plant and
the heme groups by the bacteroid
2. Leghemoglobin binds and stores oxygen, maintaining
a low oxygen level in the nodule
a. The bacteroids require oxygen, but nitrogenase
is oxygen sensitive
f. In the active symbiosis, the plant provides energy
(ATP) for nitrogenase activity, and both carbon and
nitrogen sources to the bacteroids
1. The bacteroids provide the plant with inorganic
nitrogen (ammonium)
g. The Rhizobium interaction is not stable through
sexual reproduction of the plant, it has to be
reestablished in each seedling plant
2. Nitrogen fixation by symbiotic Frankia
a. Actinomycetes of the genus Frankia are capable of
interacting with non-legumes to form nitrogen fixing
nodules (actinorhizal nodules)
1. More than 200 species of woody plants in over 25
genera form nodules
a. Plants participating in actinorhizal symbioses
can grow on sites with limited nitrogen
availability and are highly successful pioneers
2. The host-symbiont interaction involving Frankia is
much less host specific than the Rhizobium
interaction with legumes, although some Frankia
strain specificity for the host is observed
a. Many Frankia strains are poorly characterized
and these organisms are generally difficult to
isolate into pure culture
b. The sequence of events leading to the formation of
actinorhizal nodules is as follows:
1. Lectin interaction allows Frankia filaments to
bind to root hairs
2. The root hair curls and an infection thread is
formed
3. The bacterial filaments grow through the plant
cortex and cortical cells divide rapidly to form
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nodules housing Frankia filaments
4. The bacteria invade the cortical cells and form
hyphae and spores
3. The Azolla-Anabaena azollae interaction
a. Azolla is an aquatic fern which forms a protective
cavity in each leaf, housing the nitrogen fixing
cyanobacterium Anabaena
1. The Azolla-Anabaena interaction is stable through
successive cycles of vegetative and sexual
reproduction
4. Azospirillum interactions
a. Azospirillum is a rhizosphere colonizer that enters
into mutualistic interactions with a variety of plant
species (often as an epiphyte, but sometimes
endophytically)
1. Types of mutualistic interactions
a. Nitrogen fixation by Azospirillum has been
reported by several investigators, but is still
controversial
b. Nitrate reduction and enhanced nitrate uptake
c. Production of plant growth hormones and the
resultant increases in root hair growth
d. Production of siderophores
1. Siderophore production by fluorescent
pseudomonads has been proposed to explain
disease suppresion by these organisms
e. Stimulation of nod gene inducer production in
legumes
1. The presence of Azospirillum enhances
nodulation of leguminous plants by
Rhizobium, due to increased root hair growth
(see above) and increased production of nod
gene inducers
2. Plant hosts and their colonization
a. Agriculturally significant host plants include
corn, wheat, and rice
b. Azospirillum is a nonspecific colonizer of
roots that can heavily colonize root mucigel
and penetrate the root cortex intercellular
spaces
c. Azospirillum species are actively chemotactic
and migrate rapidly through soil toward roots
and between plants when root-root contact
occurs
1. Aerial transport of Azospirillum has also
been reported
d. A variety of surface structures including polar
flagella, surface polysaccharides, and lectin-
like proteins have been implicated in root
colonization by Azospirillum
b. Azospirillum survives well in the rhizosphere but
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does not persist or travel well in most bulk soils
1. Removal of the host plant from soil results in
rapid decline in Azospirillum populations
2. Survival outside the rhizosphere is positively
correlated with soil clay, nitrogen, and organic
matter contents, and with soil water holding
capacity
a. The optimum pH for Azospirillum growth is
neutral to mildly acidic and this organism is
widespread in tropical soils
1. Survival is negatively correlated with soil
CaCO3 and sand contents
3. Characteristics of Azospirillum that may enhance
survival in soils
a. Formation of resistant cysts in temperate zone
soils and under conditions of water stress or
old age
b. In sandy soils, Azospirillum can form fibrillar
material that binds it to specific microniches
c. Azospirillum cells commonly contain large
quantities of poly--hydroxybutyrate and can
survive starvation in the laboratory for
extended periods
5. Azoarcus interactions
a. Azoarcus interacts with the roots of flood-tolerant
grasses, such as Kallar grass and rice
1. The organism penetrates the root cortex and into
the stele, spreading vertically into the plant
shoot
a. Vertical spreading may be by means of the xylem
vessels
b. This organism can sustain high rates of respiration
and nitrogen fixation at near anoxic conditions
(around 30 nM dissolved O2)
c. Members of the genus Azoarcus are phenotypically very
similar, but genotypically diverse
1. Azoarcus-specific primers have been developed,
allowing uncharacterized and uncultivated strains
to be studied
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