Transcript Biotechnology of Biofertilization and Phytostimulation

Biotechnology of Biofertilization
and Phytostimulation
I. Problem Description
A. Economic Importance
• To sustain the world population in the year 2020 it
will be necessary according to United Nations
(UN) estimates from 1989, to increase agricultural
production by 100%.
• A clear relation has been established between the
increasing yields of cereals and the introduction of
high-yielding varieties, better pest control, and the
increase in fertilizer consumption (i.e., nitrogen,
phosphorus, potassium).
• 1 kg of fertilizer produces up to 10 kg of additional
cereal, at least for the initial fertilizer application.
B. Plant Growth-Limiting Compounds
1. Nitrogen
• The most common nutrient limiting the
production of agricultural crops is nitrogen.
• Plants can utilize nitrogen only in the combined
mineral form (fixed nitrogen), such as
ammonium (NH4) or nitrate (NO3).
• Up to the 19th century, crop yields obtained in
cultivated fields were generally low.
• For both more-developed and less-developed
countries, however, capital and energy costs of
production by the Haber-Bosch process have
become significant: 500-700 million US dollars
to establish a plant and approximately 20 billion
US dollars economic cost per year.
• The increasing demand for fixed nitrogen in
modern agriculture could be solved by the
enhancement and extension of plant growth
promotion and nitrogen fixation.
• Agriculturally important legumes are estimated
to account for about one-half (80 × 106 t/yr) of all
nitrogen fixed by biological systems.
• Although legumes have had a major role in food
production throughout history, the total world
area currently cultivated with these plants is
approximately 15% of the area used for cereal
and forage grasses, the main source of food in
the modern world.
• The production of meat, alcohol, and sugar
partly depends on the availability of cereal and
forage grasses.
• To obtain high crop yield, especially when using
highly productive cultivars, it is necessary to
apply nitrogenous, phosphorus, and potassium
fertilizer in larger amounts.
• For example, a crop of irrigated sweet corn (Zea
mays) is usually fertilized with 240 kg of
nitrogen per hectare (ha) to obtain a yield of
2025 t/ha of fresh grain; irrigated wheat
(Triticum( fertilized with 120 kg of nitrogen per
hectare yields 67 t/ha of grain.
2. Phosphorus
• Long-term fertilization has improved the
phosphorus status of much of Europe's arable
land to the extent that over large areas only
maintenance application is now required.
• In other parts of the world, phosphate deficiencies
are not uncommon.
• Phosphate is supplied to cropped land at
application rates ranging from a few kilograms
phosphorus per hectare to 35 kg/ha or more.
3. Potassium
• It is one of the three major crop nutrients, with an
essential role in physiological processes, such as
water uptake, osmotic regulation, photosynthesis,
and enzyme action.
• An adequate potassium supply is necessary for
ensuring crop resistance to disease, and drought.
• Much of soil potassium is present as part of
insoluble mineral particles and inaccessible to
plants.
• Only the slow process of weathering can liberate
such potassium.
• Fertilization is required to ensure that crops get a
sufficient supply of soluble potassium.
• Usual application rates for potassium are between
40 and 170 kg/ha.
• Potassium binds to the surface of clay particles:
this reduces leaching.
4. Water
• Crops must have adequate water supply to
utilize nutrients properly.
• Where growth is severely water-restricted,
fertilization is of limited value. Water and nutrient
management, therefore, are connected.
C. Use of Microbes for Fertilization and
Phytostimulation
• Various soil microorganisms that are capable of
exerting beneficial effects on plants or
antagonistic effects on plant pests and diseases
either in culture or in a protected environment
have a potential for use in agriculture and can
lead to increased yields of a wide variety of
crops.
• Microbial groups that affect plants by supplying
combined nitrogen include the symbiotic N2-fixing
rhizobia in legumes, actinomycetes in
nonleguminous trees, and blue-green algae in
symbiosis with water ferns.
• In addition, free-living nitrogen-fixing bacteria of the
genus Azospirillum affect the development and
function of grass and legume roots, thereby
improving mineral (NO3-1, PO33-, and K+) and water
uptake.
• Other microorganisms that are known to be
beneficial to plants are the phosphate solubilizers,
plant growth-promoting pseudomonads, and
mycorrhizal fungi.
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The use of these microorganisms is of economic
importance to modern agriculture as they can
replace costly mineral fertilizers and improve
water utilization, lowering production costs, and
reducing environmental pollution, while ensuring
high yields.
Technical problems involved in the successful
inoculation of agricultural crops include:
The delivery of sufficient inoculum to the target,
The economical production of large quantities of
microorganisms,
The promotion of extended shelf life, and
The development of convenient formulations.
D. Environmental Constraints
• In the more developed countries, fertilizer use is
inefficient.
• It is estimated that only 50% of the applied
nitrogen fertilizer is used by plants, with most of
the remainder lost by either denitrification or
leaching.
• The concentration of the toxic nitrate has
increased in water reservoirs in the vicinity of
heavily fertilized fields.
• Denitrification of nitrate produces about 90%
nitrogen gas and 10% nitrous oxide, the latter
being a greenhouse gas with energy reflectively
180 times that of carbon dioxide.
E. Political Decisions
• Until recently, subsidies and legislation in Europe
were designed to increase agricultural production,
assure farmers a fair income, and to keep food
prices at a reasonably low level.
• Today, food production in the Western World is at a
sufficient level.
• Moreover, the excessive use of chemicals has
resulted in health hazards (e.g., owing to leaching
of toxic NO3 into groundwater and volatilization of
N-oxides into the environment).
• The European Union has adopted its Common
Agricultural Policy with price cuts for key products,
incentives for a reduction in chemical input.
• Farmers are faced with environmental taxes and
the need to produce less yield per hectare.
II. Role of Biotechnology
A. Biotechnological Approaches
• Basically two kinds of approaches can be taken for
the application of microbial fertilizers or
phytostimulators.
• First, a large number of strains are screened on
selected crop plants under laboratory or greenhouse
conditions, (e.g., for their capabilities to improve
germination, seedling vigor, root elongation, root
branching, nitrogen fixation, and legume nodulation).
• Selected strains are further tested in pots in soil and
finally under field conditions.
• The best strain(s) will be developed into a
product.
• Bradyrhizobium and Rhizobium inoculants were
developed in this way.
• Another approach consists of trying to
understand why certain strains exert beneficial
effects.
• This understanding will provide notions for
improvement of strains and screening
procedures and of the inoculant production or
storage process.
• A clear advantage of the latter approach is that it
will result in qualitatively superior products.
• However, the disadvantage is that this approach
is so expensive that it is not feasible for most
agroindustrial products.
B. Use of Specific Microorganisms
1. Bradyrhizobium and Rhizobium
a. Mechanism: Biological nitrogen fixation (BNF)
accounts for 65% of the nitrogen currently used
in agriculture and will be increasingly important
in future crop productivity, especially for
sustainable systems, small-scale operations,
and marginal land utilization.
• Rhizobium and Bradyrhizobium bacteria are
responsible for most of the BNF.
• These bacteria are able to invade the roots of
their leguminous host plants, where they trigger
the formation of a nodule.
• In this organ the bacterium develops into a
differentiated form, the bacteroid, which is able
to convert atmospheric nitrogen into ammonia.
• The latter compound can be used by the host
plant as a nitrogen source.
• The host plant provides the bacteroid with
dicarboxylic acid carbon sources.
• This plant-bacterium symbiosis is host-specific in
the sense that on a particular host plant only one
or a limited number of rhizobia are able to
generate nitrogen-fixing nodules.
• For example, pea, vetch, and lentil can be
nodulated only by R. leguminosarum bv. viciae,
where as clover is nodulated by the very similar
R. leguminosarum bv. trifolii.
• Economically, the most important of these
symbioses is the combination soybeanBradyrhizobium.
• The latter bacterium was previously known as R.
japonicum.
b. Results.: Inoculants containing cells of
Bradyrhizobium or Rhizobium have been
commercially available for a century.
• Usually these preparations contain combinations of
three to five strains.
• The major problem with the application of such
inoculants is that only 5-20% of the nodules are
occupied by the inoculant bacteria, the remainder by
indigenous (brady)rhizobia, most of which fix less N2
than the selected inoculant strains.
• A possible breakthrough in this area has been
reached by Tikhonovich et al. who bred a new pea
cultivar that can be nodulated only by the efficient
nitrogen-fixing R. leguminosarum bv .viciae strain A1
and not by indigenous bacteria.
• The highly efficient combination of the novel pea
cultivar and R. leguminosarum strain A1 is presently
being commercialized.
• Knowledge of the molecular basis of symbiosis
has been used to increase the nitrogen-fixing
activity of inoculant R. meliloti, the guest
bacterium of alfalfa.
• The knowledge included the facts that nifA is a
major nitrogen-fixation regulatory gene and that
the genes dctABD are involved in dicarboxylate
transport from the plant to the bacteroid.
• After inoculation with modified R. meliloti
bacteria, which were provided with an extra copy
of both of these DNA fragments, the alfalfa
biomass was 12.9% higher than after inoculation
with the parental strain.
• Another study suggested that B. japonicum
inoculants may be improved by the addition of
other soil bacteria, predominantly pseudomonads,
which enhance B. japonicum-induced nodulation
and plant growth.
• The basis of this enhancement is unknown, but
biocontrol of pathogens or phytohormone
production are likely possibilities.
• Current inoculant formulations and applications
are adapted to the needs of the grower, especially
of soybean.
• Preparations of R. meliloti are available with a
constant shelf life over a 24-month period.
2. Azospirillum
• All azospirilla are nitrogen-fixing bacteria with
nitrogenase properties comparable with those of
other nitrogen fixers.
• It has been postulated that biological nitrogen
fixation by Azospirillum in association with roots
may contribute significant amounts of nitrogen to
the plant, thereby potentially saving valuable
nitrogen fertilizers.
• Greater nitrogen fixation activities were detected in
inoculated plants than in non-inoculated controls.
• Higher nitrogen fixation rates were detected near
or at flowering under conditions of high
temperature and soil moisture.
• The foregoing measurements have shown that
BNF by Azospririllum root associations in the
field contribute some nitrogen to summer
grasses and cereals (1-10 kg of nitrogen per
hectare), in itself a very positive phenomenon.
• Enhanced bacterial nifH induction was observed
in the presence of an additional carbon source
or when the oxygen tension was lowered to
microaerobic levels, indicating that both oxygen
and the availability of energy sources are
required.
• Despite their N2-fixing capability, the increase in
yield caused by Azospirillum inoculation is
mainly attributed to an improvement in root
development and thus increases in the rates of
water and mineral uptake.
• It is generally assumed that Azospirillum enhances
the root development by the production of plant
growth-promoting substances, such as auxins,
cytokinins, and gibberellins.
• Increases the number, diameter, and length of lateral
roots; enhances root hair appearance; and increases
root surface area.
• Phytohormone synthesis by Azospirillum is proposed
to influence the host root respiration rate and
metabolism and root proliferation, with a concomitant
mineral and water uptake in inoculated plants .
• Azospirillum is capable of producing indole-3-acetic
acid (IAA) by multiple IAA biosynthetic pathways.
• The production of gibberellin (GA), GA3 ,and isoGA3 in cultures of A. lipoferum was demonstrated by
gas chromatographymass spectroscopy (GCMS).
• It appears that the presence of Azospirillum in
the rhizosphere affects the metabolism of
endogenous phytohormones in the plant.
• By evaluating worldwide data accumulated over
the past 20 years on field inoculation
experiments with Azospirillum, it can be
concluded that this bacterium is capable of
promoting the yield of agriculturally important
crops in different soils and climatic regions,
using various strains of A. brasilense and A.
lipoferum and cultivars of different species of
plants.
• The picture emerging from the extensive data
reviewed is that of 60-70% successes with
statistically significant increases in yield in the
order of 5-30%.
3. Interaction of Azospirillum with the RhizobiumLegume Symbiosis
• Positive effects of combined inoculation with
Azospirillum and Rhizobium have been reported for
different legumes.
• A possible cause for this enhanced susceptibility of
the plants to Rhizobium infection following
Azospirillum inoculation could be the greater
number of epidermal cells that differentiate into
infectable root hairs.
• The effect that Azospirillum has on nodulation and
on the specific activity of nodule N2-fixation, leading
to growth promotion, may be attributed to the
following causes: early nodulation, an increase in
the total nodule number, and a general
improvement in mineral and water uptake by the
roots.
• So, Azospirillum exerts its effects through the host
plant, and not through direct interaction with
Rhizobium.
• Field inoculation with A. brasilense strain Cd
increased nodule dry weight (90%), plant-growth
parameters, and seed yield (99%) of naturally
nodulated Cicer arietinum L (chickpea).
• In Phaseolus vulgaris (common bean), inoculation
with R. etli TAL182 and R. tropici CIAT899
increased seed yield (13%), and combined
inoculation with Rhizobium and Azospirillum
resulted in a further increase (23%), whereas
plants inoculated with Azospirillum alone did not
differ in yield from uninoculated controls, despite a
relative increase in shoot dry weight.
• Azospirillum clearly promotes root hair formation in
seedling roots.
4 Azotobacter
• The nitrogen-fixing bacterium A. paspali has
been isolated only from the rhizosphere of
Paspalum notatum, a tetraploid subtropical
grass widely distributed in South America.
• Typically, N2 fixation occurs at pH 6.5-9.5,
growth at 14°-37°C.
• Oxygen is known to be a factor in influencing N2
fixation because high O2 concentrations
probably inactivate nitrogenase.
• Estimates of maximal nitrogenase activity were
obtained at Po2 of about 0.04 atm, on roots
removed from the soil and less than half of that
under anaerobic conditions or in air.
• Most of the activity was localized on the roots
and was not removed by vigorous washing in
water.
• Inoculum of A. paspali declined rapidly in
Brazilian soil, even in the rhizospheres of
Penicillium notatum, were it normally thrives
under natural conditions; decline was less rapid
in potting compost.
• A. paspali improved the growth of P. notatum by
fixing atmospheric N2 in the rhizosphere.
• Inoculation with 5 × 108 cfu ml-1 of A. paspali
under gnotobiotic conditions in petri dishes
increased root hair formation in canola roots 24
h after inoculation.
• It is reported that A. paspali fixed at least 11% of
the nitrogen utilized by P. notatum cv. Batatais
when the bacterium was under microaerobic
conditions.
• Maximal N2 fixation was obtained at a Po2 of
0.04 atm.
• Alternatively, large increase in plant growth was
obtained for a variety of dicotyledonous and
monocotyledonous plants growing in pots in
natural soil incubated with A. paspali.
• By adding inorganic nitrogen, they were able to
eliminate N2 fixation as a source of plant growth.
• They concluded that the plant growth promotion
was bacterially mediated by the production of
plant growth factors (indole acetic acid,
gibberellins, and cytokinins).
• Plant growth promotion was dependent on the
inoculum size, indicating that, for any given plant
growth condition, there is an optimal number of
A. paspali for a positive effect on the plant.
5. Mycorrhizae
• Mycorrhizae are fungi that are so closely connected
to the roots that they are considered an extension of
the root system.
• The vesicular-arbuscular mycorrhizal (VAM) fungi,
which are members of the class Zygomycetes ,
order Glomales ,form mycorrhizae with plant roots.
• The VA mycorrhizal fungi are obligate symbionts
and are not host-specific.
• They occur in about 80% of plants.
• The VA mycorrhizal fungi grow primarily inside the
root, but the network of extraradical fungal hyphae
form an extension of the effective root area of the
plant, which increases the absorption and
translocation of immobile nutrients.
• Most of the beneficial effects of VAM fungi are
related to increases in the effective root surface
area, thereby increasing the ion uptake of the
plant.
• Positive growth responses to mycorrhizal
development can be expected when the
concentration of some nutrient is extremely low
in the aqueous phase, but some solid or
unavailable form exists in reserve.
• Although it has been demonstrated that many
elements (e.g., P, S, Zn, Cu, Ca, N, K, Sr, and
Cl) can be taken up by mycorrhizal hyphae and
transported to the root, most experimental work
has been concerned with phosphorus uptake
and, to a lesser extent, nitrogen.
• Field inoculation of crop plants with VAM fungi is
very much dependent on field conditions.
• The potential for increasing plant growth and
yield by inoculation will very much depend on
the probability of natural inoculation by the
indigenous fungi and the level of available
nutrients, especially phosphorus.
• Other factors that influence successful field
inoculation will be the selection of the correct
fungal isolate for the crop host, and inoculum
type (e.g., spores, infected root pieces),
formulation, and placement.
• The VAM fungi are not considered to induce
typical defense responses in host plants.
• Nevertheless, transient increases in the activities of
the normal pathogen-response proteins chitinase
and peroxidase were detected in leek roots during
early stages of colonization by VAM fungi.
• Furthermore, soybean roots colonized by Glomus
mosseae or G. fasciculatus accumulated more of
the isoflavonoid phytoalexin glyceollin 1 than
nonmycorrhizal roots.
• Faba bean roots infected with G. intraradix
contained elevated levels of the nonflavonoid
acetylenic phytoalexin wyerone, but the amounts did
not reach those measured in host-pathogen
interactions.
• In alfalfa, during early colonization of plant roots by
G. intraradicis, isoflavonoid phytoalexin defense
response transcripts are induced and then,
subsequently, suppressed.
• Thus, although infection by mycorrhizal fungi
appears to initiate some plant defense
responses, these do not seem to reach their full
potential, which would probably have prevented
colonization.
• The role of flavonoids as signal molecules in the
establishment of the mycorrhizal plant is unclear,
but some flavonoids enhance germination and
hyphal growth of VAM fungi and promote VAM
fungal colonization of white clover roots.
• Likewise, both rhizobial nodulation factors, and
several of the flavonoids known to accumulate in
response to the nodulation factor, promoted
VAM colonization of soybean roots, suggesting a
flavonoid-mediated stimulation of mycorrhizal
colonization.
• Further indications that colonization of alfalfa
roots by mycorrhizal fungi affects flavonoid
metabolism is that nodule distribution on
mycorrhizal roots is significantly different from
that on nonmycorrhizal roots.
• Fungal colonization is limited when high
phosphorus concentrations are available.
• High phosphorus concentrations inhibit
intraradical fungal growth, possibly through
phosphorus-mediated physiological alterations
of the roots.
• Induction of plant defense genes may be one
factor in reducing colonization.
• Phosphorus, when applied to cucumber leaves,
induces the expression of chitinase and
peroxydase both locally and systemically.
6. Mycorrhization Helper Bacteria
• The symbiotic establishment of mycorrhizal fungi on
plant roots is affected especially by bacteria of the
rhizosphere.
• Some of these bacteria consistently promote
mycorrhizal development.
• This notion has led to the concept of mycorrhization
helper bacteria (MHBs).
• It seems likely that the use of MHBs can improve the
effect of mycorrhizal inocula.
• Garbaye has listed five possible explanations to
explain their activity:
1. MHBs may improve the receptivity of the root to
mycorrhizae formation (e.g., by producing auxin or by
producing plant cell wall-softening enzymes such as
endoglucanase, cellobiose hydrolase, pectate lyase,
and xylanase).
2. MHBs can interfere with the plant-fungus
recognition and attachment mechanisms,
which are the first steps of the interactive
process, leading to the symbiosis.
3. MHBs stimulates the growth of the fungus in its
saprophytic, pre-symbiotic stage in the
rhizosphere soil or on the root surface.
4. MHBs modify the rhizosphere soil (e.g., by
altering the pH or the complexation of ions).
5. MHBs trigger or accelerate the germination of
spores, sclerotia, or any other dominant
propagules specialized in the conservation and
dissemination of the fungus in the soil.
• An European consortium has been established
with the goal of testing these hypotheses,
thereby increasing the feasibility of inoculation
with the combination of mycorrhizaeMHBs.
C. Bacterial Stimulation of Water
and Phosphate Uptake
1. Water
• The abilities of plants to absorb both water and
mineral nutrients from the soil is related to their
capacity to develop extensive root systems.
• Plants are known to wilt more rapidly in water-logged
soils, as a result of decreased hydraulic conductance
in the roots.
• Inoculation of sorghum in the field with Azospirillum
led to 25-40% increase in hydraulic conductivity,
compared with the control.
• This could be explained by observed increases in the
total number and length of adventitious roots of
Sorghum bicolor, ranging from 33 to 40% over
inoculated controls.
2. Phosphate
• Phosphate deficiency can be diminished in crops by
utilization of bacteria that act directly as phosphate
solubilizers in the rhizosphere, indirectly by bacteria
that stimulate root activities; the root excreting
organic acids that help solubilize phosphate and at
the same time increase phosphate uptake, and by
mycorrhizal associations.
• Insoluble inorganic compounds of phosphorus are
largely unavailable to plants, but many
microorganisms can bring the phosphate into
solution.
• Species of Pseudomonas, Mycobacterium,
Micrococcus, Bacillus, and Flavobacterium are active
in the conversion.
• Not only do the microorganisms assimilate the
element, but they also make a large portion soluble.
• Inoculation of plants with A. brasilense significantly
enhanced (30-50% over controls) the uptake of
H2PO-4 by maize in hydroponic systems and by 1030% in the sorghum and wheat field.
• The increases in phosphorus uptake could be
derived in this case by increased root respiration.
• In inoculated plants, respiratory energy is the
driving force behind biosynthetic reactions and
transport processes.
• Maize root cell-free extracts from seedlings
inoculated with A. brasilense, at a concentration of
107 cfu/plant, contained elevated levels of enzymes
related to the tricarboxylic acid cycle, the glycolysis
pathway, and the breakdown of organic phosphate.
• Enzyme activity increases of 13-62% over the
uninoculated controls were observed.
D. Prospects of Microbial
Fertilization of Specific Major Crops
1. Rice
• Nitrogen is the key input required for rice
production.
• Super high-yielding rice genotypes with potential
grain yields of 13-15 t/ha require a nitrogen supply
of about 400-700 kg/ha.
• Over the past two and a half decades, rice farmers
have become increasingly dependent on chemical
fertilizers as a source of nitrogen.
• However, spiraling increasing costs, limited
availability and low-use efficiency demand an
increasing nitrogen supply aided by
microorganisms.
• It is in this context that BNF-derived nitrogen
assumes importance, because the submerged
soils on which more than 85% of the world's rice
is grown provide two of the most favorable
conditions for BNF: namely, optimum oxygen
tension and a constant and regular supply of
carbon substrate.
• Diazotrophs can be broadly divided into two
existing BNF systems:
(1)those that supply exogenous BNF, such as
phototrophic cyanobacteria in symbiosis with
Azolla, and heterotrophic and phototrophic
rhizobia in symbiosis with aquatic Sesbania and
Aeschynomene species;
(2) indigenous nitrogen-supplying diazotrophs,
including heterotrophic-phototrophic bacteria or
cyanobacteria in soil-plant-flood water.
• Azoarcus is a slightly curved gram-negative, rodshaped diazotroph isolated from the root interior
of Kallar grass.
• The cells fix nitrogen micro-aerobically, grow
well on salts of organic acids, but not on
carbohydrates, and on only a few amino acids.
• This bacterium is able to systematically infect
roots of both Kallar grass and rice.
• Nitrogen fixation by Azoarcus is extremely
efficient (i.e., specific nitrogenase activity was
one order of magnitude higher than values found
for bacteroids).
2. Sugarcane
• The BNF associated with this crop plays an
important role in its yield and the energy
balance.
• In Brazil, approximately 10 billion L of ethanol
are produced annually.
• This permits the replacement of 200,000 barrels
oil per day and, therefore, has a major influence
on the economy of the country.
• Although sugarcane accumulates large
quantities of nitrogen in its tissues (100-250 kg
ha-1 yr-1( , in Brazil the sugarcane crop rarely
responds to nitrogen-fertilizer application, even
when growing on soils with very low nitrogen
availability, and other crops, such as maize,
normally need considerable nitrogen fertilization.
• This observation stimulated researchers to
investigate this phenomenon and results
suggested that plant-associated BNF could be
playing an important role in nitrogen nutrition of
this crop.
• It was confirmed that the associative BNF can
contribute nitrogen at more than 150 kg ha-1 yr-1,
which can represent more than 60% of the total
nitrogen accumulated by the plants.
• A long-term experiment was shown that the total
nitrogen balance of the soil-plant system
indicated that the BNF contribution to the crop
was between 39 to 68 kg ha-1 yr-1 , which
represented up to 70% of the total nitrogen
accumulated by the plants.
• In the last decade, two new nitrogen-fixing genera
were identified and, because of their occurrence
principally within plant tissues, they have been
called endophytes ,instead of endorhizosphereassociated bacteria, a term used until recently for
root interior.
• Diazotrophic endophytes have an enormous
potential for use because of their ability to colonize
the entire plant interior and locate themselves
within niches protected from oxygen competition by
most other bacteria or other factors so that their
potential to fix nitrogen can be expressed at the
maximum level.
• These properties may be the reason for the high
nitrogen fixation observed in sugarcane plants.
• Among the endophytic diazotrophs found
associated with sugarcane are Acetobacter
diazotrophicus and Herbaspirillum seropedicae.
• A. diazotrophicus has been found mainly
associate with sugar-rich plants, such as
sugarcane, sweet potato, and Cameroon grass,
that propagate vegetatively.
• In addition, it was recently isolated from coffee
plants in Mexico.
• The species H. seropedicae is much less
restricted than A. diazotrophicus because it has
been isolated from many other graminaceous
plants, including oil palm trees and fruit plants and
seems to be transferred mainly through the seeds.
E. Colonization
• To function as a biofertilizer or as a phytostimulator,
a microbe must be present at the right site and the
right time at the place of action.
• This process, called colonization, can be considered
as the delivery system of the microbe's beneficial
factor(s).
• It is shown that the presence of flagella is required
for efficient potato root colonization by
Pseudomonas fluorescens biocontrol strain
WCS374.
• The role of flagella in colonization may be due to
their function in chemotaxis towards root exudate
nutrients.
• It is shown that Azospirillum mutants impaired in
motility and chemotaxis exhibit a strongly reduced
wheat root colonization ability.
• The second factor shown to play a role in
rhizosphere colonization of potato is the O-antigen
of the bacterial cell surface component
lipopolysaccharide (LPS).
• More recently, a gnotobiotic system was developed
to screen random transposon mutants for their
ability to colonize the 7-day-old tomato root tip after
inoculation of germinated seedlings with a 1:1
mixture of one mutant and the parental strain P.
fluorescens WCS365 at day 0.
• The results showed that mutants unable to
produce amino acids or vitamin B1 are defective
in root tip colonization and, also, when applied
alone.
• Apparently, the root produces insufficient
amounts of these compounds to allow normal
growth of the mutant cells.
• Another factor that was correlated with efficient
colonization was growth rate.
• Several poorly colonizing mutants appeared to
grow more slowly, as tested in laboratory media,
than the parental strain, suggesting a causal
relation.
• Root colonization also depends on growth on
major exudate carbon sources
• Interestingly, most of the mutants that appeared
to be defective in tomato root colonization are
nonmotile, lack the O-antigen of LPS, are poor
growers, or are auxotrophic, confirming the
results previously mentioned.
• It is concluded that pseudomonads sense a
stimulus (from the plant?) which, through the
two-component system, activates a bacterial trait
that is crucial for colonization.
III. Conclusions, Future Directions,
and Prospects
A. Present Situation
• Inoculation of crops with microbial fertilizers or
phytostimulators has been successfully
applied.
• The best example is (Brady)rhizobium, which
has been sold for a century as an inoculant.
• A recent example is that the Dutch seed firm S
& G Seeds BV sells radish seeds only in the
form of seeds coated with Pseudomonas
biocontrol bacteria (i.e., the product Biocoat).
• For inoculation to become more successful,
several major bottlenecks have to be
overcome:
1. Results in the field and, to a lesser extent, in
greenhouses are not always consistent.
2. Inoculant microbes tend to loose competition
with the indigenous microflora.
3. Shelf life can be a problem, especially for
nonsporulating bacteria
B. Application of Biotechnology to Improve
Microbial Inoculants
• The major bottlenecks for the successful
application of inoculant microbes is our poor
understanding of which bacterial traits are involved
in the beneficial effects of inoculant bacteria ,and
how these traits are influenced by environmental
factors. Several topics that require further
elucidation:
1. Molecular Rhizosphere Physiology
• The challenges for the next decades is to
understand how microbes grow and survive in situ.
• What factors are limiting growth in the rhizosphere
(e.g., nutrient limitation, toxic products)?
• What is the influence of abiotic factors, such as
temperature and drought?
• Which genes are specifically expressed under
rhizosphere conditions?
• Which of these have essential functions in
growth and survival?
• Can these genes be used to increase the rate of
appearance of bacterial beneficial activity
immediately after rehydration of planted seeds
or for increasing the shelf life of coated seeds?
2. Colonization and Beneficial Traits
• For optimization of the success of inoculation it
is essential to understand which traits are
involved in these processes and how their
expression is influenced by environmental
factors.
3. Competition of Inoculant Microbes with
Indigenous Biotic Factors
• Several results indicate that the success of
inoculation severely suffers from competition of
inoculant microbes by biotic factors.
• Soybean nodules resulting from seeds treated
with inoculant Bradyrhizobium are, in only 520% of the cases, occupied by inoculant
bacteria.
• The colonization of wheat roots be P.
fluorescens strain WCS365 is over 100-fold
inhibited by field soil in comparison with the use
of X-ray-irradiated field soil.
4. Better Inoculant Strains
• Once the mechanisms of action of beneficial
bacteria are known, screening specifically aimed
at such a mechanism can be developed.
• Similarly, once we understand more of the
influence of environmental factors, we can
screen for better strains by choosing better sites
for sampling the bacteria.
• Considering that only about 1% of the soil
bacteria can be cultivated, progress can also be
expected in the cultivation of bacteria that so far
could not be cultivated, and the subsequent
screening for beneficial isolates.
• The performance of inoculant bacteria can be
improved by genetic modification.
5. Endophytes
• Some bacteria exert their beneficial effect inside
the plant (e.g ,.Rhizobium and Azoarcus(
• Up to the moment of internalization, endophytes
are subject to the same competitive factors as
other bacteria.
• However, once inside, the competition from
other bacteria is absent or strongly decreased.
• Therefore, the use of endophytes for inoculation
may be advantageous.
6. Environmental Factors
• An understanding of these factors is of crucial
importance.
• Fundamental research can reveal some of the
important factors, as illustrated by the following
example.
• In cases where sugarcane plants respond to
nitrogen fertilizer application, the same response
has sometimes been observed by substituting
the nitrogen fertilizer by molybdenum
application, which may be because this
micronutrient is essential for the synthesis and
activity of nitrogenase
C. Application of Biotechnology to Create
Novel Combinations of Crops and Beneficial
Microbes
• During the last two decades many new nitrogenfixing bacteria have been isolated and identified,
including species of the genera Azospirillum,
Herbaspirillum, Acetobacter, and Azoarcus.
• The greater part of these diazotrophs have been
isolated from tropical regions, especially in
Brazil, and they have been the main source for
groups in the world working with associative
diazotrophs.
• Other associative nitrogen-fixing bacteria have
been identified, but probably because of their
low number, or restricted occurrence, they are
not well explored.
• The interest in the association of diazotrophs
with graminaceous plants reinforces the
importance of the biological nitrogen fixation
process to sustainable agriculture systems
where low inputs of nitrogen fertilizers are
desirable.
• Rice suffers from the mismatch of its nitrogen
demand and its nitrogen supply.
• Fertilizer nitrogen in flooded rice soil is highly
prone to loss through ammonia volatilization.
• The possibility of nitrogen-fixing rice has been
discussed for a long time.
• An old dream is to engineer rice to establish a
symbiosis with Rhizobium.
• Alternatively, DNA fragments encoding the nif
and fix genes should be transferred to and
expressed in rice in such a way that the oxygensensitive nitrogenase complex would be
protected from oxygen.
• Moreover, it is very likely that we do not know all
factors required to create a nodulating rice plant.
• Rather, the recent success obtained with
Azoarcus in nitrogen fertilization of rice makes
the latter approach a much more promising
alternative.
• Herbaspirillium rubrisubalbicans and
Burkholderia spp. are also nitrogen-fixing
endophytic bacteria found in association with
sugarcane, cereals, and other plants of
agronomic importance.
• H. rubrisubalbicans was recently identified among
Pseudomonas rubrisubalbicans strains, a species
considered a mild phytopathogenic agent caused
mottle stripe disease in some susceptible varieties
of sugarcane grown in countries other than Brazil.
• Although it was thought that H. rubrisubalbicans
would be restricted to sugarcane, it was recently
isolated from rice plants and fruit plants.
• Burkholderia, a novel nitrogen-fixing bacterium,
has been isolated from several plants, including
sugarcane, sweet potato, cassava, cereals, and
more recently, fruit plants.
• The role of these two new endophytic nitrogenfixing bacteria in their associations with plants is
not yet known, although they may exercise the
same functions as the other endophytes.