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Biotechnology of Biofertilization and Phytostimulation

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Title: Biotechnology of Biofertilization and Phytostimulation


1
Biotechnology of Biofertilization and
Phytostimulation
2
I. Problem Description
  • 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.

3
  • B. Plant Growth-Limiting Compounds
  • 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.

4
  • 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.

5
  • 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.

6
  • 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.

7
  • 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.

8
  • 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.

9
  • 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.

10
  • 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.

11
  • 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.

12
  • 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.

13
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.

14
  • 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.

15
  • B. Use of Specific Microorganisms
  • Bradyrhizobium and Rhizobium
  • 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.

16
  • 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 soybean-Bradyrhizobiu
    m.
  • The latter bacterium was previously known as R.
    japonicum.

17
  • 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.

18
  • 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.

19
  • 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.

20
  • 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.

21
  • 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.

22
  • 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
    iso-GA3 in cultures of A. lipoferum was
    demonstrated by gas chromatographymass
    spectroscopy (GCMS).

23
  • 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.

24
  • 3. Interaction of Azospirillum with the
    Rhizobium-Legume 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.

25
  • 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.

26
  • 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 14-37C.
  • 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.

27
  • 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.

28
  • 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.

29
  • 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.

30
  • 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.

31
  • 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.

32
  • 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.

33
  • 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.

34
  • 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.

35
  • 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
  • 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).

36
  • 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.

37
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.

38
  • 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.

39
  • Inoculation of plants with A. brasilense
    significantly enhanced (30-50 over controls) the
    uptake of H2PO-4 by maize in hydroponic systems
    and by 10-30 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.

40
D. Prospects of Microbial Fertilization of
Specific Major Crops
  • 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.

41
  • 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
  • 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.

42
  • Azoarcus is a slightly curved gram-negative,
    rod-shaped 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).

43
  • 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.

44
  • 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.

45
  • 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
    endorhizosphere-associated 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.

46
  • 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.

47
  • 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.

48
  • 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
    11 mixture of one mutant and the parental strain
    P. fluorescens WCS365 at day 0.

49
  • 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.

50
  • 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.

51
III. Conclusions, Future Directions, and Prospects
  • 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).

52
  • For inoculation to become more successful,
    several major bottlenecks have to be overcome
  • Results in the field and, to a lesser extent, in
    greenhouses are not always consistent.
  • Inoculant microbes tend to loose competition
    with the indigenous microflora.
  • Shelf life can be a problem, especially for
    nonsporulating bacteria 

53
  • 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
  • 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?

54
  • 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.

55
  • 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 5-20 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.

56
  • 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.

57
  • 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.

58
  • 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

59
  • C. Application of Biotechnology to Create Novel
    Combinations of Crops and Beneficial Microbes
  • During the last two decades many new
    nitrogen-fixing 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.

60
  • 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.

61
  • Alternatively, DNA fragments encoding the nif and
    fix genes should be transferred to and expressed
    in rice in such a way that the oxygen-sensitive
    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.

62
  • 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
    nitrogen-fixing bacteria in their associations
    with plants is not yet known, although they may
    exercise the same functions as the other
    endophytes.
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