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 Compounds1. 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.

• 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:

1. The delivery of sufficient inoculum to the target,

2. The economical production of large quantities of microorganisms,

3. The promotion of extended shelf life, and

4. 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 Microorganisms1. Bradyrhizobium and Rhizobiuma. 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 soybean-Bradyrhizobium.

• 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 iso-GA3 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 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.

• 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 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.

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, 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).

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 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.

• 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 non-motile, 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 non-sporulating 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 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.

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 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.

• 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 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.

• 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.