I The use of micro-organisms in agriculture
Biofertilizers: Present and future use of transgenic micro-organisms
Luis Gabriel Wall
Laboratory of Biochemistry, Microbiology and Biological Interactions in Soil (LBMIBS), Department of Science and Technology, University of Quilmes, Bernal, Argentina
Biofertilizers are living microbial preparations which enhance or promote plant growth, relatively to a control without inoculation. A huge amount of research literature has been produced in the last 20 years concerning plant growth-promoting rhizobacteria (PGPR) related subjects, describing different micro-organisms acting on different plants, and proposing different mechanisms to explain the plant growth promotion effect. However, we still do not know which of the different in vitro mechanisms of biofertilizer action are responsible for the positive effects in the field. Biofertilizer technology has significantly developed in the market. The nature of multiple mechanisms discovered for PGPR actions and the possibility of genetically modifying a particular strain concerning a particular plant growth-promoting activity suggest that the use of genetically modified organisms such as biofertilizers will be an area of multiple and diverse possibilities of action in the near future. The study of the microbial ecology of this scenario and its dynamics will certainly improve the development of biofertilizer technology for the future of agriculture.
One of the big challenges for the future of humanity is to produce enough food in a sustainable way. Another big challenge is to produce bio-fuels to replace those non-renewable types of fuels for which resources will be exhausted some day. Crop plants play a key role for solving both of these challenges. Besides water, crop production is limited by the availability of the main nutrients in soil, such as nitrogen (N) and phosphorus (P). Soil micro-organisms are key elements in biogeochemical cycles of elements on our planet (Buscot and Varma, 2010).
From an evolutionary point of view, many plant/micro-organisms interactions have been selected which produced mutual benefits for the interacting organisms. Plants are primary biomass producers through photosynthesis and those photosynthates can be partially released into the soil via root exudates or via root and/or plant debris degradation. In this way, soil organic matter is increased and can be used by heterotrophic organisms as substrates to grow. It is reasonable to think that some microbes have succeeded in the history of evolution because of their capacities to improve plant growth, to assure their own source of food or substrates needed to grow. Most of these kinds of microbes live in the rhizosphere, the part of the soil which is influenced by the release of substances from the plant (Dessaux et al., 2010).
From a utilitarian point of view, these kinds of micro-organisms can be used to improve plant growth to assure food production. If these micro-organisms facilitate plant nutrition, their action would be valuable in terms of sustainability of the processes, because it would diminish the need for chemical fertilizers, whose production depends on non-renewable energy sources.
Taking all of these ideas into account, biofertilizers are defined as industrial products based on culturable micro-organisms that were isolated from the soil or rhizosphere of plants and which have been proven capable of modifying, and improving, plant development through a collection of different mechanisms of action. A product is characterised as a biofertilizer following an experimental test where the behaviour of a plant inoculated with a suspension containing a huge amount of cells of a particular micro-organism is compared to a control situation where the plant grows without the addition of this micro-organism. This experimental test can be performed either in vitro or in vivo. In vitro means growing plants hydroponically or using a controlled substrate, in pots in growth chambers or in greenhouses. In vivo means that the test is performed in soil, either in greenhouses or in field conditions. In vivo results may differ from in vitro results because the microbial background is different, and it is almost impossible to verify and compare the microbial background in soils with experimental in vitro conditions, simply because we still do not know precisely how to characterise the total microbial diversity existing in soil. Culturable micro-organisms are about 1% of the total existing micro-organisms in soil (Staley and Kanopa, 1985; Torsvik and Ovreas, 2002). So, regardless of the characterisation result of a micro-organism as a biofertilizer after different in vitro tests, the biofertilizer activity should be proven in soil, in field conditions, because the plant microbe interaction must function in the presence, and influence, of the huge diversity of other micro-organisms living in soil.
There are different modes of interactions between biofertilizers and plants (Gray and Smith, 2005), considering the degree of association between micro-organisms with plant roots in a gradient of root proximity and intimacy as follows:
- 1. micro-organisms living in the soil near the root, utilising nitrogen and carbon metabolites leaking from the root (rhizosphere)
- 2. micro-organisms colonising the rhizoplane (root surface)
- 3. micro-organisms colonising the root tissue inhabiting intercellular spaces (endophytes)
- 4. micro-organisms living inside cells in specialised root structures or nodules (symbionts).
Cases 1-3 do not induce any particular root structure and the micro-organisms are considered to be in a looser associative interaction with the plant compared to a more complex integrated association including the development of specialised root structures as in the last case of symbiotic associations. In all cases, biofertilizers should reach and colonise the rhizosphere to act on the plant through interaction with its root. It has been shown that root colonisation is part of the mechanism needed to produce plant growth promotion (Lugtenberg and Kamilova, 2009).
Besides the degree of association with the root tissue, at least two different modes of action can be recognised in biofertilizers’ activity: direct and indirect mechanisms. Direct mechanisms imply the supply of a nutrient or the release of microbial substances which enhance plant growth or development. Indirect mechanisms are those which suppress or inhibit a deleterious situation for regular plant development, as for instance, a disease caused by a pathogen (Vessey, 2003; Glick et al., 1999).
For study purposes and organisation of the available knowledge on this subject, considering the different scenarios of plant-microbe interactions and the different mechanisms of plant growth promotion, different definitions are used to organise the concept of biofertilizer:
- 1. Free-living/non-symbiotic micro-organisms considered to be plant growth-promoting rhizobacteria (PGPR) (Kloepper and Schroth, 1978). These biofertilizers are also considered extracellular (Gray and Smith, 2005). To clarify different kinds of PGPR, different authors have proposed different definitions which try to separate them according to their mode of action:
- - Plant growth-promoting bacteria (PGPB). These microbes promote direct plant growth by enhancing different mineral nutrition or by regulating plant development implying a phytohormone-like way (Bashan and de-Bashan, 2010; Verma et al., 2010).
- - Biocontrol PGPB. These microbes are mainly antagonists to different plant pathogens. They improve indirectly plant growth by releasing the disease state of the plant (Bashan and Holguin, 1997). This particular group of micro-organisms has received some attention in the last years because of their economic implications (see Chapter 2).
- - Plant stress homeostasis-regulating bacteria (PSHB). This group of micro-organisms was quite recently proposed to highlight cases were the plant growth promotion takes place within an abiotic stress condition (i.e. water stress, salt stress) (Cassan et al., 2009; Bashan and de-Bashan, 2010)
- 2. Micro-symbionts or intracellular plant growth-promoting micro-organisms, as defined by Gray and Smith (2005). These associations are more visible in the plant because they induce specialised root structures where the plant microbe interaction takes place. All of these interactions are at least important for a main nutrient supply for the plant (nitrogen or phosphorus), but other positive concurrent mechanisms for plant growth promotion cannot be disregarded as part of the activity of these micro-organisms:
- - N2-fixing rhizobacteria:
- ? rhizobia-legume simbiosis
- ? Frankia-actinorhizal plant simbiosis.
- - Micorriza fungi:
- ? arbuscular mycorriza fungi (AMF)
- ? ectomycorrhyza fungi (EMF).
- - N2-fixing rhizobacteria:
What is new about this since the previous OECD consensus document dedicated to biofertilizers, published in 1995? Back then, 48 out of 51 pages referred to symbiotic biofertilizers, describing mostly rhizobia, Frankia and mycorrhiza, which are symbiotic cases. The remaining three pages of the document referred to free-living micro-organisms as “future biofertilizers”. Since 1995, many articles have been published related to these new groups of micro-organisms generally named or referred to as PGPR. Different mechanisms of action have been described and are still a matter of intensive research. Most of this knowledge has been recently reviewed (Glick et al., 1999; Vessey, 2003; Bashan et al., 2004; Gray and Smith, 2005; Barriuso et al., 2008; Lugtenberg and Kamilova, 2009; Verma et al., 2010; Bashan and de-Bashan, 2010).