Abstracts 2003 : Scroll down the page to read all the abstracts from the 2003 Inoculant Forum or use the Index below to find a specific abstract. A rhizosphere perspective on inoculant
efficacy Email: Clapperton@agr.gc.ca Abstract The presence of the root defines the rhizosphere, while the intimate interactions between the plant and soil biota within the soil habitat characterise the rhizosphere. Root exudates are the substrate or fuel for the intense microbial (bacteria, fungi, algae, protozoa, nematodes and arthropods) activity within the rhizosphere. Thus, it is the quantity and quality of the exudates and condition of the soil habitat will determine the colonisation potential of the rhizosphere. Each plant species leaks a unique carbon and nitrogen signature of carbohydrates, amino acids and organic acids that determines the primary colonisers of the microbial community. The composition of the exudates also affects the availability of nutrients in the immediate vicinity of the roots influencing the establishment and growth of the plant. An increase or decrease in the nutritional status of the plant can further alter the quality of the exudates affecting the microbial diversity and populations in the rhizosphere. Root biomass and architecture, fertiliser type, tillage and cropping history also affect root growth and patterns of exudation and the populations and diversity or rhizosphere colonising organisms. Together, these factors determine whether rhizosphere interactions and processes will have a positive, neutral or beneficial affect on plant growth. Microbial inoculants placed with or in the immediate proximity of the seed must be able to successfully establish and multiply along the growing root, and integrate into the rhizosphere community, or alter and/or overwhelm the community to give the desired growth effect. The diversity of crops in a rotation, perhaps even the sequence of crops in a rotation not to mention the cultivar will affect the efficacy of an inoculant regardless of the crop and soil management techniques. We suggest the greatest success will be achieved in developing inoculants that contain cool temperature competitive strains, and a diversity of species that are more tailored to a particular crop species or type. Technical considerations aside, one size fits no one. There is also a need to work with plant breeders to develop appropriate inoculants, that work in synergy with the crop traits, particularly rooting. Regional Report on Inoculant Research
- Alberta 1Lacombe Research Centre, 6000 C & E
Calgary Trail, Lacombe, Alberta, Canada T4L 1W1 Contact: claytong@agr.gc.ca, lupwayin@agr.gc.ca, and ricew@agr.gc.ca Abstract Legume inoculation provides suitable rhizobia in the rhizosphere at the time of nodule initiation. Large numbers of rhizobia on the seed or in the soil favor survival before planting, which in turn promotes rhizobia multiplication in the rhizosphere and early nodulation. Inoculant research in Alberta conducted over the past five years includes: Inoculant delivery systems, granular inoculant rate and placement of granules, granular inoculant mixtures with fertilizer at various periods of time in the mixture, granular inoculant rate and carrier type (peat granule vs. clay), inoculant strain and cultivar interactions and the effects of rhizobia inoculation on endophytic bacteria in subsequent crops. AAFC researchers at Lacombe (Clayton) and Beaverlodge (Lupwayi)have conducted most of the above-mentioned trials. Others that have worked with inoculant in field pea systems include Alberta Agriculture, Food and Rural Development (AAFRD) that studied the yield potential and constraints of field pea production. There has been some industry activity that have put out some small plot trials occasionally such as Agrium and Agricore that mostly looked at formulations as well as placement to some degree. Currently the research activity to study inoculants in legume production have decreased. Industry has been developing and studying inoculant efficacy in private trials. Phosphate inoculants – prairies
and northern plains Abstract Extensive research conducted with phosphate-solubilizing microorganisms by researchers around the world since the 1940’s has resulted in very few commercial products. One reason for the lack of commercial applications from much of this research is the lack of consistent and demonstrated efficacy relevant to farmer’s needs and cropping practices. Farmers in the North American northern prairies have used phosphate (P) inoculant products based on the soil fungus Penicillium bilaiae since 1991. Researchers at the AAFC research station in Lethbridge isolated the fungus in 1981. Hundreds of small plot field research trials have been conducted by public and private institutions with P. bilaiae on a wide range of crop plants across the North American northern prairies since 1985. In general, the fungus increased crop vegetative growth, P uptake and grain yields when P was limiting to crop growth in these small plot field research trials. Inoculation with P. bilaiae was also shown to be compatible with various rhizobia species and mycorrhizae. Over 400 controlled farmer applied split-field trials have also been conducted with P bilaiae across the northern prairies since 1988. Inoculation with P bilaiae increased average grain yields by 7.6% with a 95% confidence limit of 1.2% in these split-field trials. Phosphate inoculants based on the fungus P. bilaii consistently increase the availability of phosphate to farmer’s crops in the North American northern prairies as evidenced by increases in P uptake and grain yield in P responsive conditions. BREEDING FOR BETTER NITROGEN FIXATION IN
GRAIN LEGUMES: HOW DOES RHIZOBIUM FIT IN? 1 University of Minnesota, St Paul,
MN, USA Abstract Nitrogen (N 2) fixation by leguminous plants accounts for almost 50% of the nitrogen used in agriculture, but must increase further as the world’s population surges toward 10 billion by the year 2040. Giller (2001) suggests that rates of 1-2 kg N 2 fixed ha -1 growing season day -1 are possible in all legumes, but current rates are clearly less than that, and in some countries may even be declining. Improvement in N 2 fixation by grain legumes will require genetic improvement in the host, understanding of host-Rhizobium interaction and careful strain selection, and more attention to environmental stress effects. Most of the elements needed for breeding programs to improve N 2 fixation in grain legumes are already established. Genetic variation in nodulation and N 2 fixation has been identified in almost every legume studied to date; heritability estimates for traits associated with improved nodulation and N 2 fixation are all adequate for gains in N 2 fixation through plant breeding. A variety of breeding approaches have been tested with success, and initial progeny evaluation can be simply undertaken. Despite this, few breeding programs include N 2 fixation as a routine activity, and almost none attempt to pyramid genes for desirable N 2 fixation traits in legumes of interest. Only in Brazil are such activities given the attention they warrant. The result is that soybean in Brazil currently yields an average of 2567 kg ha -1, but still derives 69 to 94% of its nitrogen needs from fixation. For soybean alone, the resultant saving in fertilizer costs is estimated at $ US 1.95 billion annually, and is a significant factor in reducing production costs. By comparison, corn/soybean rotations in the midwest USA are said to be unsustainable. However, even in Brazil, recently developed soybean cultivars appear to have declined somewhat in N 2 fixation, and only recently have marker systems been identified that could help in pyramiding desirable traits. Where do Rhizobium fit into this picture? Clearly, breeding activities should be undertaken using inoculated plants, and with gains from selection for N 2 fixation distinguished from those due to improved N harvest index. Rhizobium strain selection for modern-day grain legumes must also be revisited, and host/strain interaction, in particular examined. Such interactions may derive from multiple domestication events or differences in germplasm base. Host and rhizobia need to be matched, with as much emphasis given host/strain compatibility, and soil and rhizosphere colonization, as has previously been given to “strain competitiveness” alone. Much greater emphasis also needs to be given host and strain selection for conditions of stress, particularly pH. Inoculant Research in the US – Northern
Great Plains Soybean, field pea, lentil, chickpea, and alfalfa are the principal legumes grown in the states of Montana (MT), South Dakota (SD), and North Dakota (ND). Inoculant research focuses on evaluating field responses to nitrogen-fixing bacteria. Current projects in soybean address inoculation rates (MT, ND), inoculant x seed treatment compatibility (SD), inoculant formulation and placement (ND), and comparison of commercial and experimental inoculants (ND). In North Dakota, an annual comparison of commercial field pea inoculants is conducted and a multi-site project to study inoculant x seed treatment compatibility will begin in 2003. A multi-site trial in 1999-2000 evaluated the response of field pea to inoculant formulations and to N and P fertilizer levels, formulations, and placement. Dry bean and chickpea inoculation trials have been conducted, but were discontinued. Nitrogen “Credit”ability in the Northern Great Plains Garry Hnatowich, Senior Research Agronomist, Philom Bios Abstract One of the most frequently posed questions by producers, involved in legume production in the northern Great Plains, is “How much nitrogen will this crop contribute to my subsequent crop?” The premise behind this query is a desire to quantify the “nitrogen credit or fertilizer nitrogen replacement value” attributed to the pulse crop in rotation. The general assumption being that nitrogen fixation provides, or contributes, a considerably net contribution of plant available nitrogen to subsequent non-legume crops. A general belief exists in the mind of producers that, during the year of production, a legume crop is contributing significant nitrogen benefit to the residual soil reserve. Growers witness that pulse residue decomposes quicker than cereal or oilseed residue, and intuitively recognize, this as a reflection of the lower C:N ratios attributed to pulse crops. They further recognize, and benefit, from rotating legumes with a non-legume crop with reduced nitrogen fertilizer inputs. Provincial/State recommendations reflect a nitrogen credit to pulse residue, typically ranging in value between 0 – 40kg N/ha, depending upon the legume crop and yield produced. Soil testing laboratories reflect, but differ, in their fertilizer nitrogen recommendations of the legume in rotation towards the following non-fixing crop. The purpose of this pressention is to explore the nitrogen and non-nitrogen benefits attributed to pulse production. Are nitrogen credits warranted? Do they have merit and how do we define and attribute non-nitrogen related benefits of legumes in rotation? The role of genetics in selection of rhizobial
inoculant strains Abstract Molecular genetic approaches have yielded an enormous amount of information about the interactions of rhizobia with their legume hosts, to the extent that we now have a very good understanding of how nodulation takes place, and how nitrogen fixation inside legume nodules is regulated. However, there have not been corresponding advances in the understanding of, and improvement of,. inoculant strain performance in the field. Some of the improvements that are desirable in inoculant strains include enhanced nitrogen fixation ability, greater competitiveness, applicability of strains to a broad range of host cultivars, soil conditions, and climates, and (possibly) good survival in the soil. The two basic approaches that can be taken to develop better inoculant strains are strain selection and genetic engineering. While genetic engineering of strains that fix more nitrogen and are capable of competing better in the root environment has been shown to be quite feasible, there is potentially a problem with consumer acceptance of such products. Thus it may in the short term be more practical to look for ways of speeding up selection of suitable inoculant strains. Laboratory studies using molecular techniques may provide a short-cut to obtaining information that will allow rational selection of inoculant strains. Strain identification using economical methods is an important aspect of such a selection program. Genetic tools can also be employed to identify characteristics that contribute to inoculant performance and to develop screens for these attributes. My presentation will focus on various strain identification techniques such as plasmid profiling, RAPD and RFLP studies, as well as on identifying factors that might contribute to strain competitiveness. The roles of catabolism of specific carbon compounds, of motility and chemotaxis, and of production of antibiotics and bacteriocins will be discussed. I will also address simple strategies that might be used to select for better strains by using different cultivation techniques and different assays of strain performance. Compost as an Alternative Carrier for Microbial
Inoculants Abstract Pulse crop production in the Canadian Prairies, and especially in Saskatchewan, continues to increase in importance. For example, it is estimated that the Saskatchewan pulse crop could increase by almost 3-fold (to 8 million acres) during the next 15 to 20 years (Saskatchewan Pulse Growers, 1999). Likewise, tame forage production (which includes the legumes alfalfa and sweet and red clovers) has been steadily increasing (42% during the period from 1991-97 relative to the previous five-year period) and is expected to increase further as producers look for alternative ways to diversify production (SAF, 1996). Given that many of the soils in western Canada do not contain sufficient numbers of native rhizobia to establish effective nodulation of pulse crops (Hynes et al.,1995), it is no surprise that there is a significant demand for high quality rhizobial inoculants. Traditionally, peat has been the carrier of choice for rhizobial inoculants and, though several other formulations are now commercially available, peat is still considered to be the most dependable carrier and is the standard by which other carriers are judged. Despite its many attractive characteristics (e.g., high water holding capacity, high buffering capacity, and natural nutrient supplying power), in many countries the use of peat is hindered by its unavailability or high cost. Though historically this has not been the case in Canada, heightened environmental awareness and concern over the effects of peatland development on the conservation and protection of wetland wildlife habitats, the protection of rare or unusual species, the release of carbon gases and its relationship to global warming, and water quality issues (CSPMA, 1996) have resulted in rising costs and concerns within the inoculant industry over the future availability of inoculant-quality peat. In addition, the suitability of peat as a carrier for inoculants is affected by past environmental conditions (those under which the peat was initially formed) as well as by present conditions in the bog – including the possibility of contamination from acid rain and polluted drainage waters. Moreover, peat can (at best) be considered only a very slowly renewable resource – one that is currently being mined at a rate faster than it is being regenerated in active bogs. These factors, together with general concerns over environmental quality and the sustainable use of our natural resources have led to renewed interest in the development of new and creative inoculant formulations. The development of granular compost-based inoculants represents one such innovation. Both compost and peat are humus-rich organic materials that provides an excellent medium for sustaining large microbial populations and are nontoxic, biodegradable, and non-polluting. Unlike peat however, compost is a readily renewable resource available from a rapidly growing number of suppliers (Antler, 1996) and is available at a substantially lower cost than that of peat; i.e., compost is generally available at a cost of $20–$40 per tonne ($0.02–$0.04 per kg) as compared to peat which costs an average of $200–$250 per tonne ($0.20–$0.25 per kg). In addition, unlike peat, compost has the potential to yield a “custom” carrier – that is, by adjusting the feedstock, the quality and properties of the final compost can be varied and controlled. Here, we discuss the use of compost as a carrier for rhizobial and other microbial inoculants. Strategies for the development
of inoculants for increased yield of legumes and grasses as well as plant
disease control Abstract Previously we reported the construction of a stably maintained, broad host range plasmid (pHUTFXPAR) carrying genes that enhance nodulation competitiveness through trifolitoxin production and efficient nitrogen fixation by oxidation of the H 2 evolved from nitrogenase (Kent et al. 1998. Appl. Environ. Microbiol. 64:1657-1662). pHUTFXPAR was inserted into Rhizobium leguminosarum bv. phaseoli 127K105a by conjugation. Three field experiments were conducted to test the ability to improve seed yield on Phaseolus vulgaris by strain 127K105a(pHUTFXPAR). The first trial was carried out during the 2000 planting season at a University of Wisconsin field site. The second and third trials were performed simultaneously at two Madison locations in 2002, All the field experiments were conducted with 12 replicates of three treatments including uninoculated and inoculation with either 127K105a or 127K105a(pHUTFXPAR). The pooled dry seed weight from both years and both locations show significant yield increases from the plants inoculated with 127K105a(pHUTFXPAR). The percent yield increase is of 18% and 12% when the dry seed weight from plants inoculated with 127K105a(pHUTFXPAR) is compared to the uninoculated the 127K105a treatments , respectively. Trifolitoxin inhibits strains of Agrobacterium as well as Rhizobium. As a result, we have recently used trifolitoxin producing strains to reduce crown gall disease on grape. The number of galls formed on grape stems was reduced to zero when the Agrobacterium pathogen was co-inoculated with a trifolitoxin-producing Rhizobium strain in greenhouse experiments. Experiments to test the field efficacy of this system are planned for 2003. As legumes are often planted in rotation with grasses, farmers who cultivate grasses are often familiar with the application of inoculants. Through a series of field experiments over multiple years and multiple sites, several bacteria were identified that significantly increase the yield of maize (Riggs et al. Austr. J. Plant Physiol. 28:829-836). Subsequent work has suggested that these strains can increase the yield of soybean as well. The ability of these strains to increase plant productivity by growth hormone production or nitrogen fixation is under investigation. Most of these strains are endophytes. How plants regulate the extent of endophytic colonization is now being studied. 1 Current address: University of Nevada Las Vegas, Department of Biological Sciences, Las Vegas, NV 89154 USA 2 email address: triplett@wisc.edu Inoculant Research in Manitoba Abstract Inoculant research in Manitoba in the past 5 years has focused mainly on N-fixing products. Some research has also been carried out on phosphorus solubilization and microrhizal fungi. Crops studied include dry bean, pea, lentil, alfalfa, wheat and canola. Tests were conducted under dryland, irrigated and laboratory conditions. Inoculants were studied to determine the value of inoculants in providing nutrition and or other positive effects on crop growth such as improved moisture management. Studies have also been conducted to determine the effects of competing products used on legume crops such as fungicides, bacteriocide and insecticide treatments. There have also been studies conducted by private companies on proprietary products that would include representatives of the various inoculants mentioned but due to confidentiality, they are unable to disclose the nature of these studies. Inoculant Research in Saskatchewan 1 Saskatchewan Agriculture Food and Rural Revitalization, 125 - 3085 Albert St., Regina, Saskatchewan S4S OB1 Canada Abstract Inoculant research in Saskatchewan in the past 5 years has focused mainly on N-fixing products. Some research has also been carried out on phosphorus solubilization and sulfur oxidization. Crops studied include chickpea, dry bean, pea, lentil, alfalfa, wheat and canola. Tests were conducted under dry-land and irrigated conditions. Inoculants were studied with regard to the following variables: nitrogen fertilizer, carrier, formulation, seed treatment fungicides, soil temperature, soil moisture, polymer coatings, and placement. A number of studies also compared effects of inoculant use on subsequent non-inoculated crops. This paper provides a list of reported inoculant research carried out in Saskatchewan in the past five years. Significant research and development of inoculant products is done each year in Saskatchewan by inoculant corporations. This research is listed if it has been reported in public forums. Promoting Propagation of Arbuscular Mycorrhizal
Fungi Abstract Arbuscular mycorrhizae ( AM) are symbiotic associations formed between plants and soil fungi. It is generally accepted that AM are mutualistic associations in which both organisms are benefited with the partnership. AM fungi mycelium form a network around and inside plant roots promoting bi-directional nutrient movement by which plant photosynthates flow to the fungal network and inorganic nutrients move to the plant. AM fungi are ubiquitous in the soil and can form symbiosis with most terrestrial plants. Extraradical mycelium produced by AM fungi can spread throughout the soil surrounding the root system increasing the plants ability to absorb water and nutrients. Phosphate ions have very small diffusion coefficients and P-depletion zones are formed around the roots systems. AM fungal network is especially important for phosphorus nutrition since fungal mycelium can grow beyond the P-depletion zone of roots, allowing P-transport to the plant to continue. In addition to promoting water and nutrient uptake, AM can induce drought and disease resistance, and increase soil carbon content, and improve soil porosity by increasing soil particle aggregation. Synergy between AM and other soil microorganisms such as N-fixing or plant growth promoting rhizobacteria might increase positive response of plants. AM is one type of mycorrhizae important to crops such as flax, corn, wheat and barley. Some crops, for example canola, do not form this type of association and might affect the condition of fungal network for the following crop. In the field, there are several factors that can affect plant response to mycorrhizal fungi colonization such as a host crop dependency to mycorrhizal colonization, tillage system, fertilizer application, and inoculum’s potential of the mycorrhizal fungi. Nowadays, low-input sustainable systems are being promoted because of environmental and economic reasons, hence increasing the role of AM in agriculture. Management of AM populations can become a very important tool in nutrient cycling. This will demand improved management of natural AM populations, which might suffice, or alternatively the use of commercially available AM inoculum might be needed. Natural AM populations can be affected by several factors including tillage systems and crop rotation. In a field study in Manitoba, AM colonization of flax roots was affected by preceding crop and in some cases by tillage system, affecting crop yield. Because of the obligate symbiotic status, the arbuscular mycorrhizal fungi need imperatively the presence of a plant to grow and proliferate. As such, the maintenance of AM fungal collections and the development of mycorrhizal inoculum require methodologies and infrastructures quite different from those used with other microbial collections. Traditionally, AM inoculum is produced in pot-cultures under growth chamber or greenhouse conditions. Spores, colonized root segments or soil containing fungal propagules (spores, hypha, root sections) can be used as starting inoculum. Pure cultures can be obtained with pot-culture technique by using a single spore as inoculum. The development of a substantial collection of AM fungal inoculants require a tremendous amount of work, expertise, and working time for the watering, fertilization, regular repotting, regular verification of pure culture status of pots etc. During the last 2 decades, several companies developed commercial mycorrhizal inoculants. Most of them used the traditional pot-culture methodology for the propagation of their basic product. The inoculum is then mixed with a substrate, often compost or peat moss with inert material such as perlite or vermiculite. Aeroponic methods and bioreactor approaches based on the root-organ culture have also been developed by some industries but their processes remain confidential. In the past decade, the root-organ culture technique has been adapted for AM fungi cultivation. It consists in the propagation under axenic conditions inside Petri dishes of excised roots beside which surface sterilised germinating spores or colonized root segments are deposited. Such monoxenic cultures (root + fungus) can proliferate and be maintained for several months. The fungal material produced is perfectly adapted for morphological studies as for molecular and biochemistry investigations. It provides the user with clean material deprived from any contaminant. In Canada, there are a couple of producers that can supply commercial AM mycorrhizal inoculant. Abroad, there are many companies that can supply AM inoculum. Also, there are commercial research collections which provide users with small amount of fungal material under the form of a substrate containing colonized root segments together with spores and hyphae or of sieved fungal material isolated from the rhizosphere of plants grown in pot-culture or from root-organ culture. Furthermore, several laboratories from governmental and academic institutions maintain strains of AM mycorrhizal fungi under cultivation using mainly the traditional pot-culture propagation methodology and sometimes the root-organ culture methodology. Several strains covering a large number of species are available there but not listed in official databases. Exchanges are usually made under the umbrella of research collaborations. There is no official listing of those smaller collections reserved for research activities. Interest in AM fungi propagation in agriculture is increasing due to its role in the soil nutrient cycle and promotion of plant health and crop yield in low-input system, which could help to solve economic and environmental concerns. Propagation of natural AM populations can be achieved by using different agronomic practices such as minimum tillage systems and mycorrhizal crops in the rotation. However, there are agronomic systems that will require the use commercial AM inoculants available in Canada and abroad. PGPR/Prospects For New Inoculants Abstract Root colonizing bacteria (rhizobacteria) that exert beneficial effects on plant development have been defined as plant growth promoting rhizobacteria (PGPR). These organisms may enhance plant growth via direct or indirect mechanisms that are not yet well understood. Direct promotion of plant growth by PGPR may result from the synthesis of phytohormones that stimulate root development, fixation of atmospheric nitrogen, solubilzation of phosphate, or from the enhancement of nutrient uptake Some PGPR promote the growth of plants indirectly by suppressing the growth of plant pathogens via the production of antibiotics, siderophores, extracellular enzymes, or by inducing systemic resistance. Molecular tools are providing sensitive means for resolving the importance of these mechanisms for plant growth promotion and biological control and for understanding the rhizobacteria/root interaction at a fine scale. Although significant control of plant pathogens or direct enhancement of plant development has been demonstrated in the laboratory and in the greenhouse, results in the field have been less consistent. Because of these and other challenges in screening, formulation and application, PGPR have yet to fulfill their promise and potential as commercial inoculants. As our understanding of their diversity, host specificity, mechanisms of action, colonization ability, and formulation and application requirements increases, there is renewed expectation that the potential of these organisms to sustainably enhance plant growth in agricultural, horticultural and agroforestry systems will be realized. This presentation will review recent progress in our understanding and application of PGPR as inoculants and highlight outstanding challenges to their development as commercial inoculants. Aspects of the progress in my laboratory in isolating and characterizing new PGPR from Saskatchewan soils and in determining the role of seed and seedling exudates in the interaction between seedlings and microbial populations will be presented. An economic assessment of the global
inoculant industry Abstract The inoculant industry represents a relatively small but potentially important part of the increasingly competitive global agri-food sector. Inoculants—as either a substitute or compliment to the use of commercial or non-commercial fertilizers—have the potential to increase the productivity and profitability of field crops, enhance food production of vital staple foods, support social progress in many underdeveloped countries and moderate environmental effects of commercial agriculture. Fertilizers, especially nitrogen and phosphates, are one of the most important inputs used in the global agri-food industry. Between 1960 and 2000, the world use of nitrogen increased from 12 to 81 million tonnes N, a seven-fold increase in 40 years (despite a substantial fall in demand in the formerly centrally planned economies after 1990). Meanwhile, phosphate fertilizer consumption rose relatively less, but still reached 33 million tonnes in 2000. The inoculant industry—initially involving nitrogen-fixing rhizobia bacteria—has been around for about 100 years but has increased in importance as new formulations have been developed and marketed to growers in the past few decades. Inoculants have significant potential to create global economic and welfare gains, as the technology is highly effective with legumes, which are estimated to contribute about 20% of worldwide food protein. This is especially important for consumers in less well-developed nations, where legumes comprise a significant share of nutritional requirements. Meanwhile, the recent development and introduction of phosphate-fixing Penicillium fungus inoculants has broadened the potential market. The new inoculants show significant incremental gains in efficiency when applied to wheat and canola crops, especially in marginal growing areas. They are currently applied on only a small area, but have significant potential for growth. The industry, while still relatively small, is beginning to have a measurable effect on the global agrifood economy. The preliminary estimate is that all inoculants combined are being used on more than 40 million acres worldwide, which represents about 10% of the total global acreage devoted to legume crops. A preliminary analysis suggests the following economic impacts are likely being realized:
Economic analyses offer some insight into the impact of new or evolving technologies, but they can at times be too narrow. In this case, there is evidence that while inoculant technology has the potential to contribute to the commercial success of producers around the world, it also could contribute directly to stabilizing and extending production of vital protein crops in marginal areas, thereby improving the welfare of many who are not normally beneficiaries of new technology. Equally important, the technology has the potential to lessen global agriculture’s dependence on commercial nitrogen and phosphate fertilizers, which require significant quantities of energy to produce. This could both contribute to a lessening of pressure on global energy markets and minimize production of environmentally damaging greenhouse gases. Inoculant Research in Eastern Canada. Abstract In Eastern Canada, the main contributors to inoculant research and development are universities (Laval, Mc Gill, Mc Master, Waterloo), federal (AAFC, Sainte-Foy and Ottawa) and provincial (Prince Edward Island Department of Agriculture, INRS) research centres, as well as industry (Agribiotics Inc., Bios-Agriculture, Mikro-Tek and Premier Tech). With regard to nitrogen fixation, Sinorhizobium meliloti is the one of most studied rhizobial species. Studies focus on the diversity of native S. meliloti in soils, the manipulation of genes to improve competition for nodulation, molecular and physiological studies on carbon metabolism related to stress tolerance (starvation). As well, the recent sequencing of the S. meliloti genome and current project to identify some two thousand genes whose functions are unknown will provide knowledge potentially useful in developing superior inoculants strains. One study evaluates the suitability of bradyrhizobia that nodulate native legumes for potential use in soybean inoculants. Also, signal molecules involved in plant-microbes exchanges are being employed in an attempt to improve Bradyrhizobium japonicum nodulation of soybean in cool soils. Agribiotics and Bios Agriculture currently produce commercial inoculants for soybean. With a view towards introducing Kura clover (originating from Eurasia) as a new forage crop, the diversity and phylogeny of Rhizobium leguminosarum bv. trifolii and of various clover species are being studied. Cold-adapted rhizobia associated with native legumes of arctic and sub-arctic regions, such as Mesorhizobium sp (from Astragalus) and R. leguminosarum bv. viciae (from Lathyrus) have been selected for their potential to improve nitrogen fixation at low temperature. With regard to inoculant production technology, research has focused on the potential of organic wastes as low cost culture media for the production of rhizobia. AAFC Sainte-Foy is one of the two laboratories analysing the quality of commercial inoculants in Canada. Rhizobia are also studied for purposes beyond nitrogen fixation. Their potential in soil bioremediation is being investigated since they are capable of degradating aromatic pollutants (PCBs). Rhizobia also have potential for use as PGPR (plant growth promoting rhizobacteria) with non-legumes (eg. lettuce, maize-soybean rotation). The stimulation of plant growth is also studied with other bacterial species such as Pseudomonas, Serratia and Bacillus. For example, the potential role of ACC (1-aminocyclopropane-1-carboxylic acid) deaminase in stimulating plant growth by lowering plant ethylene level under stress is being investigated. Improving plant phosphorus nutrition by inoculation with phosphate solubilizing microorganisms (eg. Rhizobium, Penicillium) and mycorrhiza is also being investigated with wheat and potato. Research on VAM is focussed on the development of efficient symbioses for phytoremediation and agro-forestry (eg. poplar), and on the role of mycorrhizae in biocontrol (orchid). Increased knowledge on taxonomy, diversity and is being acquired by the development of molecular techniques (e.g using DNA micro-satellites). Development of cultivation techniques and studies on nutrient exchanges are underway using split-root systems. Commercial products (Mikro-Tek and Premier-Tech) containing ecto- or endo-mycorrhizae are dedicated mainly to horticulture and nurseries. Finally, studies on plant and soil microbial community structure are conducted with the aim to select beneficial associations compatible with crop rotations. As well, these studies will enhance our understanding of the antagonistic response of established populations to the successful insertion of microbes applied by inoculation . Maintaining Inoculant Quality Standards 1 Beaverlodge Research Farm, Box 29,
Beaverlodge, Alberta, Canada T0H 0C0 Abstract Several factors contribute to quality in a legume inoculant. These include large cell numbers of a superior rhizobial strain, freedom from significant contamination, a formulation that is effective and easy to apply, adequate shelf life of the product, proper packaging, and clear labeling with instructions for use on each package. Evaluation of inoculant quality by enumerating the viable rhizobia present is usually an accurate index of inoculant potential. Numerical considerations are of such significance in determining the effectiveness of inoculant that the need for quality control cell enumeration systems is widely recognized. The basic aim of legume inoculation is to provide the maximum number of suitable rhizobia in the rhizosphere at the time of nodule initiation. Large numbers of rhizobia on the seed or in the soil favor survival before planting, which in turn promotes rhizobia multiplication in the rhizosphere and early nodulation. Proper inoculation with even the best quality inoculant does not always result in a demonstrable increase in nodulation or yield. Nevertheless, increasing the number of viable rhizobial cells applied per legume seed will usually increase nodulation and nitrogen fixation, especially under stress conditions. Stress conditions are not uncommon in Great Plains agriculture. Some pulse growers have considered mixing granular inoculant with fertilizer in order to make their seeding operation more efficient. The effects of such a practice on the survival of rhizobia and response of peas to inoculation were examined in laboratory and field studies. The results suggest that high-quality granular inoculant can probably be mixed with fertilizer and applied together, but the mixture should preferably be applied on the same day. The rhizobial cells in a good-quality powdered peat inoculant have been estimated to constitute only about 0.13% of the total volume of the inoculant. There is clearly room for a further 10-fold increase in cell number. Such increase in rhizobial cell numbers may be reached with further research effort, but manufacturer use of relatively simple quality control programs is the most direct and immediate way to guarantee high-quality inoculant products reach the market. In Canada, rhizobial inoculant products are classified as supplements under the Fertilizers Act and are subject to registration and regulation. Information required for registration includes a list of constituent materials, a guarantee of the minimum number of viable rhizobia, the product label, and efficacy data. Regulation is accomplished through the Canadian Legume Inoculant and Pre-Inoculated Seed Testing program. Implementation of this testing program in 1975 resulted in an initial rapid and significant increase in the quality of inoculant products offered to the Canadian farmers. This high quality of inoculant products has been maintained since then. The importance of legislated regulation of inoculant quality is obvious, and must be continued if high quality of legume inoculants and pre-inoculated seed products is to be maintained. Environmental Factors Influencing Symbiotic
Nitrogen Fixation Under Field Conditions Abstract Nitrogen is one of the major limiting nutrients for most crop and other plant species in most environments. From several indications and by many estimations, the world will no doubt face severe food shortages in the not to distant future. This is in part due to excessive population growth and the negative environmental impact associated with population growth. Populations in developing (and less developed) countries are assumed to account for 90% of the projected world’s population, many of whom live on marginal land. Given that enhanced agricultural production will require the utilization of large areas of land that are now considered to be marginal or non-arable, alternate strategies are needed to meet the future needs of humans. Several diverse biological associations contribute to N 2 fixation in soil systems, but in most agricultural systems, the majority of biologically-fixed N occurs via the symbiotic interactions of legumes and the soil bacteria of the genera Rhizobium, Bradyrhizobium, Sinorhizobium, Allorhizobium, Mesorhizobium, and Azorhizobium. Together these symbiotic partners fix an astounding 90 Tg N per year. However, the soil environment is under a constant state of change, and as such, can be relatively stressful for both macro- and microsymbionts. Fluctuations in pH, nutrient availability, temperature, and water status, among other factors, greatly influence the growth, survival, and metabolic activity of soil microorganisms and plants. While microbes, plants, and other soil inhabitants have evolved to adapt to the ever changing and often inhospitable soil environment, it has been known for a long period of time that virtually any environmental factor known to negatively influence the growth of rhizobia or the host plant itself has a dramatic impact on symbiotic N 2 fixation. These factors can independently negatively-influence the nodulation process itself, and thereby indirectly affect nitrogen fixation, or directly influence plant growth and vigor during post-nodulation events and influence the efficient functioning of the enzyme complex nitrogenase. Environmental factors known to influence symbiotic nitrogen fixation under field conditions include: soil water levels; soil aggregates; salinity; pH; macronutrients such as N, Ca and P; soil temperature; micronutrient status, rhizobial densities, inoculation success, and the presence of competing micro and macroorganisms. In this session I will review these factors known to influence symbiotic nitrogen fixation under field conditions, and discuss possible means to overcome many of these limiting factors. A Look Back & Forward Abstract Symbiotic nitrogen fixation is an old system if we consider the relatively short time in which it has been identified, studied and utilized to enhance crop production. The applied effort of utilizing inoculants has focused on strain selection, product formulation and application without manipulation or enhancement of the functioning symbiotic system. The inoculant industry has developed from initial products introduced in the early 1900’s, through multiple federal, university and private industry sources to a select few companies in North America today. A significant change in inoculant carriers and formulations has occurred during this period with a range from inexpensive organic and inorganic solid matrix carriers to the newer developed stable liquid formulations. When a producer assesses the inoculant price, quality and convenience of application, it is most commonly the latter that is the primary factor in selecting an inoculant. The current pulse crop products represent truly a joint research and development effort between the public and private sectors, wherein a quality product was made convenient. The change in pulse management, inoculant forms and planting equipment has altered significantly the practice of inoculation. The characteristics of a quality inoculant have primarily been defined by high numbers of effective and efficient strains in the product. Many stress factors affect the applied cells and, therefore, assays or systems that estimate the health and vigor of the applied population and that assesses their ability to survive stresses may be more meaningful than laboratory population estimates. Nodulation and crop yield are the primary inoculant parameters assessed, however, the soil nitrogen balance, carryover nitrogen and ground water ecological factors may become even more important in the future. Strain competitiveness and nodule occupancy remains as a constraint in placing more efficient strains in position to increase total nitrogen fixation. Do we need to re-define what inoculants are intended to accomplish, and in fact better assess their benefits and quite possibly extend their beneficial features? Basic research is currently discovering how the microbes and the plants interact via their molecular signal language. Understanding of the root hair binding and specific host selection processes, flavonoids, nod factors and quorum sensing gene regulation systems are key basic science advances, and applications or modifications of these systems should enhance the inoculant field performance. We are rapidly identifying via genomics the genes of regulation, not only for cell performance but also cell adjustments and survival. As science in this field advances, the newfound knowledge will likely be able to alter or regulate the “choke points” in the overall system to enhance the microbial inoculants performance, resulting in improved crop production. Agronomic Benefits of Inoculation of Crop
Plants with Beneficial Microorganisms in the Great Plains Abstract Agriculturists have been utilizing inoculation of plants with beneficial soil microorganisms probably for millennia. However the understanding of the interaction between host plants and beneficial microorganism and quantification of the benefits are a much more recent phenomena. Beneficial microorganisms can improve plant growth by a variety of mechanisms. They can increase the supply or availability of nutrients to host plants (biofertilizers) or displace or inhibit plant pathogens and weeds (biopesticides). This review looks at the agronomic benefits of biofertilizing microorganisms commonly used in inoculants in the Great Plains of North America, as well as the potential of utilizing newly-research or under-utilized microorganisms in new inoculant products. Modes of action of biofertilizing inoculants include (1) increasing the supply of mineral nutrients to the host plant (e.g. N 2 fixers, phosphate solubilizers, sulphur oxidizers), (2) modifying assimilate partitioning and growth patterns of the host plant to increase the absorptive surface area of roots and volume of soil explored (e.g. phytohormone producing bacteria, arbuscular mycorrhizae), and (3) aiding the establishment or functioning of other beneficial microorganisms (“helper” bacteria). By far the most long exploited and most widely used biofertilizing inoculants used on the Great Plains of North America are the N 2-fixing bacteria (generally referred to as rhizobia) that infect forage and grain legumes. The benefits of rhizobia to the accumulation of N in general in legume crops on the Great Plains are well established. However, it is very difficult to predict with accuracy the amount of fixed N which will be supplied to a crop by rhizobia in a season given the influences of host and microsymbiont genotypes, soil N levels, temperature regime, water availability, inoculation efficacy, and competitiveness of native rhizobia populations. The use of associative N 2-fixing bacteria (endophytic, rhizoplanic, and rhizospheric) has been, and continues to be, widely investigated. However no strains are currently registered for use in Canada, and although many inoculants containing putative associative N 2-fixer are available in the USA, consistent, widespread agronomic benefits of these products are difficult to obtain. Both rhizospheric bacteria and fungi have been implicated as phosphate solubilizers. Penicillium bilaii is a rhizospheric fungus widely used in the Canadian prairie in a number of crops species. Although the fungus has been shown to solubilize P in culture, promotion of plant growth can not always be related to increased P uptake in host plants. Phosphate solubilizing bacteria are common components of microbial inoculants available in the USA. Sulphur oxidizing bacteria show promise as a means of increasing sulphur availability and stimulating growth in some S-rich crops such as canola. Although there is much research on microbial siderophores production in the rhizosphere for biocontrol activities, it is unclear as to how much siderophore production contribute to increased iron absorption by host plants. Outside the clear effects of rhizobia in N accumulation in legumes, the second most well characterized benefit of rhizospheric organisms on host plant growth is the positive effects on root architecture and functional absorptive surface area. These effects are media most commonly mediated by phytohormone production by the microsymbionts. Commonly these phytohormones result in increases in specific root length and increases in root hair production. The wide-spread symbioses between crop plants and arbuscular mycorrhizae effectively lead to a much increased absorptive surface area for the host. Although such effects of rhizospheric organisms on root morphology are common, difficulties in assuring introduced species are competitive in the rhizosphere to indigenous organism is a limiting factor to their greater use. Although the beneficial effects of rhizobia to legume production are well established, the expanded use of microorganisms in inoculants to increase nutrient accumulation in crop plants in the Great Plains is challenged by the variability of responses to these organisms. No doubt a greater understanding of rhizosphere ecology is necessary for more consistent responses and wide-spread use of microbial inoculants in this region. Formulations in the Field Fran Walley 1*, George Clayton 2, Yantai Gan 3, and Guy Lafond 4 Department of Soil Science 1, University of Saskatchewan 2, Lacombe Research Centre 3, SPARC, Indian Head Research Farm 4, AAFC. Abstract Over the years, a number of studies have been conducted in Saskatchewan, and across western Canada, to examine the impact of rhizobial inoculant formulation on the success of various Rhizobium/legume associations. The introduction of granular inoculant formulations in the ‘90’s stimulated a new flurry of research activity and throughout the latter portion of the decade, a number of projects were conducted to compare the efficacy of this ‘new’ formulation to liquid and peat-based powders. Coincidentally, at the same time that the new granular formulations were being introduced into the Prairie market, we also saw the introduction and rapid expansion of desi- and kabuli-type chickpea production in Saskatchewan that served to further stimulate inoculant research activity. As a consequence, we have amassed a considerable body of research data for a variety of pulse and legume forage crop in which comparisons have been made between various inoculant formulations. Results from this research will be summarized and discussed with reference to impact on current farming practices. Despite the many research programs that have been conducted in the relatively near past, farmers still have many unanswered questions when it comes to choosing an inoculant formulation for their own field use. Some of the pressing questions will be posed and future research needs will be identified and discussed. Plant growth-promoting inoculants in Australian
agriculture Abstract Australian agriculture encompasses a wide range of farming systems, reflecting the diversity in agro-ecological conditions found throughout the continent. Common throughout the systems are trends to increase intensification to boost production and profitability. Yet many soils are constrained in their ability to sustain high levels of agricultural production, mostly from factors associated with low fertility (especially C, N and P), root disease, and extremes of pH. Soil microorganisms associated with plant roots provide many of the services that underpin the sustainability of agro-ecosystems. The exploitation of such microbes as plant growth promoting inoculants is being pursued as a means to increase productivity in a sustainable fashion. The inoculation of legume species with N 2-fixing symbiotic rhizobia represents the most widespread and important use of inoculants in Australian agriculture. Pasture or crop legumes are widely used in rotation with cereals, providing opportunities to control weeds, bolster soil organic matter and fertility, and act as disease breaks. Based on available data, approximately 1.5 M ha of legumes are inoculated with rhizobia each year, at a cost of A$5-6 M. The practice of inoculation with rhizobia can be very cost effective, as it costs less than A$4 /ha yet has the potential to provide an extra A$200-300 worth of N/ha. Industry data shows that 80% of the inoculants are for grain legume application and 20% for pasture legume species. Annually, the 2 M ha of grain legumes sown (half of which are lupins) fix in the order of A$150 M worth of N. Although substantially less pasture legume seed is inoculated, pastures fix in excess of A$1 B worth of N annually – most of which is attributable to carry-over benefits of historical inoculation. Over 90 species of legumes are inoculated with 40 different strains of rhizobia. The Australian Legume Inoculant Research Unit plays a key role in ensuring growers have access to strains of the highest quality and efficacy. Although rhizobia for N 2-fixation dominate the seed-inoculant industry, a variety of other microorganisms with various functional attributes are being developed. After 10 years research, pilot-scale production of the fungus Penicillium radicum began in 2001 by Australian Seed Inoculants Pty Inc (Pr70 Release). The inoculant increased the growth and yield of cereal plants in field trials across a range of soil types. Mobilization of insoluble soil phosphorus (P) is thought to be a key mechanism driving growth promotion. Many Australian soils have high P-fixing characteristics that lower the efficiency of P-fertilizer application so that repeated use of high-grade P fertilizers is required to sustain production. By mobilizing insoluble soil P, Pr70 Release may be able to increase the phosphorus efficiency of current fertilizer use and access the P ‘banked’ into the soil from historical application. The feasibility of commercializing a strain of Trichoderma koningii in Australia is being investigated. The fungus reduced wheat take-all and sharp eyespot diseases in field-testing across Australia, China and the USA. Initial formulations were developed following close collaboration between the CSIRO and China Agricultural University. A formulation was commercially available for broad-acre agricultural use in China in 1999-2000. There is scope for the development of microbial inoculants for a range of other production systems. For example, inoculants to promote early growth and vigor of pasture plants are being investigated. Development of New Microbial Formulations Philom Bios Inc. - 318-111 Research Drive - Saskatoon , Saskatchewan, Canada S7N 3R2 Abstract A key constraint to successfully commercializing beneficial microorganisms is overcoming difficulties in formulating a viable, cost-effective and user-friendly final product. The live nature of the active ingredient (i.e., the microbial agent) signifies the importance of formulation in maintaining the microbial cells in a metabolically and physiologically competent state, in order to obtain the desired benefit when applied. Formulation typically consists of the active ingredient in a suitable carrier and additives that aid in the stabilization and protection of the microbial cells during storage, transport and at the target zone. The development of new microbial formulations is a challenging task. Regardless of whether the product is new or improved, the product must be stable during storage and transportation, easy to handle and apply, enhance activity of the organism in the field and be cost-effective and practical. Therefore, several critical factors including user preference have to be considered before delivery of the final product. Challenges in formulating a microbial product can be further exacerbated by strain to strain variability, which necessitates discrete research protocols for individual strains that adds further to the research and development cost. Furthermore, often the same organism has to be formulated in different forms to suit different climates, soil-types and user preferences. A notable other challenge in microbial formulations is the limited availability of published scientific information, arising partly from commercial secrecy, and a lack of effort and resource allocation for formulation research. This lack of adequate information highlights the need for research collaborations between academia, government-based research units and industry. Notwithstanding these challenges, there are several strategies that can be explored to deliver a commercially viable formulation. This presentation will focus on some of these strategies, their principles and techniques to formulation. The development of specific examples of inoculant formulations at Philom Bios Inc. will be discussed.
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