Abstracts 2005 : Scroll down the page to read all the abstracts from the 2005 Inoculant Forum or use the Index below to find a specific abstract. Poster Abstracts:
Dreaming of the Perfect Inoculant
E-Mail : catroux@dijon.inra.fr Abstract The social demand for safer agricultural products and food is increasing in developed countries and governments are releasing new regulations in order to protect the environment and natural resources against chemical pollutions and contaminations. In this way, the management of soil microorganisms involved in biogeochemical cycles (C, N, P), in plant health and in soil bioremediation may offer new possibilities. Academic scientists and companies support this idea, but despite important scientific investments in soil microbial ecology, a limited number of microbial inoculants are on the market. They represent a small percentage of the chemicals sold (except legumes inoculants and Bt products). Now, combining available knowledge in soil microbial ecology, in molecular ecology and in signaling between microorganisms and plants, has open news avenues to dream of the perfect inoculant. This presentation will analyze the limitations encountered when seeking for the perfect inoculant. The production and use of a new inoculant is under the control of three partners with very contrasted interests: the scientist, the manufacturer and the farmer. This may slow down the technological transfer between science and practical use. To produce an inoculant, the first step is to select a strain or a mixture of strains able to produce a biological effect. Some point may be critical as the strain isolation which is generally performed at temperatures far from the soil conditions. Another point to improve is to select on the biological efficacy but also on competitiveness and maybe to change the selection strategy. The second step is to process an inoculant efficient in field conditions, safe, with a good shelf life, able to be processed at low cost and satisfying the regulations. "New" technologies mixing microorganisms and signal molecules are promising as complex mixtures of strains with complementary effect. But they require more investments to be fully reliable. The third step is for the farmers to buy a product easy to use, with a good reliable efficacy in field conditions, with a long time effect and as cheap as possible. But they need also to use it properly. Are we sure that the strain selection has taken into account all the characteristics required by farmers? In conclusion, some proposals to improve the process will be given... in order to continue to dream of the perfect inoculant. Plant Inoculants Canada Inc. Abstract WHAT PIC is ABOUT In 2003, the Canadian inoculants industry, together with several non-Canadian inoculant companies that do business in Canada, joined together to form an industry association, Plant Inoculants Canada Inc. (PIC) whose primary purpose is to represent the interests of the industry in a variety of fora, the most important of which is working proactively with Canadian federal regulatory agencies to resolve regulatory issues facing the industry BOARD of DIRECTORS The Board consists of nine senior managers and academics from the inoculants industry. The Chair and President is Peter McCann, President of Brighton BioConsulting, formerly CEO of Ag-West Biotech Inc., Saskatoon. OUR MISSION Recognizing the value of plant inoculants to growers and environmental sustainability, and with the support of, and on behalf of, the inoculants industry, PIC will act to interface between the industry and government agencies, researchers, and the public. PIC goals include the facilitation and coordination of the development of industry positions on regulatory issues, and to ensure understanding and support for the industry and its objectives. PIC also works at the interface between the industry and the public to promote the use of biological fertilizers and ensure understanding and support for the industry and its objectives MEMBERSHIP The 10 founding members of Plant Inoculants Canada Inc. include Canadian and US inoculant producers, both large and small, research scientists and academics: Membership is open to any group or individual whose primary interest lies in the research, development, manufacture and marketing of inoculants for agricultural crops and for home and garden use. New members are welcomed, so as to provide PIC with the widest possible base of support. There are three classes of membership, Corporate Members, generally fully commercial companies with sales in excess of $1,000,000 per year; Associate Members, companies and other organizations with sales of less than $1,000,000 per year and Individual Members, generally research scientists in government and university laboratories. Annual membership fees are $2,000, $500 and $50 respectively. CURRENT ACTIVITIES
Regulation of Research Trials
under the Fertilizers Act and Regulations Abstract Pursuant to the Fertilizers Act and Regulations, individuals wishing to conduct scientific research on novel supplements must either notify or obtain written authorization from the CFIA prior to releasing the supplement into the environment. Research trials that are conducted in adequately contained facilities are exempt from regulation under the Fertilizers Act. Conversely, all field trials and trials that are not contained such as those conducted in commercial greenhouse facilities are subject to regulation and must meet all the requirements of the Fertilizers Act prior to trial establishment. Research trials may be eligible for notification, a streamlined regulatory process aimed at supplements that pose no or negligible risk, alternatively they may require a written research authorization. The Fertilizer Section, CFIA is currently revising the guidelines to both notifications and authorizations for supplement research (Trade memorandum T-4-103). This review has been initiated to streamline the regulatory process and to enhance the compliance of research trials conducted in Canada with the requirements of the Fertilizers Act and Regulations. The proposed changes to this document will be discussed. Registration & Testing
of Inoculants in the U.S. Abstract Unlike Canada, the United States does not have a unified national approach for the registration of inoculants. This process is implemented on a state-by-state basis. Each state is able to request compliance with a variety of different regulations. This may include parameters such as efficacy testing, labeling or licensing. Conversely, field testing is regulated on a national level, but only if the organism of interest is genetically modified. In most cases, traditional inoculants will not fall into this category. In general, the ability to test and commercialize an agricultural inoculant in the United States is much less regulated than in Canada. It is important though to become familiar with all of the applicable rules and regulations in the state where the inoculant will be sold. Sulfur Inoculants Abstract Sulfur (S) is an essential plant nutrient involves in the synthesis of amino acids and many organic compounds. In recent years S deficiency is becoming more prevalent due to increasing use of high analysis fertilizer, use of low S fuels, increasing crop removal and decreasing use of S as pesticides. Soluble form of S fertilizer is widely used during growing season to meet crop S demand but this readily soluble S is liable to leaching loss. Many farmers began to use elemental S to reduce input cost although elemental form of S must be oxidized to plant useable sulfate form for plant uptake. This conversion of S form in soil is generally mediated by soil microorganisms. This arises the concept of using microorganism as S-oxidizing tool and may reveal huge potential as microbial inoculant. Use of plant growth promoting rhizobacteria (PGPR) as microbial inoculant for better crop production is nothing new to researchers. However, little information is available on successful application of S-oxidizing PGPR as inoculant for agricultural crops. Few studies of using S-oxidizing bacteria under growth room conditions were reported but the successful attempt to use S inoculant is still lacking. Our multi year multi sites field studies with canola PGPR showed that the bacterial inoculant, which is also a S-oxidizer significantly enhanced canola yield. Seed analysis showed that the inoculant did not change the seed quality traits like oil, protein, oleic acid, linolenic acid and glucosinolate content of canola seed. Thus, this research has developed a bacterial inoculant that not only improves canola production but also retains all the seed quality aspect of canola. Furthermore, S-oxidizing PGPR was found compatible with Rhizobium species. Leguminous crops like soybean and peas have also been shown to respond to this type of S inoculant. For example, soybean yield and root nodule number has been increased when S inoculant was used in association with Bradyrhizobium. Hence, this naturally occurring S-oxidizing PGPR may be used as inoculant for canola, soybean and other crops. The low input cost of PGPR inoculant along with better crop yield can provide agronomic benefits to farmers in an environment friendly manner, nevertheless, ease of application and adequate product shelf life could be the most significant factors for the inoculant to be commercially successful. Evolving Strategies for
the Practical Use of Free-Living Beneficial Bacteria in Agriculture
and Floriculture Abstract Plant growth-promoting rhizobacteria are naturally occurring bacteria isolated from rhizospheres that when inoculated onto crops or into soil colonize plant roots and increase plant growth or reduce damage to plant diseases. Over the past decade, the theoretical basis of PGPR that has been reported in numerous scientific publications has led to development of several commercial products containing PGPR. We discuss four strategies for developing such products and give examples where we have tested the products or where test results have been published in refereed scientific manuscripts. In all cases where the products are formed using purified cultures, the PGPR are spore-forming bacilli. The first strategy is formulation of individual strains, and examples are Kodiak®, containing Bacillus subtilis strain GB03, and Yield Shield®, containing B. pumilus strain GB34 (also designated as strain INR7 in the literature). Strain GB03 in Kodiak enhances root growth and produces the antibiotic iturin that inhibits Rhizoctonia solani and Fusarium spp. on cotton and bean (Phaseolus). Treatment of soybean with Yield Shield leads to protection against R. solani via induced systemic resistance elicited by strain GB34. Another product development strategy is to formulate simple mixtures, and an example is BioYield® that contains strain GB03 and B. amyloliquefaciens strain GB99 (also designated as strain IN937a in the literature). BioYield enhances growth of transplanted vegetables and melons. Complex mixtures of over 40 strains of bacilli PGPR are also formulated into products. Equity®, Naturize Outdoor Plant Food ®, Naturize Indoor and Potted Plant Food®, and PGA® each contain the same mixture of 47 strains of bacilli. These products differ in the final type of formulation and the presence of other ingredients known to exert effects on plant roots (humates and kelp), impregnation onto granular fertilizer, or mixing with water-soluble fertilizer. Each of these products enhances root and overall growth of seedlings and transplants of several vegetable and floriculture crops. Another product development strategy uses continuous anaerobic fermentation of manure with the addition of proprietary microbial stimulants. This approach develops a stable, multi-trophic community that includes culturable and nonculturable, aerobic and anaerobic, Gram-positive and Gram-negative bacteria, protozoa, actinomycetes, and cyanobacteria together with their metabolites. Soil Builder® and AgBlend® are examples of this approach, and these products promote root and overall growth of various plants. Future research is needed to determine if PGPR can be useful tactics for integrating into sustainable agricultural practices for reducing nitrate leaching and phosphate run-off; for increasing crop production under conditions of salt or drought stress; and for enhancing the quality of produce. Arbuscular Mycorrhizal
Fungi & Phosphate:
Apples & Oranges Abstract Arbuscular mycorrhizal (AM) fungi appear as a fundamental element of ecosystems, as they have co-evolved with the soil-plant system for 400 million years. Early research has revealed that AM fungi have a large and profound beneficial impact on the physiology of their host plants. Better productivity, increased hormone production, shoot to root biomass ratio, root epidermis and cortex cell life span, as well as enhanced photosynthetic activity were reported in mycorrhizal plants. Much of these effects were attributed to better plant phosphorus nutrition. Thus, these fungi became known as important plant phosphorus providers working for photosynthesis-derived carbon and energy within the framework of an infection controlled by phosphorus availability. But if these fungi are so beneficial to plants, tax payers rightly wonder, why does the agricultural industry utilize AM fungi only marginally after 50 years of costly research? The objective of this presentation is to answer this question and to correct the errors of the past. There are a number of reasons explaining the lack of interest of agriculture for the mycorrhizal symbiosis; most are related to an erroneous understanding of the mycorrhizal symbiosis. (1) Controlled condition experiments comparing mycorrhizal and non-mycorrhizal plants may have created unreasonable expectations on AM fungi derived benefits to plant growth. (2) AM fungi were perceived as a mere biological farm input which, when added to plant roots, improve phosphorus uptake by crops. This oversimplified perception of AM fungi has certainly been a deterrent to the use of AM fungi in crop production especially considering that phosphorus fertilizers are relatively inexpensive and so easy to use. (3) The general failure of mycorrhizologists to recognize what are the fundamentally important roles of AM fungi in soil, led to the exodus of agronomists from the field of mycorrhizal research. The name of this type of association, « mycorrhizae » - literally « fungal root » -, carries a misleading concept for soil fungi, and has contributed to the persistence of the erroneous conception of the AM fungi as plant extensions. (4) Finally and most importantly, science has so far failed to provide the industry with means to consider and manage AM fungi in agricultural soils. AM fungi are now revealing themselves as an important group of soil fungi rather than components of plant root systems. These fungi should not be considered as a monolithic group of « fungal root extensions » for the recovery of phosphorus. AM species are clearly distinct organisms with specific functional characteristics. AM symbioses are presented as the products of interactions between the soil environment, and the plant and fungal genotypes, which can exist and even benefit crops in phosphorus-rich soils. AM fungi are a photosynthesis-driven mechanism to improve soil environmental quality and nutrient cycling efficiency, thus the way to minimize seepage of nutrients from agricultural soils. AM fungi are not mere providers of phosphorus to plants growing in nutrient poor soils, but rather, they are an important component of the soil-plant system without which no cropping systems will be sustainable. Challenges in
Commercialization, Formulations & Marketing
of AMF Inoculants for Agronomic Crops Abstract Arbuscular mycorrhizal fungi (AMF) are known to increase the root absorption zone and provide the foundation for a healthy soil structure. This results in increased uptake of nutrients, especially phosphorus and contributes to the plant’s overall resistance to environmental stresses, caused by soil salinity, high pH and soil-borne pathogens. In the past, mass production of these mycorrhizal inoculants was difficult to achieve and limited the marketing of these organisms to crops grown in containers for the horticulture market. A few years ago, research has improved the technology for producing these fungi and has increased manifold the number of AMF propagules generated. Not only are the units more productive but the inoculant is more efficient, homogeneous, disease-free and versatile. This breakthrough enabled PTB to look at new markets, like agronomic crops that require larger quantities of inoculants than in horticulture. From the research to the marketing of inoculants is a step by step procedure for either the horticultural segment or agronomic crops. The research and market definition have to be correctly planned in order to develop the right product compatible with the current cultural practices. Product knowledge is the key for educating the future users of the potential benefits of AMF. Teamwork between sales, distribution and research groups out in the field and in the market place allows for adjustment in the product definition and developing future alliances. Examples of benefits and efficiency data obtained with AMF for agronomic crops will be discussed.Seed Coatings and Inoculants Abstract The success of rhizobial inoculants depends to a large degree on the survival of the symbiont before the seed is planted and the ability to compete with indigenous strains. Seed coating was originally developed as a means of increasing the establishment of pasture species that were seeded by aircraft onto noncultivated ground. Since then seed coatings have been widely adapted to become multifunctional coatings in use in production agriculture. A main emphasis is still to ensure that a higher percentage of the seeds planted become plants especially under adverse conditions. Seed coating provides a protective benefit to the rhizobia resulting in improved shelf life. Data will be presented relative to plant establishment, nodule occupancy and emergence as a function of water availability. Innovations and trends in seed coating will be discussed including the use of seed coatings for fluid seeding, temperature controlled polymers, biologicals other than rhizobia, seed sizing and genetically engineered rhizobia. Herbicide Inoculant Interactions Abstract Herbicides represent a major chemical input into most legume production systems and their impact on the legume-Rhizobium association has been extensively discussed in the literature. Despite considerable research, few general conclusions can be drawn regarding the impact of herbicides on symbiotic dinitrogen fixation, owing largely to the seemingly inexhaustible combinations of herbicides, crops, crop varieties, Rhizobium species and strains, and environments – and the interactions amongst all of these factors. Irrespective of the complexity, current research and literature suggests that we must be aware of the potential for herbicide-inoculant interactions to occur, and this awareness should guide our decisions regarding appropriate inoculant strategies to maximize benefits of the legume-Rhizobium association. Effect of Jazz (Trifloxystrobin and
Metalaxyl), Crown (Carbathiin and Thiabendazole), Allegiance FL (Metalaxyl),
Vitaflo 280 (Carbathiin and Thiram), and Apron Maxx (Fluodioxonil and
Metalaxyl) fungicidal seed treatments on Nitrogen Fixation of Chickpea,
Lentil, Pea, and Dry Bean. Abstract Limited information is available on the effects of Jazz, Crown, Allegiance FL, Vitaflo 280, and Apron Maxx fungicidal seed treatments on dinitrogen (N 2) fixation of chickpea (Cicer arietinum), lentil (Lens culinaris Medikus), pea (Pisum sativum), and dry bean (Phaseolus vulgaris). To determine if Jazz, Crown, Allegiance FL, Vitaflo 280, and Apron Maxx have a negative effect on the ability of the Rhizobium to fix N 2 from the atmosphere on chickpea, lentil, and pea, experiments were established at two locations in Saskatchewan in 2002 and 2003. Treatments were i) Rhizobium applied to the untreated control, ii) Rhizobium applied simultaneously to fungicide, iii) Rhizobium applied sequentially to fungicide and, iv) non-inoculated Rhizobium untreated control. Two Rhizobium strains were used. Percent nitrogen derived from the atmosphere was determined by the 15N natural abundance method. Jazz, Crown, Allegiance FL, Vitaflo 280, and Apron Maxx had no negative effects on the ability of the Rhizobium to fix N 2 from the atmosphere. Understanding Gene Function in Rhizobia Abstract The genome of the alfalfa symbiont Sinorhizobium meliloti strain 1021 contains 6,691,694 basepairs. Informatic analysis of this sequence has identified over 6,200 protein coding genes. We estimate that about 2-3000 proteins are involved in the basic biological activities that are required for growth of the bacterial cell in a mineral salts medium. These proteins are similar to proteins found in all other organisms and are involved in processes such as cell wall/membrane synthesis and metabolic processes including amino acid, DNA and RNA synthesis, and energy metabolism. The functions of the over 2,500 of proteins are totally unknown. It is likely that many of these proteins are involved in the life of the bacteria in the soil and the at plant-root interface. We predict that many proteins will be involved in the metabolism of carbon, nitrogen, phosphorous and sulphur-containing compounds. Other proteins could be involved in detoxification of biotic and abiotic compounds, or in interactions with other organisms. I will describe methods we are developing to investigate the biological roles of these proteins of unknown function (PUF proteins). We have made a library of the S. meliloti genome and find that many of these genes are not expressed in normal mineral salts medium with glucose or succinate as carbon sources. I will describe how we are using this library to identify conditions where these puf genes are expressed and how the expression information allows us to predict the functions of the PUF proteins. This work is funded by Genome Canada through the Ontario Genomics Institute and by the Ontario Research and Development Challenge Fund (ORDCF). Nodulation
Specificity & Success
in the Kura Clover-Rhizobia Symbiosis E-mail: philippe.seguin@mcgill.ca Abstract Kura clover (Trifolium ambiguum M.B.) is a perennial rhizomatous forage legume whose use is currently limited by establishment difficulties in part attributable to nodulation problems. It has long been reported to have very specific rhizobial requirements. It is not nodulated by rhizobia that nodulate other Trifolium species and its rhizobia usually do not effectively nodulate other Trifolium species. Specificity was also reported among various ecotypes and ploidy levels within T. ambiguum (i.e., diploid, tetraploid, and hexaploid). These observations are, however, based on a limited number of rhizobia most originating from only two regions in the Caucasus. Soils outside the natural distribution range of Kura clover (i.e., the Caucasus and Eastern Europe) rarely contain indigenous rhizobia that can effectively nodulate this species. Therefore, when Kura clover is introduced in other regions, rhizobia must also be introduced. However, of the limited number of strains currently available, most have a limited effectiveness. Use of these strains often results in slow nodulation and low N 2 fixation, which limit plant growth. In addition to nodulation limitations, another factor that may have slowed Kura clover utilization is a concern with the ability of Kura clover rhizobia to ineffectively nodulate related species of clover including white clover (T. repens L.). This undesirable characteristic combined with the highly competitive nature of Kura clover rhizobial strains currently used could be detrimental to white clover production. It is thus essential not only to identify rhizobial strains with a greater effectiveness with Kura clover, but also strains that will not be detrimental to white clover. Our work in the last five years has addressed many of the above-mentioned points. We first characterized and evaluated 12 newly isolated North American strains and 6 previously available strains, including those used in commercial rhizobial inoculants. Diversity observed among the 12 North American isolates was limited. These were however often more efficient than strains used in commercial inoculants. In a growth chamber study, they resulted in 45% greater foliage dry matter (DM) than the best commercial strain tested. Some of the strains currently used in commercial inoculants failed to effectively nodulate Kura clover plants. North American isolates tested in field trials (i.e., CT1-1, CT1-2, and WI4-4) overall increased total DM accumulation by 27% when compared to commercial inoculant strains. While these strains appear to have potential they still produced less DM than non-inoculated N-fertilized controls. This thus lead us to evaluate 128 rhizobia isolated from Kura clover center of origin (i.e., Azerbaijan, Armenia, and Northwest Iran) using the three ploidy levels of Kura clover, red clover (T. pratense L.), and white clover plants as trap hosts. Diversity among the 128 isolates was large and similar for rhizobia grouped according to their geographic origin or original host plant. Isolates did not cluster according to their original host. N odulation specificity of the Kura clover-nodulating rhizobial isolates studied was less complex and not as clearly delineated as previously reported. Some strains originally isolated from Kura clover could effectively nodulate more than one ploidy level of Kura clover and even one or both of the two other Trifolium species evaluated. Three isolates (ARH2-1, ART1-2, and IRT10-1) formed effective nodules on both Kura clover and white clover, however none promoted plant growth of both species to levels currently obtained with highly effective strains. Rhizobial isolates that are highly effective with both species remain yet to be identified. Contribution
of Biological Nitrogen Fixation to the N Nutrition of Grain Crops in
the Tropics: The Success of Soybean (Glycine Max L.
Merr.) in South America Abstract The high cost of mostly imported N fertilizers in South America has necessitated an approach to crop production that emphasizes biological nitrogen fixation (BNF). We will discuss the contribution of symbiotic nitrogen fixation to the production of soybean (Glycine max (L.) Merrill) in South America, with an emphasis in Brazil and Argentina. Soybean was introduced into South America at the end of the 19 th century, but significant commercial crop expansion only began in the 1960s. Today the area under cultivation accounts for almost half of world soybean production. BNF has always been a significant concern in this production system with programs for both strain selection and the identification of plant genotypes with superior symbiotic performance beginning in the late 1960s, and still a major research focus. The expectation is that strain selection matched to genotype improvement should permit yields of 4,000 kg ha -1 or more, without the need of N fertilization. Nowadays most soils cropped with soybean have high Bradyrhizobium populations from previous inoculations, but also exhibit significant diversity. This includes variation in morphological, physiological, genetic and symbiotic traits resulting from adaptation to the soils, as well as horizontal gene transfer. More efficient and competitive strains have been identified and selected from this population. Positive responses to reinoculation are reported in soils with 10 3 cells g -1 of soil or higher, such that in 74 field trials performed in Argentina yield was enhanced by a mean of 14% in comparison to the non-inoculated treatment, while in 29 field experiments performed in Brazil reinoculation increased yield by 8%. In Brazil, rates of nitrogen fixation in soybean can exceed 300 kg of N ha -1, providing from 69 to 94% of total plant N. Benefits due to the release of N to the following crop have also been reported. Starter- or post-flowering N fertilization has not resulted in increased yield, emphasizing the efficiency of the biological process. The increased use of micronutrients and especially fungicides in contact with the inoculants is an ongoing problem, with the survival of Bradyrhizobium on the seed sometimes drastically affected. High soil temperature and low soil moisture contents may also limit biological nitrogen fixation in the tropics, but no tillage systems can appease these stresses. Most importantly, the strategies reported here may be applied in developing or industrialized countries, by large or small landholders, and for subsistence or cash legume crops. Key words: Bradyrhizobium, inoculants, nitrogen fixation, nodulation, soybean. Best Crop Management
Options to Enhance Nodulation in Annual Pulses Abstract The acreage of annual pulse crops (namely field pea, lentil, chickpea, and dry bean) has expended rapidly in the past ten years in western Canada, and the production of field pea and lentil, for example, has increased by > 10 folds. Adaptation of best crop management options will ensure the optimization of symbiosis and enhance N 2-fixation. In soils of western Canada, indigenous rhizobial populations are usually low, and the formation of symbiosis largely depends on the application of appropriate rhizobial strains at planting. Studies conducted in western Canada have shown that carriers, formulations, and delivery systems of rhizobial inoculants directly affect the symbiosis, whereas the intensity of N 2-fixation of the symbiosis is related to soil texture, moisture availability, starter N, and stubble type prior to growing a pulse crop. Use of inoculants increased the number of nodules and nodule weight in field pea and lentil by 5 to 160% compared to the respective, non-inoculated checks. Application of low doses of starter N lowered nodulation but it encouraged vigorous plant growth during vegetative period, resulting in increased biomass and infrequently increased seed yields. Soil-applied granular forms of inoculants outperformed seed-applied peat-based inoculants by 3 to 60% in pea and lentil (measured by plant biomass and seed yield). With the same rhizonial strains, lentils grown on fine-textured clay soils produced significantly more nodules, greater nodule weight, and higher seed yields than the crop grown on medium-textured loam soils. When soil moisture is continuously available in the later part of the growing season, chickpea plants inoculated with granular inoculants continued N-fixation, resulting in 8 to 21 days of delay in maturity compared to the plots receiving 30 to 80 kg N ha -1 of fertilizer. The long-season chickpea matured 2 to 7 days earlier when grown on barley stubble compared to summerfallow. Development and use of best crop management options is the key to enhance nodulation in these pulse crops. Key words: soil type, moisture availability, soil texture, nodulation, stubble type, inoculant formulation. Alternate
Crops On The Prairies - Lupin and Faba Bean: New Crops with
New Needs Abstract Narrow-leafed lupin (Lupinus angustifolius L.) and zero-tannin faba bean (Vicia faba) have the potential to meet the growing demand for vegetable/plant protein. Preliminary screening tests, in 2002 and 2003, have identified faba bean and lupin as being adapted to the Parkland and Peace regions of Alberta. Extensive agronomic testing of these two crops began in 2004 to determine optimum production requirements. The three-year agronomic research program is supported through Alberta Agriculture, Food and Rural Development – LFARI (Lupin and Faba Bean Agronomic Research Initiative) and ACIDF Emerging Pulse Opportunities – Tannin Free Faba Beans and Lupin: “The Alberta Protein Advantage”. Successful commercial production of these crops requires the identification of production practices that will ensure high quality faba bean and lupin supplies. There are two major components of the research program. One part of the program is investigating optimal seeding rates, responses to weed competition, nitrogen and non-nitrogen benefits to subsequent cereal crops. The other section of the program is studying a multitude of agronomic factors ranging from: varietal evaluation, post emergence broad leaf herbicide crop tolerance, inoculant strain efficacy, seed-seedling-foliar disease identification and control, seeding date and seed size effects along with phosphorous, potassium and sulfur nutrient requirements. Despite a challenging growing season we achieved successful, but variable, lupin and faba bean seed production. The 2004 growing season began with cold soils, followed by extreme rainfall events in July. Harvest was plagued by rain, snow and frost resulting in a short harvest season. Despite the challenges, all research sites throughout the Parkland and Peace regions were harvested with acceptable seed quality. Narrow-leafed lupin yields ranged from 1000 kg/ha to 3500 kg/ha. Although, lupin production was successful and shows great potential, there are a number of production challenges related to current lupin agronomy, which need to be address. These challenges include low emergence rates, blister beetle pest pressure, pod shattering losses at harvest and no registered herbicides. Zero-tannin faba bean yields ranged from 1000 kg/ha to 7000 kg/ha. Faba bean produced higher seed yields than either field pea or lupin in good growing environments. In growing environments with reduced rainfall, faba bean seed yields were similar or lower compared to field pea, indicating faba bean’s higher moisture requirements. Some of the most interesting findings in this research project were observed in the inoculant strain efficacy tests. In this study, nodulation and yield responses of zero-tannin faba bean and narrow-leafed lupin to eleven inoculants were investigated. Faba bean yields appeared to be unaffected by the various inoculants tested. Lack of faba bean response from inoculation may have been due to previous field pea production. Two of the inoculants tested in the study were lupin specific inoculants developed, for this test, by the Nitragin Co. ( Milwaukee, Wisconsin). The lupin specific inoculant (Nitragin Lupin type H) contained rhizobium lupini and was provided in a granular form (Soil Implant) and in a peat based form. Regardless of formulation, the lupin specific inoculants increased lupin yields by 20-40% compared with the other inoculants tested. Based on these first year results, the rhizobium lupini(type H) inoculant appears to be critical for the production of high and stable narrow-leafed lupin yields. Overall, the lupin and faba bean research projects in 2004 were a tremendous success despite the weather. Success on a “bad” year suggests a bright future for these crops in the Parkland and Peace regions of Alberta. Development of “Alberta Made” agronomic packages for these crops and removal of agronomic barriers will allow for rapid commercialization of zero tannin faba bean and lupin in the future. Inoculants in South
America, Industry Perspective Abstract The use of inoculants in South America is very significant and the volumes produced in South America exceed the total volume of those produced in North America due to the high usage rates in the major soybean markets. Soybean inoculant dominates any discussion of South American inoculants. Each market is regulated to some extend with certain markets being highly regulated as in the case of Brazil. Inoculant technologies are on par or exceed those that experienced in North America for soybean crops as it relates to inoculant concentration guarantees, shelf life, and stated compatibility with fungicides. Pre-inoculant technology is advanced in certain countries, especially Argentina. Micronutrient usage as a seed treatment along with inoculants is highly common in Brazil. Based on acreage development in soybeans, the inoculant industry is expected to continue to grow with only those companies at the higher end of the technology paradigm able to compete long term. Inoculants
Plus Chemical Enhancers Abstract Microbial inoculants have been used for over a century. During that time there has been substantial progress in inoculant technologies. Currently, a combination of forces (increased fossil fuel prices, environmental damage associated with N fertilizer, increased understanding of microbial genetics and plant microbe communications) conspire to make the future for “biofertilizers” a very bright one. Research over the last two decades has shown that bacteria are skilled and frequent communicators. The rhizobia-legume nitrogen fixation system has come to be the best understood example of plant-microbe communication, with the two partners exerting profound effects on each others gene expression and, in the case of plants, organ development. This inter-organismal communication is the first step in the establishment of the symbiosis. It involves the excretion of flavonoids and related compounds from the plant, which activate the microbial nod genes. The bacteria produce Nod factors (lipo-chitooligosaccharides – LCOs) in response, and these activate nodulin genes in the plant roots. We have shown that this signal exchange is disrupted by low temperatures, and some other environmental factors, and that adding flavonoid-like compounds can overcome part of this inhibition. We have recently found other set of plant-to-microbe signals that are more effective than the flavonoid-like compounds. We also found that inoculants producing more LCOs caused accelerated seedling emergence in the field. This effect was present before any effects on nodulation should have been manifest. Subsequent research showed that treatment with LCOs increases plant photosynthetic rates and growth. Finally, we have recently been searching for other bacteria-to-plant signals, in this case, from plant growth promoting rhizobacteria, and have found a set that, at least upon first assessment, seem to show activity similar to the LCOs. New
Patented Growth Promoter Technology to Enhance Early
Season Soybean Development & Grain
Yield Abstract New soybean growth promoter technology based on the U.S. patents 5,549,718 and 5,646,018 has been developed and field tested. The natural bacterial promoter compound has been purified, formulated and assayed via HPLC to provide field efficacious concentrations ranging from 0.000001 to 0.001% w/v of the active lipo-oligosaccharide compound (nod factor). Purified material has been applied as a liquid formulation to soybean seed at the time of planting. Multi-year field evaluations have demonstrated an early season growth enhancement that includes improved stand, early season vigor, earlier root nodule formation with both applied and indigenous Bradyrhizobium japonicum independent of soil temperature, earlier canopy closure and improved grain yield. A liquid formulation has been commercialized (Optimize TM) that utilizes Cell-Tech ® as the carrier, and is custom applied on seed at a retail facility to ensure uniform application and grower convenience. Cell-Tech included as the carrier, with Bradyrhizobium japonicum, ensures sufficient nodulation on fields with or without a history of soybean production. The liquid formulation is applied to soybeans at the rate of 125 ml/45.4 kg seed. The material may be combined with compatible fungicides and applied 30 days before planting. The Optimize formulation in the U.S. has increased plant stand compared to the control from 70.8 to 76.4%, vigor (1 to 9 relative scale) from 4.8 to 6.5, nodulation from 21.1 to 24.4 nodules per plant, decreased days to canopy closure from 70.4 to 67.4, and increased grain yield by 343 kg/ha (10.1%). Four trials in Argentina in 2003 provided an average grain yield increase of 402 kg/ha (12.2%). The Use
of Legume/Rhizobial Signals for Yield Enhancement in
Non-Leguminous Crops Abstract Over the last 10-15 years, considerable advances have been made in understanding the biochemical and physiological processes involved in infection, nodulation and nitrogen fixation within legumes. This work has elucidated the role of isoflavanoids and lipo-chitooligosaccharides (LCOs or nod-factor) as critical signaling molecules between the plant and the bacterium, responsible for triggering the start of these processes. This has opened avenues for the development of improved rhizobial inoculants which incorporate these molecules and lead to earlier emergence, nodulation and increased yield. Building on earlier work at McGill University, Agribiotics has recognized a unique agricultural and horticultural role for the LCO technology. LCO in foliar application to major non-leguminous crops has the potential to produce significant yield increases – when applied at specific plant development stages. In this presentation, we will summarize, for the first time publically, the evidence from three years of field and greenhouse studies which demonstrates the potential of this technology for yield induction. This work has led to the filing of new patents and the development of a completely new technological platform within the Company. Potential Use
of Rhizobium as PGPR with Non-Legumes Abstract The beneficial effect of the symbiotic association between rhizobia and legumes is well known. In the last 10 years, many studies reported that rhizobia can also form beneficial association with other economically important grain (maize, rice and wheat) and vegetable (lettuce and radishes) crops. There is also evidence that rhizobia are natural endophytes of some crops and that they can colonize roots of several plants. Rhizobia also show in vitro pgpr characteristics, and the mode of action of PGPR activity in association with plants has been established in few reports. However, when screening rhizobia for beneficial effects, some isolates are neutral or show a deleterious effect. Thus, the selection of rhizobia with both PGPR activity and efficient symbiotic nitrogen fixation should be advantageous in crop rotation or intercrops systems using legumes and non-legumes. Some examples of rhizobial strain selection for clover-wheat and maize-soybean rotations will be presented. N-Fixation
Non Legumes Abstract Several lines of evidence will be presented for nitrogen fixation in wheat provided by Klebsiella pneumoniae 342 (Kp342). Kp342 enters the interior of many plants in very high numbers compared to other enteric bacteria tested strains and was originally isolated from a nitrogen efficient line of maize. Recent work has shown that the number of endophytic bacteria within the plant is regulated by plant defense responses. Kp342 also enhances plant growth by a means independent of nitrogen fixation. Evidence will be presented on the mechanism of this growth enhancement. The mechanism of entry of Kp342 into plants is also being studied. To further understand the plant's response to endophytic colonization, proteomics is being used to identify those proteins whose levels change in response to endophytic colonization. Finally, a discussion on the progress toward nitrogen fixation in agronomic grasses will be presented. Rhizobium Strain
Selection: Differing Needs for Agricultural Plants
and in Revegetation Settings Abstract In 1800 there were more than 7 million ha of prairies in Minnesota; now less than 0.5% of that remains. Prairie reestablishment activities undertaken by state agencies, non-governmental organizations, and private groups commonly involve a number of legume species, but inoculation of these species with rhizobia poses several problems not common in an agricultural setting. These include lack of suitable inoculant strains and formulations, delays between seed inoculation and legume germination, differences in fertilization, and the effects of freezing and thawing on Fall-planted seed and inoculant. We have trapped more than 700 rhizobia for Amorpha canescens, Astragalus canadensis, Chamaecrista fascilulata, Dalea purpurea and D.candida, Desmanthus illinoensis, Desmodium canadense and Lespedeza capitata from Department of Natural Resources sites in Minnesota, and have identified inoculant-quality rhizobia for each host. When these isolates were used in a prairie situation the legumes considered derived from 30 to 100% of their nitrogen from fixation, with 57-65% of the rhizobia recovered from Dalea having a genetic fingerprint identical to that of inoculant strains. Cross inoculation between Amorpha and Dalea hosts and their rhizobia has been observed in both natural prairies and in revegetation settings, with the fast-growing rhizobia of the prairie legumes found in this region exhibiting significantly more promiscuity in symbiosis than was anticipated. It is intriguing that rhizobia recovered from Dalea and Desmanthus in prairie plantings in Minnesota have been identified as belonging to the species R.gallicum and R.giardinii. Both rhizobia have only previously been recorded as microsymbionts for Phaseolus vulgaris. Use of winter wheat as a cover crop, and as an alternate carrier for inoculant rhizobia is being studied, with differences evident between wheat cultivars in rhizobial survival and multiplication in the rhizosphere after planting. Barriers, at the Level
of Plant Breeding, Preventing the Release of Symbiotically
Efficient Cultivars. Abstract Symbiotic nitrogen fixation in crop legumes is a complex interaction of Rhizobial strain (or related species), host and environment. Despite extensive research efforts globally, few superior nitrogen (N) fixing cultivars are available. The few successful outcomes from breeding programs are reviewed to demonstrate that the barriers of complexity, inappropriate methodology and experimental approach, which hinder the development of symbiotically efficient cultivars, can be overcome. Growing legumes in soils with high nitrate and ammonium availability causes crop growth to rely on N uptake, not N fixation; and stress reduces N fixation drastically. Superior cultivars for bean (Phaseolus vulgaris), soybean (Glycine max), chickpea (Cicer arietinum) and pigeon pea (Cajanus cajan) have resulted from a range of strategies but with common themes. These are screening for the best N fixing genotypes in low N soils in the presence of an effective strain; screening for high N fixation in the presence of nitrate; screening for high nodulation; and promiscuous fixation in the case where strain choice cannot be controlled. One case study involved seeking improved fixation in stress by a feedback mechanism. Superior N fixation is found from effective nodulation and fixation (many large nodules, long duration nodules, supernodulation, and highly efficient strains). Selecting genotypes in low N fields ensures highest reliance on N fixation and increases the probability of detecting superior N fixation. The strategy of selecting for superior N partitioning involves selecting for greatest crop and N biomass at pod-fill, high N harvest index and crop harvest index. Superior nitrate tolerance is found by selecting the best N fixing host and strain combination in the presence of moderate or high nitrate concentrations. High replication in screening trials is required to reduce variation because N fixation trials require low N soils and yields are lower than in conventional trials. For nodulation, the successful techniques are measuring nodule mass and number. For fixation, the best two practical measurements appear to be biomass and harvest index, followed by rapid screening methods such as xylem and shoot ureides in soybean. EFFECT OF CELLS NUMBER ON SOYBEAN
NODULATION E-mail : rjcampo@cnpso.embrapa.br. Abstract Proper inoculation with liquid- or peat- based inoculant is not warranty of adequate nodulation in soils of first-year cropping and under different stress conditions. Nevertheless, increases in the number of viable bradyrhizobia cells may result in higher nodulation and N 2 rates under such conditions. This study aimed at providing the maximum number of viable bradyrhizobia cells in the soybean rhizosphere. The trials were set up for three years, following all technical recommendations and using a complete randomized block design with six replicates, in first-year cropping areas (<100 bradyrhizobia cells/g soil) of Brazil, under five ecological conditions. Different amouns of peat-based inoculant were used to provide different concentrations of viable Bradyrhizobium. Peat inoculants had a minimum of 1x10 9 cells/g and were composed of two strains of Bradyrhizobium. Nodulation (nodule number and dry weight), increased with the number of cells applied, however, the optimal concentration of cells varied with the ecological condition from each site, and with the strains used. In Luziânia, 500,000 cells were necessary to achieve the highest nodulation, but in Cristalina and Taciba nodulation was still increasing when applying 2.1 and 1.2 million of cells, respectively. In Jaciara and Lucas do Rio Verde, 700,000 cells of strains SEMIA 587 plus SEMIA 5019, and one million of cells of strains SEMIA 5079 plus SEMIA 5080 were demanded to achieve the highest nodulation. An analysis of all results obtained indicated that in general a concentration around 1.2 million of viable cells/seed is necessary to optimize nodulation in soybean. Mycorrhizal inoculum production
technology for increasing the productivity of forest plantations Abstract Although numerous studies have demonstrated that mycorrhizal fungi could improve the growth and survival of many forest plants, the use of inoculation to utilize mycorrhizal technology in plantation forestry is not extensive in Canada. Effective utilization of mycorrhizal biotechnology in forest plantations and agricultural practices depends on the availability of high quality inoculum, improved technology for application of inoculum and an understanding of benefits of using mycorrhizal inoculum for plant development. Large-scale exploitation of mycorrhizal inoculum in improving plantation yields requires demonstration of economic and environmental benefits and acceptance of the inoculation process, which is compatible with the existing aspects of production system. Symbiotech Research Inc. has developed Treeboost, an ectomycorrhizal fungal inoculum that has the potential to improve afforestation and reforestation activities in Canadian Prairies, particularly in difficult sites. This poster reports the various forms of ectomycorrhizal inocula that are currently available. This includes a new technique for inoculum production, an explanation of their production processes and an evaluation of the potential of using different inoculum in commercial nurseries.Illinois Bundleflower:
Response to Inoculation and Microsymbiont Biodiversity Abstract Agriculture in the Midwestern USA is increasingly characterized by fewer crops in the landscape. In this context, attempts are made seeking for alternative land uses and new crops. The inclusion of nitrogen-fixing legumes in agroecosystems can provide environmentally friendly supply of this nutrient, mitigating agriculture-related environmental degradation. Illinois bundleflower [Desmanthus illinoensis (Michx.) MacMillan], a perennial herb native to the central and Southeastern USA, enhances overall biomass yield in mixed legume/grass pastures, and is a valuable alternative grain and forage crop. However, inoculant-quality rhizobial strains have not been identified for this plant. We trapped Desmanthus illinoensis rhizobia from sites within the native range of Illinois bundleflower, and determined the effect of inoculation in growth chamber, greenhouse, and field. The microsymbiont genetic diversity within the geographic range of the host was also addressed in this study. Three plant accessions showed striking responses in plant dry matter production to inoculation at both Salina ( Kansas) and Becker ( Minnesota) in the seeding year. Toward the northern extreme of the range for Illinois bundleflower, at Becker, only a Minnesotan adapted ecotype overwintered and, to our surprise, inoculation was a requirement for plant persistence. The biodiversity of Illinois bundleflower rhizobia was studied using rep-PCR fingerprinting, host range, cultural characteristics of isolates, and 16s rRNA gene-sequence analysis. Unexpectedly, inoculant-quality strains selected in this study clustered with Rhizobium giardinii. Illinois bundleflower thus becomes the second reported host for this species, and the only host to date on which it is effective. Influence
of Pulse Crops on Arbuscular Mycorrhizal Fungi in a Durum-Based Cropping
System Abstract Pulses are an important component in crop rotations in southern Saskatchewan. Besides their capability to fix nitrogen, pulse crops establish a symbiotic relationship with arbuscular mycorrhizal (AM) fungi, which have been shown to increase nutrient and water uptake through hyphal extensions in the soil. Incorporating strongly mycorrhizal crops in a rotation may increase inoculum levels in the soil and benefit the growth of a subsequent crop. This 2-year study was designed to evaluate the impact of including pulses in crop rotations on the soil AM inoculum, mycorrhizal root colonization, and N and P uptake in a crop of durum wheat, grown the subsequent year. Preceding crops of pea, lentil and chickpea were compared to canola and durum. Plant development was monitored and soil sampling was done at emergence, at the 5-leaf, flag leaf, anthesis and physiological maturity growth stages of durum wheat. In 2004, preceding crop had an effect on the soil AM inoculum, as revealed by differential (P<0.0001) AM root colonization ranging 6 % after canola to 31 % after lentil, at peak development. Durum wheat P content varied with preceding crop even though no significant difference between treatments was detected in soil available N and P levels. Durum wheat P uptake was correlated with AMF colonization. We conclude that a preceding crop can influence a subsequent crop’s growth through modification of the soil AM fungal inoculum. The
Lupin Industry in Alberta: The Pioneering Years Abstract Lupin seed has the potential to meet a growing demand for vegetable/plant protein. A research program has been initiated to test this new crop in Alberta. Preliminary screening tests, in 2002 and 2003, of Lupinus angustifolius, narrow leafed lupin cultivars from around the world, identified cultivars from Eastern Europe as being adapted to the Parkland region of Alberta. More extensive agronomic testing of these cultivars began in 2004, with the goal of determining optimum production requirements. Factors tested at three sites in north-central Alberta included lupin seeding rates, lupin response to weed competition, and a range of weed control strategies. Successful seed production (2.2-3.5 t ha -1), achieved in an absence of weed pressure and under good growing conditions, was obtained with actual plant populations of 100-150 plants m -2 (preliminary data). Possible weed control strategies (early applications of graminicides, and broadleaf weed control with reduced rates of metribuzin) were also identified. However, unforeseen challenges, some of which appear to be unique to Alberta, were also encountered. These challenges include low emergence rates, Blister Beetle pest pressure, and pod shattering losses at harvest. Research will be extended in 2005 and 2006 to address these issues, including testing seed treatments, insecticide applications and alternative harvest management tools. With the cooperation of scientists to overcome the agronomic hurdles, and government agencies to work on herbicide registration, there is a bright future ahead for the lupin industry in Alberta. Azospirillum brasilense field
inoculation of cereal crops the Pampas region, Argentina Abstract Beneficial effects of inoculating with Azospirillum brasilense strains on crop productivity has been widely described. However, field inoculation of grain crops in extensive production systems is not well documented. Our objective was to evaluate the effects of on-seed inoculation with a liquid formulation of Azospirillum brasilense on early growth and grain production of 211 wheat (Triticum aestivum L.) and 45 maize (Zea mays L.) farmer´s crops in the Pampas region, Argentina. The study was performed during the 2002/03, /03 and /04 growing seasons under regular crop management practices (N and P fertilization, fungicide treated seeds, high yielding genotypes, etc.). The inoculant contained 10 8 cfu ml -1 of Azospirillum brasilense strain Az39 provided by INTA (National Institute for Agricultural Technology, Argentina). In both crops, the seeds were inoculated before planting with a liquid formulation produced by Nitragin Argentina S.A. (Pilar, Buenos Aires, Argentina) at the rate of 10 ml kg -1. In most of the sites, the inoculation with this liquid formulation of Azospirillum brasilense increased the root and shoot early growth and the grain number of the crops. In 81 % of the wheat field trials, on-seed inoculation increased grain yields by 347 kg ha -1 (11%). Also in most of the corn field trials, the inoculation treatment increased grain yields by 394 kg ha -1 (5%). These effects were independent of the crop management practices (N and P fertilization, on-seed fungicide treatments, etc.) and production areas within the Pampas. In general, more response to the inoculation was described in the absence of major crop growth limitations (i.e. drought, diseases, etc.). Similar results were also described in similar field trials performed in Paraguay and USA. We conclude that the use of this liquid formulation providing Azospirillum brasilense contributes increasing wheat and corn grain yield in production systems from the Pampas region, Argentina. Sinorhizobium
melilotitpiA1 mutants
have a novel plant phenotype Sinorhizobium meliloti is a Gram negative soil bacterium capable of forming nitrogen fixing nodules on alfalfa plant roots. Mutants of a related species (Rhizobium leguminosarum) that are unable to catabolize the methyl-pentose rhamnose, are not as competitive for nodule formation as wild-type. To test if this is true for rhamnose catabolism mutants of S. meliloti, a Tn5 mutagenesis of wild-type strain Rm 1021 was carried out and mutants were screened for the ability to grow on rhamnose as a sole carbon source. One of the isolated mutants, SRmA185, grew slower than wild-type on rhamnose as a sole carbon source and did not grow on glycerol. The mutation was characterized and shown to interrupt the gene tpiA1. This gene is annotated as encoding for a triose phosphate isomerase. The genome of Rm 1021 also has a gene tpiA2 which is annotated as encoding for a triose phosphate isomerase. To test the hypothesis that tpiA1 and tpiA2 encode triose phosphate isomerase enzymes, cell free extracts were generated from Rm 1021, tpiA1 and tpiA2 mutants cultured with several types of growth media. The results support both tpiA1 and tpiA2 encoding triose phosphate isomerases. Enzyme activity above background was observed for tpiA1 mutant cell free extracts cultured with erythritol, which is evidence for a second triose phosphate isomerase induced by erythritol (tpiA2). To test if tpiA2 can compensate for the lost triose phosphate isomerase in tpiA1, a tpiA1 - strain was plated on glycerol media supplemented with minimal amounts of erythritol. The resulting restoration of growth on glycerol suggests that tpiA2 can substitute for tpiA1 under some circumstances. Nodulation competition experiments showed that tpiA1 mutants were as competitive as wild-type. During experimentation it was observed that growth of tpiA1 - inoculated plants appeared greater than those inoculated with wild-type. Plant growth experiments were replicated four times and on average, plants inoculated with tpiA1 mutants showed a 19±3.5% increase in dry weight.
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