Dulce n. Rodriguez - Inoculantes para leguminosas
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Los días 20 y 21 de mayo de 2014, la Fundación Ramón Areces organizó el Simposio Internacional 'Microorganismos beneficiosos para la agricultura y la protección de la biosfera' dentro de su programa de Ciencias de la Vida y de la Materia.
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Dulce n. Rodriguez - Inoculantes para leguminosas
- 1. Legume Inoculants International Symposium: Beneficial Microbes forAgriculture and Biosphere Protection Fundación Ramón Areces DN Rodríguez-Navarro & FJTemprano IFAPA, Centro Las Torres-Tomejil Junta de Andalucía
- 2. Definition of Microbial inoculant: An inoculant is a biotechnological product based on microbes (bacteria, fungi or virus) formulated to render a commercial product. They are mostly used in Agriculture with different purposes: increase crop production and crop protection. Mycorriza-VA Biofungicides Biopesticides Bioremediation Rhizobium
- 3. Scheme to obtain a commercial microbe inoculant Sampling and selection Isolation: pure cultures Identification and characterization Selected strains PRESERVACION Biological test (in vitro, in vivo) Field experiments or Scale-up procedures Industrial Multiplication Formulation and storage conditions Environmental impact and toxicity Distinct analitycal methods REGISTRATIONREGISTRATION COMERCIALIZATIONCOMERCIALIZATION
- 4. Especificity of the host Especificity of the host N2-fixation efficiencyN2-fixation efficiency CompetitivenessCompetitiveness There is not an universal rhizobia able to nodulate with all legumes. Most of the rhizobia have a narrow host-range. N2-fixation can vary from ineffective (Fix-) to highly effective (Fix+) strains The ability of the inoculant´s strain to form the majority of nodules in the presence of specific soil rhizobia Symbiotic properties of rhizobia
- 5. Field experiments: selection of Trifolium (left) and soybean (right) inoculants Greenhouse experiment controlled conditions: selection of bean (left) and G. soja (right) strains.
- 6. • Lack of native specific rhizobial populations for the crop • Absence of extreme conditions: acidic soil , temperature, etc. • Low content of mineral soil-N Environmental conditions favorable for legume inoculation successEnvironmental conditions favorable for legume inoculation success
- 7. (1) Number of nodules / plant Inoculant Number of nodules (1) DW nodules (mg) Yield (Kg/ha) Grain N (Kg/ha) USDA 110 52.1 b 269.9 a 4.010 ab 234.4 a SMH12 71.0 b 235.9 b 4.335 a 245.8 a A 8318 99.2 a 274.1 ab 3.239 ab 162.5 b A 8425 118.7 a 291.8 a 3.529 ab 164.1 b HH 29 103.0 a 270.2 ab 3.454 ab 171.4 b TN 0 c 0 c 3.162 b 174.4 b T 0 c 0 c 1.683 c 82.3 c Response of soybean cv. Osumi to inoculation with S. fredii inoculants. Location: Las Torres Experimental Station. Alcalá del Río-Seville, SW-Spain
- 8. Effect of inoculation on soybeans growing in a soil devoid of specific rhizobia populations. Guadalquivir Valley (Sevilla)
- 9. Inoculant NNOD (1) DW nodules (mg) Yield (Kg/ha) Grain N content (Kg/ha) USDA 110 59.2 a 221.9 a 5.176 a 296.3 a SMH12 61.4 a 230.8 a 4.683 a 237.0 a A 8318 61.5 a 151.3 a 4.578 a 287.0 a A 8425 62.2 a 150.5 a 5.421 a 302.4 a HH 29 63.2 a 185.5 a 5.140 a 282.4 a TN 41.4 a 141.5 a 5.085 a 295.0 a T 50.0 a 193.7 a 5.380 a 301.5 a (1) Number of nodules / plant Response of soybean cv. Osumi to S. fredii inoculants. Location: Guadajira Experimental Station, Badajoz-Spain. 2001
- 10. Non-effect of inoculation on soybeans growing in a soil containing specific rhizobia populations. Guadiana Valley (Badajoz)
- 11. Inoculant No. Nodules studied % inoculant ocupancy SMH 12/SBP 120 6.7 A8318/SBP 126 3.9 A8425/SBP 120 0.8 HH 29/SBP 113 17.7 Competition of S. fredii inoculants against soil bradyrhizobia populations. Soybean, cv. Osumi SBP, soil bradyrhizobia populations ?
- 12. Solid and Liquid legume inoculantsSolid and Liquid legume inoculants formulationsformulations
- 13. Different carriers for solid-inoculants formulations Several clays: perlite, sepiolite , talk, etc.
- 14. Nitragin Combined is a mixture of • Liquid Inoculant: Cell Tech • Fungicide: Protreat-2 PEANUT INOCULANT Liquid inoculant recommended for soil- inoculantion Number of bacteria per seed: >2000 Strength of the pellet (IRF): 89% Nodulation: % nodulated plants
- 15. LIQUID INOCULANTSLIQUID INOCULANTS EASIER TO ELABORATE AND HANDLE BY FARMERS LOW-COST PRODUCTION LESS PROTECTION OF RHIZOBIUM ON SEEDS FORMULATION: INDUSTRIAL PATENTS. LACK OF INFORMATION
- 16. 1. 109 cells/g of viable rhizobia. 2. Less than 106 contaminant organisms per gram inoculant. 3. Must include data on plant infection tests. 4. Specification on package should contain the above information as well as: Specify the crop for which it is meant The manufacturer’s details Storage and transport conditions Composition of the carrier Expiry date Recommended dose and instruction on how to apply the inoculant. Quality Control protocol The inoculants being supplied for N2-Africa project are expected to meet the following criteria:
- 17. SMH12 (S.fredii) 0 1 2 3 4 5 6 7 8 9 10 11 0 10 20 30 40 50 60 70 80 90 100 días logbacterias/ml USDA110 (B.japonicum) 0 1 2 3 4 5 6 7 8 9 10 11 0 10 20 30 40 50 60 70 80 90 100 días logbacterias/ml Vincent-manitol Vincent-glicerol Álvarez-manitol Álvarez-glicerol Bergersen-manitol Bergersen-glicerol Liquid Inoculant (Microbial cultures + Preservatives) Las formulaciones líquidas presentan una menor supervivencia sobre las semillas. Búsqueda de sustancias protectoras frente a la desecación Glycerol days days
- 18. Inmovilization of microbial cells in calcium alginate beads by cross-linking technique
- 20. Thanks for your attention
- 21. 4406 4839 5016 5220 3800 4000 4200 4400 4600 4800 5000 5200 5400 Rendimiento (kg / ha) Gráfico: Diferencia de rendimientos (kg / ha) Trat.1 Trat.2 Trat.3 Trat.4 Soybean cv Asgrow A4613 yields (Kg/ha) Treat.1 Uninoculated control Treat. 2 Phosphate-Fertilizer: 85 Kg/ha Treat. 3 P-Fertilizer+B. japonicum (E109) Treat. 4 Fertilizer+B. japonicum+A. brasilense (AZ39)
Notas do Editor
- The top master of commercial microbe inoculants are those based on rhizobial strains; dating back to 1896 when liquid formulations were commercialized in USA. In words of Prof. Catroux: rhizobial inoculants are the best success example of the Applied Microbiology. Other important microbe-inoculants for Crop Production (mainly destinated to cereals) are those based on Azospirillum strains, there is a top review 1994 by Okon & Labandera-González where reported the success and constraints of 20 years worldwide Azospirillum inoculation under field conditions. In this Symposium, tomorrow we will have a conference about: Inoculants for cereals (Y. Bashan). BCA´s: the first bacteria Agrobacterium radiobacter strain K84, was registered with EPA for control of crown gall in 1979. Ten years latter, the fungus Trichoderma harzianum ATCC20476 was registered with EPA for control of a plant disease. New formulations are being worldwide used and marketed for different target pathogens and pests, nowadays being an emergent topic in Crop Protection. Again, tomorow we will discuss their used after the conference of Prof. E. Montesinos. Mycorhizal inoculants have lasted more to being released into the market. In this Symposium we also have an expert on this commercial formulations. In addition to Prof. Barea. Commercial inoculants based on microorgansms aimed to environmental bioremediation/soil contamination, I really dont´n kown the market.
- Esquema: Sampling and strain selection sources: Rhizospheric soil or supresive soils; plant-organs: nodules, stems, fruits, etc. Sampling: preferentially at different sites and geographical locations. Selection: selective media containig several compounds as tricalcium-phosphate (P-solubilizers), quitin, different metabolites as precursors or inducers of certain enzymatic activities, etc.) 2. Purification: commonly from individual colonies, this procedure will need several steps or re-stricking 3. Preservation: Frozen at –80º C, lyofilization… Some fungi isolates used as biocontrol agents (against other fungi, insects and mycoherbicidal) may lost their virulence in artificial lab. media; so that, they should be preserved on vegetal material, like fruits or seeds. Mycorrhizal fungi also need the presence of the plant-host to grow and multiply and, commonly inoculants were a mixture of roots, mycelia and the substrate . Up-to-date mycorrhiza inoculants are based on spores cultures. (Tomorow we will have a conference about these microorganisms) Industrial phases: once the biological material (active ingredient) has been selected and evaluated for its potential as an inoculant, the following steps: industrial multiplication, formulation of the end-product, etc. should be accomplished with the collaboration of Industry, due to the high cost, the better known of the market and, of the registration requeriments. Researchers will define methods to study the environmental impact, those methods to assess the potential toxicity and, to track the inoculant; finally the QC program should be carefully designed. Formulation: the efficacy of these biological products, which contain living microorganisms, is significantly influenced by the formulation constituents and the production process. Formulation is used not only devised for dispersal, but also as: “reservoir” of nutrients for the bioagent, may alter the “shelf live” of the end-product and other biological processes, besides it should fit with the conventional methods of application. Distinct analitical methods: this matter has two needs, first: the simple recognition of microorganisms marketed in commerce and secondly, the protection of patented or others intellectual properties, i.e. biocontrol strains. To cover this aspect reserachers from the Weizmann Inst. of Seciences proposed/recommended a “Bar-code” system for differntiating a large number of products. The system consists in a set of two universal “non-sense” (non-coding) nucleotide sequences, that can be read by universal PCR primers; the universal primers are large enough that few mutational changes will still allow it to be recognized by a PCR probe. Finally, the current regulatory requeriments make the registration of microbial inocualnts (mainly BCAs) a time consuming, costly and often prohibitive exercise.
- Especificity of rhizobia: most of the rhizobia strains show a norrow host range, however there are some rhizobial species particulary promiscous, as an example S. fredii which is able to form nodules with more than 100 legumes. Infectivity: capacity to form nodules, obviously. For legume inoculants production, N2-fixation efficiency should be the main characteristic of selected rhizobia. Nevermid the selection of an effective organism is the first step in the development of any microbial control agent or biofertiliser. Other traits, as competitive ability/capacity to form nodules, under diifferent constraints conditions, is a general desirable (useful) character; competitiveness not only to form nodules (rhizobia) but also to survive and colonize the rhizosphere or the target organ, in the case of other microbial inoculants (biocontrol agents).
- The strain selection process involve two steps. Firstly a large number of “potential rhizobia inoculants” can be tested under bacteriologiacal controlled conditions in greenhouse. Secondly, the symbiotic performance of the selected strains, should be tested under field conditions, in different locations during several seasons
- Some of these conditions can be applied to diferent types of microbial inoculants, i.e. extreme conditions may negatively affect most of the microbes either or both their establisment and function. Azospirillum inoculants gave a better fiels performance in soils with reduced fertility.
- The more important feature is that all inoculated plots raised yied higher than the N-fertilizer treatment.
- In this field experiment there was no response to inoculants (yield increase), due to native bradyrhizobial populations. These populations were good N2-fixers (see yields of non-fertilized plots) as well as very competitive. In fact, we studied the competition ability of the Sinorhizobium inoculant-strains. The maximum occupancy of nodules by an inoculant was of 7% (SMH12)
- The Competitive ability of an strain may vary with differents factors: the plant genotype, edaphoclimatic conditions: soil pH, temperature, etc. The intrinsic competitive capacity % SMH12 nodules/cv Kochi: 17.5
- Soild and liquid legume inoculants formulations. Left: experimental appearence. Right: commercial products
- Esterilización por Radiación gamma (25 KGy). La empresa: Ionmed Esterilización, S:A Clays are widely used as microbial inoculants carriers and have a long story of usage in various formulations, applied as granules, suspensions, and powder. Clays can act as a desiccant providing excelent storage for dried inoculants due to large surface area, pore size distribution and, total porosity. In addition water can be controlled to provide moisture for biologically active formulations. Besides, clays can absorb or distribute dispersing and suspending agents. Clays increase the rhizobia survival in soils (60 days) due to the creation of protective microhabitats accessible to bacteria but inaccessible to predators En ocasiones, como con la bacteria Pseudomonas chlororaphis, agente de biocontrol de enfermedades fúngicas en cereales ligadas a al semilla, se emplean formulaciones que emplean aceites para suspender el microorganismo (Hokeberg y cols., 2001).
- Some legumes as Vigna unguiculata or Arachys hypogaea have a better response to soil-applied Inoculants than seed inoculation (H. Burity group, EMBRAPA, Recife-Brasil). Some forage legumes are pre-inoculated and coated giving “pellets” as en-products directly to be sown
- Liquid inoculants formulations used broth cultures mainly in water but also in mineral or organic oils. Seeds are either dipped into the inoculant before sowing or an applicator evenly sprays the liquid inoculant on the seeds. Liquid formulations simplify production and application for the farmers and may have some advantages since they use low-cost material and are easily attainable by small producers (Singleton et al. 2002; Albareda et al. 2008). Liquid inoculants allow contacting directly seeds and microorganisms and consequently increasing the survival of bacteria on plant roots. However, bacterial survival rates on liquid formulations decrease because this technique does not provide a protective environment for microorganisms and the number of bacteria distributed in each seed is quite heterogeneous. In addition, microorganisms are not sufficiently protected against environmental conditions and contamination during storage, transport, and application into the soil (Bashan et al. 2002). The use of liquid inoculants mainly requires a correct storage, without losing their efficiency and cell viability. A stable population of rhizobia on liquid formulations can be stored for 3 months (Albareda et al. 2008); whereas, PGPR on liquid formulation could show a decrease on the number of living cells (Bashan et al. 2002; Haggag and Singer 2012).
- Quality Control systems for “biological agents” often need to be much more comprehensive and specialised than those used for synthetic chemical insecticides or fertilizers, where a simple chemical analysis can be used to assess quality. Usually, the QC protocols can be divided into three areas: QC during production; QC of the end-product and, follow-up the product quality during storage. So, given the crucial role of QC in microbial inoculants production still being a matter of concern that the definition of standardised protocols for the QC of these products are not yet widely agreed or accepted. As un example of QC of legume inoculants the N2-Africa project defined this protocol. The recommended quality assurance tests to be conducted on inoculant samples (for N2-activities) are: Viable rhizobia cell plate count on yeast extract mannitol agar (YMA) containing Congo red, using the drop plate or spread plate methods Enumeration of contaminants by plating on peptone glucose agar (PGA) and Plant infection test (MPN).
- Actually liquid formulations are widely used, as the begining of the microbial inoculants industry. So much care should be take with the culture media formulation, as some C source-utilization decrease the pH, i.e. glycerol Some enterprises marketed Liquid formulations for soil-application, although the “preservatives” used still a matter of industrial secret. These must be related with compounds that avoid desiccation, nutrients and, supplements that increase the survival during the storage by protecting membranes and controlled secondary metabolites production. In some liquid formulations contain “stickers”.
- CD support
- El objetivo del ensayo de campo (Isla Verde, Córdoba-Argentina), fue evaluar la interacción entre la fertilización fosfórica y el empleo de inoculantes microbianos. El tratamiento 4: fertilización con P y co-inoculación, presenta un incremento del 18%, respecto al Testigo. La inoculación con B. japonicum+ Fertilizante (3) representó un incremento del 14% respecto al Testigo La Fertilización con P (2), produce unos incrementos de casi el 10%