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 <div class="title"><h3>4.24.2 - Optimizing rhizobia inoculants for legumes</h3><br><h3 class="ch-owner">Dylan P. Harding, University of Guelph, Canada</h3></div>
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<p>Suggested citation for this chapter.</p>
<p>Harding,DP. (2022) Optimizing rhizobia inoculants for legumes. In Farmpedia, The Encyclopedia for Small Scale Farmers. Editor, M.N. Raizada, University of Guelph, Canada. http://www.farmpedia.org</p>
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       <h3 class="title-bg">Background</h3>
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          <p>Rather than having to purchase synthetic nitrogen fertilizer, farmers can take advantage of certain types of bacteria (“nitrogen fixing bacteria” or “rhizobial/Rhizobium” bacteria) that inhabit the root organs of legume plants (e.g. beans) where they can convert naturally occurring atmospheric nitrogen gas (N2) to nitrogen fertilizer (ammonia). There is a wide variety of nitrogen-fixing bacteria in nature, and certain varieties will be better at forming a symbiotic relationship with a particular legume plant than others (Wielbo, Kidaj, Koper, Kubik-Komar, & Skorupska, 2012). A compatible nitrogen-fixing bacteria strain for the desired crop must be present for nitrogen-fixation to happen. Most soils already possess nitrogen-fixing bacteria, and on a given soil some legumes will be able to form relationships with the native bacteria and others will not. When legumes are not able to form successful relationships with native bacteria, inoculation with a bacterial strain that is known to form successful relationships with the desired crop can often be beneficial.</p> 

<p>Inoculation is usually performed by coating legume seeds with a powder containing a small population of the desired rhizobial bacteria just before planting. Inoculants are commercially available and generally must be purchased because on-farm production is beyond the capacity of small scale farmers in most cases, however in some situations the introduced bacteria may be able to persist in the soil and will only need to be acquired once. Microbial inoculants usually cost approximately $1.20 to $6 (US) per acre. If inoculation is successful, the cost of the inoculant is generally much less than purchasing an equal amount of nitrogen fertilizer to that which that the bacteria will produce.</p>
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       <h3 class="title-bg">Determining Need for Inoculation </h3>
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         <p>Symptoms of nitrogen deficiency in legumes  (yellowing and tip drying of older leaves) suggest soil conditions are not ideal for nitrogen fixation, which can sometimes be caused by a lack of compatible rhizobium bacteria in the soil. As also noted in the previous chapter, legume nitrogen fixation can be estimated by gently digging up the root system and counting the number of root nodules. Nodules have the appearance of small beads on the roots. Nodules that are actively fixing nitrogen appear red (inside and/or out) and this colour in particular, along with nodule number and size, indicates the total level of nitrogen fixation per plant. To gauge whether there may be a lack of ideal rhizobial strains, nodule observations from fields with low-yielding legumes should be compared with fields with high-yielding crops of the same species. If nitrogen fixation is determined to be poor, it could be because there are no compatible nitrogen-fixing bacteria for the legume species in the soil. Soil acidity or molybdenum deficiency can also cause problems with nitrogen-fixation (see Chapters 5 and 8 for further information).</p>

<p>Some legume varieties will form successful relationships with only a few rhizobium species whereas other legumes can form relationships with a wide variety of rhizobia. The ability to form successful nitrogen-fixing relationships with a wide variety of rhizobial species is referred to as “promiscuity”. Promiscuity varies between different legume species and also between different cultivars of the same species, as shown in Table 6.1. For example, in Sub-Saharan Africa, soybeans will generally form relationships with only a few strains of rhizobial bacteria, so they will generally require inoculation (Giller, Murwira, Dhliwayo, Mafongoya, & Mpepereki, 2011). However, though soybean is not generally considered promiscuous, certain varieties have been observed to demonstrate this quality more than others (Mpepereki, Javaheri, Davis, & Giller, 2000). Cowpea on the other hand is a highly promiscuous legume that will form successful relationships with native bacteria in most soils and thus will generally not benefit from inoculation (Chianu, Nkonya, Mairura, Chianu, & Akinnifesi, 2011; Guimaraes et al., 2012).</p> 

<p>The effect of a rhizobial inoculant on a particular crop and soil will vary widely. Seed providers should provide information about the promiscuity of their seeds (and whether or not inoculation is recommended) but this may not always be the case. As with many of the interventions discussed in this book, the most effective way to determine if a given combination of legume, inoculant, and soil will improve yields is through the establishment of a small test plot. Such a test should be setup so that one half of a farmer’s field is planted with inoculated seeds and the other half with non-inoculated seeds. The management of the plot should be as consistent as possible throughout the entire area, and ideally the farmer should not know which half is which to encourage even treatment. If improved nodulation and yield response is shown to be associated with the inoculated legume seeds, a larger purchase of inoculant for wider field use can be considered</p>
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       <h3 class="title-bg">Additional Benefits and Challenges to Rhizobial Inoculation</h3>
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          <p>As noted above, most soils already host a varied population of microbial species and a given inoculant may not be able to survive in a soil if it cannot compete with native bacteria (Mathu et al., 2012). Additionally, saline soil conditions can destroy many rhizobial species and these conditions may also interfere with successful inoculation (Ventorino et al., 2012). Many rhizobial species are also sensitive to acidity (Guimaraes et al., 2012). When salinization and/ or acidic soil is a reality, tolerant rhizobial strains (if available) should be introduced to enable ongoing N fixation.</p>

<p>High temperatures, especially during storage and shipping, can damage inoculants, decreasing or eliminating their effect (Chianu et al., 2011). Low-tech food storage techniques (see Chapter X: Food Storage) could be used to protect inoculants between delivery and use if such practices have been established. Temperature control during shipping is also a major challenge to the distribution and widespread practice of legume inoculation in Africa. Given the sensitivity and storage difficulties of rhizobial inoculants, it is usually only practical for a farmer to purchase enough inoculant for one planting at a time. For this reason, small package sizes (approximately 25 g, or enough to inoculate ¼ acre of land) are ideal so that farmers can purchase the right amount of inoculant for their current plantings (Chianu et al., 2011).</p>  

<p>It is also worth noting that improved drought tolerance has been observed in several legumes species when reliant on biological nitrogen fixation rather than fertilizer (Antolin, Yoller, & Sanchezdiaz, 1995; Frechilla et al., 2000; Kirova, Tzvetkova, Vaseva, & Ignatov, 2008; Lodeiro, Gonzalez, Hernandez, Balague, & Favelukes, 2000)</p>
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       <h3 style="background: #FBB03B;padding: 15px;font-weight: 600;color: #000;font-size: 22px;margin:unset;text-align:center;">Practical Tips and Further Information</h3>
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          <p>Given the sophisticated materials required to make a rhizobial inoculant, NGOs or local farm groups are encouraged to contact a commercial company or other suppliers in order to obtain these materials (see below). Once obtained, the bacteria must be coated onto legume seeds. A method for the preparation of inoculant seed coatings is outlined in Appendix 3 of the May 2010 N2 Africa Workshop report (Saidou Koala et al., 2010). An adhesive should be used when inoculating seeds to ensure lasting contact between the seed and its coating (Saidou Koala et al., 2010). This adhesive can be prepared using locally available materials such as gum Arabic, granular sugar or sugarcane molasses.</p>  

<p>See Table 1 of “Workshop Report: Training of Master Trainers on Legume and Inoculant Technologies” for correct ratios of adhesive, water and inoculant/ mineral mix, available online at http://www.n2africa.org/sites/n2africa.org/files/images/images/N2Africa_Workshop%20Report-Training%20of%20Master%20Trainers%20on%20Legume%20and%20Inoculant%20Technologies.pdf (page 16). The preparation of the seed coating will also require plastic bags without holes, or for larger volumes of seed, a bucket with a tight-fitting lid. The application rate recommended by N2 Africa for seed inoculation is 10 g of inoculant per kilogram of seed for most common legumes (soy, bush bean, climbing bean, cowpea, ground nut) (Saidou Koala et al., 2010).</p> 

<p>In central East Africa seed inoculants can be sourced from MEA Fertilizers located in Nakuru, Kenya http://www.mea.co.ke/Downloads/. The rhizobial species used in their Biofix product is unclear, however specific inoculants are available for beans, cowpeas, peanuts and soybeans as well as a few leguminous pasture and tree species. The approximate cost of the Biofix inoculant is $1.20 (US) per acre (Chianu et al., 2011). Legume Technology Limited of the United Kingdom has shipped their products to Nigeria in the past and could potentially ship to other African countries as well. This company offers specific inoculants for pea, broad bean, soybean and groundnut however the exact rhizobial species contained in each inoculant product is again unclear. Other African producers of rhizobial inoculants include the Grasslands Research Institute (Marondera, Zimbabwe), Madhavani Ltd., (Jinja, Uganda), and Soygro Pty Ltd. (Potchefstroom, South Africa). Inoculants have also been produced in Tanzania and Rwanda however these facilities are no longer operational. Links to providers of rhizobium inoculants are provided below.</p>  

<p>A given provider may not have an appropriate inoculant for the legume species intended for production and it should be kept in mind that substitutions between rhizobial strains are not likely to be successful. It is important to note that rhizobial inoculants can vary widely in effectiveness and thus the reputation of a provider and any available third-party information on the quality of their products should be considered before making an investment. As inoculants can be compromised by heat during shipping, the product delivery method must be considered also.</p>
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       <h1 class="title-bg">Picture Based Lesson to Train Farmers </h1>
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[[Image:5.21 page-0001.jpg|thumb|centre|Picture Based Lesson to Train Farmer|Click on the image to access a higher resolution image as well as lessons adapted for different geographic regions.]]</p>
[[Image:5.22 page-0001.jpg|thumb|centre|Picture Based Lesson to Train Farmer|Click on the image to access a higher resolution image as well as lessons adapted for different geographic regions.]]</p>
[[Image:5.23 page-0001.jpg|thumb|centre|Picture Based Lesson to Train Farmer|Click on the image to access a higher resolution image as well as lessons adapted for different geographic regions.]]</p>
[[Image:5.24 page-0001.jpg|thumb|centre|Picture Based Lesson to Train Farmer|Click on the image to access a higher resolution image as well as lessons adapted for different geographic regions.]]</p>
[[Image:5.25 page-0001.jpg|thumb|centre|Picture Based Lesson to Train Farmer|Click on the image to access a higher resolution image as well as lessons adapted for different geographic regions.]]</p>
[[Image:5.26 page-0001.jpg|thumb|centre|Picture Based Lesson to Train Farmer|Click on the image to access a higher resolution image as well as lessons adapted for different geographic regions.]]</p>
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       <h3 class="title-bg">Additional Information and Links </h3>
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          <p>See (Chianu et al., 2011) for further information regarding previous successes and failures in the establishment of rhizobial inoculant techniques in Africa (the full reference is included at the end of this chapter). </p>
          <p>For further detail on production of rhizobial inoculants in Africa see the N2 Africa report “Production and use of Rhizobial inoculants in Africa” (Bala et al., 2011) (Milestone reference number 3.4.1, available online at http://www.n2africa.org/node/165).</p>

<p>The N2 Africa organization provides useful information and videos on maximizing nitrogen fixation by legumes in Africa: http://www.n2africa.org </p>

<p>Marondera,  Zimbabwe - Soil Productivity Research Lab: http://www.drss.gov.zw/index.php?option=com_content&view=article&id=111&Itemid=129</p>

<p>Lilongwe, Malawi - Chitedze Agricultural Research Station: http://www.sdnp.org.mw/darts/research/chitedze/chite.htm
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       <h3 class="title-bg">References</h3>
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          <p>1.	Antolin, M. C., Yoller, J., & Sanchezdiaz, M. (1995). EFFECTS OF TEMPORARY DROUGHT ON NITRATE-FED AND NITROGEN-FIXING ALFALFA PLANTS. Plant Science, 107(2), 159-165. doi: 10.1016/0168-9452(95)04108-7</p>
<p>2.	Chianu, Jonas N., Nkonya, E. M., Mairura, F. S., Chianu, Justina N., & Akinnifesi, F. K. (2011). Biological Nitrogen Fixation and Socioeconomic Factors for Legume Production in Sub-Saharan Africa.</p>
<p>3.	Frechilla, S., Gonzalez, E. M., Royuela, M., Minchin, F. R., Aparicio-Tejo, P. M., & Arrese-Igor, C. (2000). Source of nitrogen nutrition (nitrogen fixation or nitrate assimilation) is a major factor involved in pea response to moderate water stress. Journal of Plant Physiology, 157(6), 609-617.</p> 
<p>4.	Giller, K. E., Murwira, M. S., Dhliwayo, D. K. C., Mafongoya, P. L., & Mpepereki, S. (2011). Soyabeans and sustainable agriculture in southern Africa. International Journal of Agricultural Sustainability, 9(1), 50-58. doi: 10.3763/ijas.2010.0548</p>
<p>5.	Guimaraes, A. A., Jaramillo, P. M. D., Nobrega, R. S. A., Florentino, L. A., Silva, K. B., & Moreira, F. M. D. (2012). Genetic and Symbiotic Diversity of Nitrogen-Fixing Bacteria Isolated from Agricultural Soils in the Western Amazon by Using Cowpea as the Trap Plant. Applied and Environmental Microbiology, 78(18), 6726-6733. doi: 10.1128/aem.01303-12</p>
<p>6.	Kirova, E., Tzvetkova, N., Vaseva, I., & Ignatov, G. (2008). Photosynthetic responses of nitrate-fed and nitrogen-fixing soybeans to progressive water stress. Journal of Plant Nutrition, 31(3), 445-458. doi: 10.1080/01904160801894988</p>
<p>7.	Koala, S., Woomer, P., Baijukya, F., Ajeigbe, H., Bala, A., Dashiell, K., Wesonga, M.</p>
<p>8.	Noordin, Q., Ngokho, P., Mukalama, J., 2010. Workshop Report: Training of Master</p>
<p>9.	Trainers on Legume and Inoculant Technologies, Kisumu, Kenya, May 2010,</p>
<p>10.	www.N2Africa.org, 24 pp.Accessed April 16, 2013 at: http://www.n2africa.org/sites/n2africa.org/files/images/images/N2Africa_Workshop%20Report-Training%20of%20Master%20Trainers%20on%20Legume%20and%20Inoculant%20Technologies.pdf</p>
<p>11.	Lodeiro, A. R., Gonzalez, P., Hernandez, A., Balague, L. J., & Favelukes, G. (2000). Comparison of drought tolerance in nitrogen-fixing and inorganic nitrogen-grown common beans. Plant Science, 154(1), 31-41. doi: 10.1016/s0168-9452(99)00246-0</p>
<p>12.	Mathu, S., Herrmann, L., Pypers, P., Matiru, V., Mwirichia, R., & Lesueur, D. (2012). Potential of indigenous bradyrhizobia versus commercial inoculants to improve cowpea (Vigna unguiculata L. walp.) and green gram (Vigna radiata L. wilczek.) yields in Kenya. Soil Science and Plant Nutrition, 58(6), 750-763. doi: 10.1080/00380768.2012.741041</p>
<p>13.	Mpepereki, S., Javaheri, F., Davis, P., & Giller, K. E. (2000). Soyabeans and sustainable agriculture - Promiscuous soyabeans in southern Africa. Field Crops Research, 65(2-3), 137-149. doi: 10.1016/s0378-4290(99)00083-0</p>
<p>14.	Ventorino, V., Caputo, R., De Pascale, S., Fagnano, M., Pepe, O., & Moschetti, G. (2012). Response to salinity stress of Rhizobium leguminosarum bv. viciae strains in the presence of different legume host plants. Annals of Microbiology, 62(2), 811-823. doi: 10.1007/s13213-011-0322-6</p>
<p>15.	Wielbo, Jerzy, Kidaj, Dominika, Koper, Piotr, Kubik-Komar, Agnieszka, & Skorupska, Anna. (2012). The effect of biotic and physical factors on the competitive ability of Rhizobium leguminosarum. Central European Journal of Biology, 7(1), 13-24. doi: 10.2478/s11535-011-0085-x</p>
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