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 <div class="title"><h3>4.24.1 - Rhizobia bacteria inoculants for legumes</h3><br><h3 class="ch-owner">Clara Kopp, University of Guelph, Canada</h3></div>
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<p>Suggested citation for this chapter.</p>
<p>Kopp,C. (2022) Rhizobia bacteria 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>Legumes (e.g. soybeans, beans, lentils, chickpeas) are a very important part of a balanced diet especially in developing countries, because they contain a lot of protein. Nitrogen (N) is a building block for proteins (Hayat et al., 2010). Other crops like corn or wheat use the nitrogen that is in the soil to build up protein and therefore need a lot of synthetic nitrogen fertilizer. However, legumes can produce their own nitrogen fertilizer by forming a symbiotic relationship with special nitrogen-fixing bacteria species called rhizobia. The rhizobia infect the root and form small spheres on the roots called nodules. Once inside the nodules, the rhizobia bacteria are able to use the nitrogen gas that makes up 80% of the atmosphere on earth to form plant available organic nitrogen out of it – specifically ammonia that is a building block for protein. The protein is then transferred to leaves and grain. As a result, legumes require minimal commercial nitrogen fertilizer (Hayat et al., 2010).</p>

<p>However to form a beneficial relationship between the legume and the bacteria the compatible nitrogen-fixing bacteria must be present in the soil, because not all rhizobia bacteria work well with all legume species (Wielbo and Jerzy, 2012). Therefore, it is often necessary to introduce the specific bacteria for the specific crop in the soil by coating the legume seeds with a small population of the beneficial bacteria - a process termed rhizobia inoculation. Then the plant roots get infected and are able to form the nodule spheres where the nitrogen fixation occurs (Hayat, 2010). Nitrogen is a building block of chlorophyll and so increased nitrogen availability increases yield and makes the leaves appear greener (Thies, 1991).</p>
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       <h3 class="title-bg">Inoculation</h3>
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          <p>First, the farmer should determine if there is a need for an inoculant. To determine if there is a N deficiency the farmer can observe the color of the leaves, because a lack of nitrogen makes a leaf appear paler, less green and more yellowish.</p>

<p>To determine if a legume is already infected by nitrogen-fixing bacteria, the farmer can carefully dig out plant roots and count the number of nodule spheres on the legume roots. Then a farmer can compare the number of spheres to high yielding legume plants. In addition, the farmer can cut open the spheres and check the color inside, because a pink color is an indicator for functional nitrogen-fixing bacteria. Active spheres are pink, because they carry oxygen to the rhizobia bacteria similar to oxygen-carrying red blood cells in humans.</p> 

<p>If nitrogen is deficient and the roots are not already infected by bacteria it makes sense to inoculate rhizobia into the soil.</p>

<p>To inoculate the seeds with rhizobia, the farmer needs to purchase a bag of rhizobia inoculant and some kind of sticky substance. This sticky substance helps to attach the bacteria on the seeds. Then one places the seeds, the sticky substance and the inoculant powder in a bucket or plastic bag after which it should be mixed until all the seeds are coated by the powder. When the seeds are then planted the bacteria infects the roots. In the end to make sure the inoculation is successful, the farmer should have a small plot, in which half is inoculated and half is not. The yield and the leaf color should differ between the two plots if the inoculant is effective</p>
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       <h3 class="title-bg">Possible Benefits</h3>
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          <p>A successful inoculation leads to higher nitrogen uptake and therefore to more chlorophyll synthesized and more photosynthesis (Hayat et al., 2010). So the farmer can achieve higher yields. The increase in yield for inoculated legumes ranges from 60% to 300% depending on crop species (Chianu et al. 2011). In addition, the input costs decrease, because there’s less need to purchase nitrogen fertilizer and usually fertilizer costs are one of the highest expenditures of a small-scale farmer (Kahindi et al., 1997). Thus, it is a cheaper and usually more effective agronomic practice to inoculate to ensure adequate N nutrition of legumes (Chianu et al. 2011).</p>

<p>Introducing nitrogen-fixing bacteria also has environmental and ecological benefits. Because no synthetic fertilizer needs to be applied, over applying of nitrogen fertilizer and nitrate leaching are avoided.</p>  

<p>In addition, if one leaves the rest of the legume plant, which is high in protein on the field, it can act as an organic nitrogen fertilizer for other plants later grown on that field. Therefore, legumes are a valuable crop to include in a crop rotation (Hayat et al., 2010).</p> 

<p>It is also has also been shown, that inoculation not only increases yield, but can also increases the stress tolerance of the legumes. For example, it could be observed that inoculation improves drought tolerance of some legume species (Yanni et al, 2016).</p>
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       <h3 class="title-bg">Common Problems and Issues</h3>
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          <p>In general, the presence of inoculant infrastructure and expertise are low in developing countries (Mutuma et al., 2014). Another problem is that the farmer needs to be able purchase the compatible inoculant for the specific legume species. As mentioned above not all rhizobia species work for all legume crops. There are some legumes that can form a relationship with many different rhizobia species and others that can only form a relationship with only one. It depends on the crop species if an inoculation with a specific species is necessary and which inoculant the farmer needs to purchase (Mutuma et al., 2014). 
Another problem caused by the poor infrastructure is the shipping and storage of the inoculant. The bacterial cells in the inoculant are sensitive to temperature. So the challenge is to secure lower (but not freezing) temperatures during storage and shipping (Chianu et al., 2011). To overcome the storage problem, low-tech food storage techniques can be used. Given the storage problem the inoculant should only be used in one growing season and not be stored for subsequent seasons (Balume et al., 2015). Therefore, it is important for small scale-farmers to be able to purchase small bags of rhizobia inoculants.
While nitrogen deficiency is not an issue for legumes after a successful inoculation, phosphorus (P) takes the role of the most limiting nutrient. Therefore, it should be noted that is it is important to secure a sufficient P supply to generate a successful outcome.</p>

<p>Micronutrient fertilizers are also critical for rhizobia to function, specifically boron (B) and molybdenum (Mo). It has been shown that the soil boron content effects the symbiotic relationship and the formation of the nodules and therefore B must not be deficient in the soil (Abreu et al., 2012).  Also soil molybdenum affects the nitrogen fixation rate as it is required for the key bacterial enzyme that converts nitrogen gas into ammonia fertilizer (Figueiredo et al., 2016). Therefore, it is important to make sure that Mo and B are not deficient. These nutrients can be added to the soil as commercial fertilizers or coated on the seeds along with the inoculant.</p>
<p>There can also be problems with the establishment of the bacteria in the soil. Specifically, if there are native rhizobia bacteria in the soil, the inoculant can have difficulties to infect the legume roots and to form nodules, because the native bacteria can outcompete the introduced inoculant (Wielbo, Jerzy, 2012). That is why the inoculation works best for a crop that is new to the region.</p>
<p>Normally if the correct bacterial strain is added to the soil, it can stay there for years. This is only true when favorable soil conditions apply. It has been shown that low pH soil or saline soil conditions can affect the time the bacteria stay in the ground (Ventorino et al., 2012). This is an issue, because inoculation is especially practical in developing countries, if it is not necessary to inoculate every year.</p>
<p>Overall, the soil pH affects the symbiotic relationship between plants and bacteria negatively (Hungria and Vargas, 2000). While the crops are usually adapted to mostly lower soil pH of tropic soils, the bacteria and especially newly introduced species are not adapted to these unfavorable soil conditions. The ideal pH for rhizobia ranges between 6 and 7. Specifically, an acid soil has an effect on the infection of the roots and the formation of nodules, which results in a decrease of the total nitrogen-fixation rate. To overcome this problem, one can either apply lime to increase the soil pH or choose a bacteria species that is more adapted to low pH values. The second attempt might not be practical for subsistence farmers due to the already limited availability of rhizobia inoculants in developing countries (Hungria and Vargas, 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, Further Information and Online Links</h3>
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          <p>Moisture blocking is a way gloves can prevent your skin from drying out and from getting too wet and dehydrating farmer's hands. By keeping the moisture from the hands inside the gloves they will prevent the skin from cracking and becoming infected (Schaffner, 2013). As well when working in wet conditions your hands can shrivel and become dehydrated if they are constantly in contact with water.</p>
          <p>First of all, to enhance the adoption rate of a rhizobia inoculant, it is important to provide farmers with background information on legumes, rhizobia bacteria and nitrogen fixation, because this information can enhance the willingness to pay for the extra treatment of the seeds (Chianu et al., 2011). 
There are studies on legume production that suggest that the main reason for the low rate of inoculation, at least in Africa, is not the price (Chianu et al., 2011). For example, a 100 g packet of Biofix (a form of rhizobia inoculant), which costs or US$1.20, is sufficient to inoculate 15 kg of common bean seed, enough to plant 1 acre. By contrast, the cost for the required nitrogen fertilizer is about US$34. Furthermore, these price ratios usually apply to the other legume species (Chianu et al., 2011).</p>
<p>Thus the main issue for NGOs or local companies, as already mentioned above, is to help to distribute/sell living inoculant and make it available to farmers. Some inoculant supplier links are noted below.</p>
<p>As mentioned above, the shipping of rhizobia poses a problem due to the temperature sensitivity of the bacteria (Balume et al., 2015). As a result, shipped commercial inoculants contain only a few living bacteria cells. Thus, it is important to find a reliable supplier and to maybe find an effective system to control the quality of the purchased product. In case of a low quality product a refund should be requested (Balume et al., 2015).</p>
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       <h3 class="title-bg">Rhizobia suppliers</h3>
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          <p>•China
https://vastland.en.alibaba.com/</p>
<p> •India
https://indogulfgroup.trustpass.alibaba.com/
http://www.indiamart.com/nivshakti-bioenergy/</p>
<p> •BASF Canada 
https://agro.basf.ca/East/Products/Product.html Under Inoculants.</p>
<p> •In Africa a potential supplier for an inoculant may be MEA Fertilizers. http://www.mea.co.ke/Organic
And they also provide information on how to inoculate with their product.</p>
<p> •In Latin America a potential supplier is Monsanto BioAg. http://www.monsantobioag.com/global/las/Pages/default.aspx</p>
<p> To get further information on how to inoculate, and information on specific legume species, NGOs are encouraged to use the provided links (below) and to contact commercial seed or inoculant suppliers.</p>
<p> In general, it is important to rely on the local resources of the specific country and to use simple low-cost and low-tech techniques and strategies. For example, it is best to use the local available resources to create a sticky substance to attach the inoculant to the seeds. Depending on the region, the sticky substance could be gum Arabic, molasses, etc. To measure the correct amount of inoculant per unit of seed, it is possible to use a bottle cap or spoon, because farmers may not have access to weight scales.</p>
<p>•Many tips and practical training manuals, not only relevant for Africa, may be found at: </p>
<p> http://www.n2africa.org.</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">References</h3>
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          <p>1.	Abreu, I., Cerda, M. E., de Nanclares, M. P., Baena, I., Lloret, J., Bonilla, I., … Reguera, M. (2012). Boron deficiency affects rhizobia cell surface polysaccharides important for suppression of plant defense mechanisms during legume recognition and for development of nitrogen-fixing symbiosis. Plant and Soil, 361(1–2), 385–395</p>
<p>2.	Balume, I., Keya, O., Karanja, N., & Woomer, P. (2015). Shelf-life of legume inoculants in different carrier materials available in East Africa. African Crop Science Journal, 23(4), 379</p>
<p>3.	Chianu, J., Nkonya, E., Mairura, F., Chianu, J., Akinnifesi, F.K. (2011). Biological nitrogen fixation and socioeconomic factors for legume production in sub-Saharan Africa : a review. Agronomy for Sustainable Development, 139–154</p>
<p>4.	Figueiredo, Marislaine Alves de; Oliveira, Damiany Padua; Soares, Bruno Lima; Morais, Augusto Ramalho de; Souza Moreira, Fatima Maria de; Bastos de Andrade, Messias Jose (2016): Nitrogen and molybdenum fertilization and inoculation of common bean with Rhizobium spp. in two oxisols. Acta Scientiarum, 18(1),  85-92</p>
<p>5.	Hayat, R., Ali, S., Amara, U., Khalid, R., & Ahmed, I. (2010). Soil beneficial bacteria and their role in plant growth promotion: A review. Annals of Microbiology, 60(4), 579–598</p>
<p>6.	Hungria, M., & Vargas, M. A. T. (2000). Environmental factors affecting N2 fixation in grain legumes in the tropics, with an emphasis on Brazil. Field Crops Research, 65(2–3), 151–164.</p> 
<p>7.	Kahindi J. H. P., Woomer P., George T., Moreira, F.M.D., Karanja N.K., Giller K. E. (1997). Agricultural intensification, soil biodiversity and ecosystem function in the tropics: The role of nitrogen-fixing bacteria. Applied Soil Ecology, 6(1),  55-76</p> 
<p>8.	Mutuma, S. P., Okello, J. J., Karanja, N. K., & Woomer, P. L. (2014). Smallholder farmers’ use and profitability of legume inoculants in western kenya. African Crop Science Journal, 22(3), 205–213</p>
<p>9.	Thies, J. E., Singleton, P. W., & Bohlool, B. B. E. N. (1991). Influence of the Size of Indigenous Rhizobial Populations on Establishment and Symbiotic Performance of Introduced Rhizobia on Field-Grown Legumes. Median, 57(1), 19–28</p>
<p>10.	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.</p> 
<p>11.	Wielbo, J. (2012). Rhizobial communities in symbiosis with legumes: genetic diversity, competition and interactions with host plants. Open Life Sciences, 7(3), 363–372</p>
<p>12.	Yanni, Y., Zidan, M., Dazzo, F., Rizk, R., Mehesen, A., Abdelfattah, F., & Elsadany, A. (2016). Enhanced symbiotic performance and productivity of drought stressed common bean after inoculation with tolerant native rhizobia in extensive fields. Agriculture, Ecosystems and Environment, 232, 119–128</p>

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