A Guide to Troubleshooting Nodulation Failure in Leguminous Cover Crops

**A Nodulation Failure Has Occurred** Pictured below are some examples of nitrogen-fixing nodules from legumes. Unfortunately, when you dig up a few legumes from your field, you can't find any of them on the roots of your growing legumes.

Answer a few questions to learn more about what may have caused this problem and what can be done to improve future nodulation outcomes. Did you inoculate? [[Yes]] No, what is [[inoculation|Inoculation]]?

Great, inoculation introduces the right rhizobial partner for your legume species to the field. It is an important first step towards efficient nitrogen fixation, especially for non-native legumes in fields where they have not been recently grown. Here are a few other considerations to help maximize your chances of successful inoculation and good N fixation. 1. Match the [[inoculant type]] to your legume species 2. Pay attention to [[expiration dates]] 3. Make sure inoculant is stored [[between 40 and 80 degrees F]]. If your inoculant quality was good, the next option to consider is [[inoculation method]].

**Inoculant Type** Most legumes are particular about which rhizobia species they will partner with. It is important to match the inoculant type to the target legume. The inoculant package will usually indicate what rhizobia species it contains and for which legume it is intended. This table provides additional information about which legumes share a common inoculant type. Image of tables of legumes

If your inoculant type was correctly matched to your legume species, consider your [[inoculation method]].

**Expiration Dates Matter** Rhizobial inoculants are a living product that experience bacterial population declines over time. Peat based inoculants have a shelf life around one year, after which bacterial populations may not be sufficient for good nodulation. Be sure to check the expiration dates on your inoculant packages and consider replacing your supply before planting if it has expired.

**Temperature Ranges for Rhizobia** Most rhizobia species show the best survival rates when they are stored between 40 and 80 degrees F. If they are exposed to temperatures above or below this range, they will experience population declines and may not inoculate successfully. If possible, store unused inoculant in a refrigerator. If not, try to find a cool, dry place out of direct sunlight. If you're confident that your inoculant has been stored under appropriate conditions, the next topic to consider is [[inoculation method]].

**Inoculation Methods** Types of commercially available rhizobial inoculants include: [[pre-inoculated seeds]] [[powdered peat]] [[liquid]] [[granular]] Explore the links to see pros and cons for each inoculant method. Or [[check out|transition]] other factors besides inoculation that can impede nodulation and nitrogen fixation.

**Pre-Inoculated Seeds**

*Photo of unpelleted (left) and pelleted with rhizobia and lime (right) subterranean clover seeds from (link: "NSW 2017";)[(openurl: "https://www.dpi.nsw.gov.au/__data/assets/pdf_file/0009/711783/Inoculating-and-pelleting-pasture-legume-seed.pdf")].* Pre-inoculated seeds can be purchased with rhizobial inoculant already applied by the seed company. Some companies will custom inoculate the seed batch when it is ordered while others inoculate large quantities of seed at once and store them until they are needed. Pre-inoculation is marketed as a time-saving benefit for farmers to skip the trouble of inoculating themselves. However, for some legume species, these time-savings result in nitrogen fixation reductions due to reduced viability of the rhizobia on the seed coat. Studies on preinoculated seeds show mixed results depending on the inoculation protocol and the species of legume ((link: "Hartley et al. 201")[(openurl: "http://www.publish.csiro.au/cp/cp12132")]). Clover seeds show particularly low viability rates with under 5% of preinoculated seeds passing the recommended threshold for rhizobia per seed ((link: "Gemell et al. 2012")[(openurl: "http://www.publish.csiro.au/an/EA03151")]). When possible, on-farm inoculant application as close to seeding time as possible can provide more control over the process and improve rhizobial viability at time of planting. [[Powdered peat|powdered peat]], [[liquid]] and [[granular]] inoculants can help encourage higher rates of nodulation success.

**Powdered Peat Inoculant** Rhizobial inoculants in a powdered peat carrier are one of the most commonly used forms of inoculant. However, they require careful attention to ensure success.

*Photo of peat inoculated (left) and untreated (right) seeds from (link: "GRDC 2014")[(openurl: "https://grdc.com.au/__data/assets/pdf_file/0020/82082/grdc-booklet-inoculating-legumes.pdf.pdf")].* One major challenge of peat is maintaining good inoculant to seed contact. Many farmers sprinkle the dry powder over their seeds before planting. The dry inoculant does not always stick well to the seeds and can be easily knocked off by tractor vibrations before reaching the soil. Applying peat inoculant as a moistened slurry can improve results over dry application ((link: "Miller et al. 2010")[(openurl: "https://www.nrcs.usda.gov/Internet/FSE_PLANTMATERIALS/publications/etpmctn12525.pdf")]). In the slurry method, dry inoculant is first mixed with water, then applied to the seeds. A cement mixer can help provide thorough coverage if available. If not, hand mixing is also effective. The amount of water required for the slurry varies based on seed size. The table below shows more detailed recommendations for ratios of seed, inoculant, and water ((link: "U of Hawaii, NifTAL")[(openurl: "https://www.ctahr.hawaii.edu/bnf/")]). The (link: "NRCS")[(openurl: "https://www.nrcs.usda.gov/Internet/FSE_PLANTMATERIALS/publications/etpmctn12525.pdf")] recommends 6.5 oz of inoculant per 100 lbs of seed. Guide to Troubleshooting Nodulation Failure

Guide to Troubleshooting Nodulation Failure

If you are seeding with a drill, slurry inoculated seeds may need time to dry in a shady place. Wet seeds can clump in the seed box and clog the flow of seed. Sticking agents or adhesives and the application of adhesive substances like gum arabic (10-20% solution) or carboxymethyl cellulose (4%) can also improve inoculant retention on seed coats ((link: "Elegba and Rennie 1984")[(openurl: "https://www.nrcresearchpress.com/doi/abs/10.4141/cjss84-063#.XQbQgohKjD4")]). If [[liquid]] or [[granular]] options are available for the desired legume and equipment for proper application is accessible, these options can provide a simpler path to high nodulation rates.

**Liquid Inoculant** In liquid inoculation, rhizobial cultures are suspended in liquid and applied either to the seeds or sprayed directly into the seed furrow at planting. Liquid inoculants can introduce higher numbers of viable rhizobia </style> <img src=https://imgur.com/lCN7vhE.jpg, style="max-width: 75%;"> </div> *Photo from (link: "GRDC 2014")[(openurl: "https://grdc.com.au/__data/assets/pdf_file/0020/82082/grdc-booklet-inoculating-legumes.pdf.pdf")].* One major limitation to liquid inoculation is availability. Since they are usually purchased in either broth or frozen form, they are more difficult to transport and store than other inoculation options. In broth form, they also have shorter shelf lives. Implementation may also be a barrier to use for some farmers who do not already own the right implements for in-furrow applications. An example of the requirements and recommendations for liquid inoculant application can be found (link: "here")[(openurl: "https://www.legumematrix.com/images/563/vault-np-soybean-tech%20sheet.pdf")]. Liquid inoculants perform best under ideal soil moisture and pH conditions. In dry conditions or in alkaline or acidic soils, [[granular]] inoculants may encourage better nodulation success. Inoculation is not the only potential inhibitor of nitrogen fixation success. Explore [[other factors|transition]].

**Granular Inoculant** Granular inoculants are small, dry particles of clay or peat that serve as a carrier for rhizobia. If granular inoculants are available for your legume species and you have access to the right equipment to apply them, they can have several advantages over other forms of inoculant. They typically have a longer life expectancy than liquid inoculants and pre-inoculated seeds and have lower vulnerability to dessication. </style> <img src=https://imgur.com/hFjhO03.jpg, style="max-width: 75%;"> </div> *Photo from (link: "GRDC 2014")[(openurl: "https://grdc.com.au/__data/assets/pdf_file/0020/82082/grdc-booklet-inoculating-legumes.pdf.pdf")].* Since granular inoculants are applied to the soil directly rather than the seed coat, problems with toxic seed treatments and antibiotic substances produced by the seeds can be avoided. When applied with a seed drill below or beside the seeding furrow, granular inoculants can also encourage the development of nodules on the lateral roots. Seed coat applications, on the other hand, usually result in nodule formation mostly on the root crown and taproot. Lateral nodules often produce more nitrogen than crown nodules, particularly later in the season ((link: "Kyei-Boahen et al. 2002")[(openurl: "https://www.researchgate.net/profile/Fran_Walley/publication/237267718_Evaluation_of_Rhizobial_Inoculation_Methods_for_Chickpea/links/53d275450cf220632f3c9bfa/Evaluation-of-Rhizobial-Inoculation-Methods-for-Chickpea.pdf")]). Even with a perfect inoculation procedure, [[other factors|transition]] can inhibit nodulation and nitrogen fixation.

**Inoculation Basics** Nitrogen fixation depends on a partnership between the legume and a bacteria called rhizobia. These rhizobia form nodules on the plant's roots and trade sugar from the plants for nitrogen in a form the plant can use. Without these rhizobia present in the soil, the legume cannot form nodules or fix nitrogen. </style> <img src=https://i.imgur.com/frPaSMA.png, style="max-width: 100%;"> </div> When you introduce a legume to a new field where it hasn't been grown before, its bacterial partner must be introduced as well. This process is called **inoculation**. Rhizobial inoculants can be purchased along with legume seeds from most seed companies in powdered peat, granular, or liquid form. Here are a few tips to help ensure inoculation success: 1. Match the [[inoculant type]] to your legume species 2. Pay attention to [[expiration dates]] 3. Make sure inoculant is stored [[between 40 and 80 degrees F]]. After obtaining high quality, living inoculant, it is also important to choose an effective [[inoculation method]].

**Soil pH** Legume tolerance to acidity and alkalinity varies across species, but most do best in a neutral to slightly acidic environment with frequent nodulation failures in highly acidic soils ((link: "Brockwell et al. 1995")[(openurl: "https://s3.amazonaws.com/academia.edu.documents/32693255/Brockwell__Bottomley_and_Thies_1995.pdf?response-content-disposition=inline%3B%20filename%3DManipulation_of_rhizobia_microflora_for.pdf&X-Amz-Algorithm=AWS4-HMAC-SHA256&X-Amz-Credential=AKIAIWOWYYGZ2Y53UL3A%2F20190812%2Fus-east-1%2Fs3%2Faws4_request&X-Amz-Date=20190812T025914Z&X-Amz-Expires=3600&X-Amz-SignedHeaders=host&X-Amz-Signature=0bd0159e15a0bdabce35420e5e5a8adfd99b38a25b6b12140c131b39b7558982")]). There is a critical threshold near 5.0 pH, below which nitrogenase activity is delayed and significantly reduced compared to neutral and more moderately acidic soils ((link: "Schubert et al. 1990")[(openurl: "https://dl.sciencesocieties.org/publications/aj/abstracts/82/5/AJ0820050969")]). Acidic conditions interfere with the rhizobia's colonization of the root hairs and impedes soil nutrient accessibility. The effect of soil acidity can be buffered by the inoculant method used. In an experiment testing this interaction, liquid inoculant was observed to fail at a pH of 5.4, and only [[granular]] was effective at pH 4.4 ((link: "Rice et al. 2000")[(openurl: "https://www.nrcresearchpress.com/doi/pdfplus/10.4141/S99-059")]). Alkaline soil itself is not typically a problem for rhizobia and experimental legumes have grown well up to a pH of 10. However, high pH soil is often associated with high salinity and reduced nutrient availability, both major obstacles to rhizobia survival and nitrogen fixation success ((link: "Bordelau and Prevost 1994")[(openurl: "https://link.springer.com/article/10.1007/BF02183092")]). [[Next|conclusion]]

**Excess Nitrogen** Nodulation and nitrogen fixation can be inhibited by field nitrogen levels higher than 40 lb/acre. Tolerance of nitrogen fixation to high soil nitrate levels varies across legume species and even among genotypes of the same species. Certain varieties have been observed to continue nitrogen fixation at soil nitrate levels exceeding 100 lb/acre ((link: "Walley et al. 2005")[(openurl: "https://www.nrcresearchpress.com/doi/pdfplus/10.4141/P04-039")]). Generally, the higher the levels of soil nitrate in a field, the lower the rate of nitrogen returns from legumes will be. Fields with low nitrogen levels stand to benefit the most from the nitrogen fixation potential of legumes. [[Next|conclusion]]

**Soil Moisture** Legumes require more moisture for nitrogen fixation than for plant growth. When the plant enters a period of moisture stress, the nodules are the first to lose water access and start to dessicate, immediately pausing nitrogen fixation ((link: "Walsh 1995")[(openurl: "https://www.sciencedirect.com/science/article/abs/pii/0038071795986444")]). Extreme moisture stress can prevent nodules from forming and induce nodule shedding in some species ((link: "Williams and De Mallorca 1984")[(openurl: "https://link.springer.com/article/10.1007/BF02161183")]). Our experiments at UTRGV suggested that watering frequency is more important than the total amount of water received during the season. Cowpeas receiving more frequent waterings produced more nodule tissue than those receiving the same total amount of water less frequently ((link: "Kasper 2019")[(openurl: "http://ezhost.utrgv.edu:2048/login?url=https://search-proquest-com.ezhost.utrgv.edu/docview/2245807027?accountid=7119")]). This is an important consideration for growers interested in legumes in semi-arid regions like the Rio Grande Valley. If supplemental irrigation is not available, nitrogen gains may be lower than expected. Excess moisture can also be a problem for nitrogen fixation if plants are submerged and oxygen supplies in the root zone are limited ((link: "Brockwell et al. 1995")[(openurl: "https://s3.amazonaws.com/academia.edu.documents/32693255/Brockwell__Bottomley_and_Thies_1995.pdf?response-content-disposition=inline%3B%20filename%3DManipulation_of_rhizobia_microflora_for.pdf&X-Amz-Algorithm=AWS4-HMAC-SHA256&X-Amz-Credential=AKIAIWOWYYGZ2Y53UL3A%2F20190812%2Fus-east-1%2Fs3%2Faws4_request&X-Amz-Date=20190812T025914Z&X-Amz-Expires=3600&X-Amz-SignedHeaders=host&X-Amz-Signature=0bd0159e15a0bdabce35420e5e5a8adfd99b38a25b6b12140c131b39b7558982")]). [[Next|conclusion]]

**Soil Temperature** Each legume species has an optimal soil temperature range for nodulation and nitrogen fixation that is not always identical to its optimal range for vegetative growth. For example, guar, a highly heat- and drought-tolerant legume, can grow well at temperatures at and above 104 degrees F, but its nodule formation and nitrogen fixation rates are greatly reduced when temperatures exceed 98 degrees F ((link: "Arayangkoon et al. 1990")[(openurl: "https://link.springer.com/article/10.1007/BF00012824")]). Temperature thresholds for other legumes are often much lower. Clover, for instance, shows limited nitrogen fixation when soil temperatures exceed 86 degrees F and common bean struggles similarly above 91 ((link: "Zahran 1999")[(openurl: "https://mmbr.asm.org/content/mmbr/63/4/968.full.pdf")]). Nitrogen fixation potential may not recover until the legume returns to its optimal temperature zone for up to two weeks ((link: "Hungria and Franco 1993")[(openurl: "http://www.bashanfoundation.org/contributions/Hungria-M/1993.-Hungria-PS.pdf")]). Low soil temperatures (55 degrees F or below) also inhibit or eliminate nodule initiation and nitrogenase activity for some legumes ((link: "Bordelau and Prevost 1994")[(openurl: "https://link.springer.com/article/10.1007/BF02183092")]). [[Next|conclusion]]

**Nutrient Deficiency** Nitrogen fixation can be negatively impacted by nutrient deficiencies including: * Phosphorus * Potassium * Sulfur * Calcium * Magnesium * Iron * Micronutrients (Boron, Cobalt, Copper, Iron, Manganese, Molybdenum, Nickel, Selenium, Zinc) The legume-rhizobia symbiosis requires some of these nutrients at higher rates than the plant or free-living bacteria require alone ((link: "O’Hara 2001")[(openurl: "http://www.publish.csiro.au/ea/pdf/ea00087")]). Co-inoculation with mycorrhizae can sometimes improve nitrogen fixation, due to mycorrhizae’s contributions to plant nutrition as a phosphorus scavenger ((link: "Chalk et al. 2006")[(openurl: "https://www.sciencedirect.com/science/article/abs/pii/S0038071706002495")]). In field tests of mycorrhizae, however, improvements in nitrogen fixation depended on the status of the native mycorrhizal population with limited increases when native fungi were already present ((link: "Ortas 2003")[(openurl: "https://www.tandfonline.com/doi/abs/10.1081/PLN-120016494")]). [[Next|conclusion]]

**Salinity** Although rhizobia usually survive at extremely high levels of salt, many legumes have low resistance to salinity, limiting N fixation in saline soils ((link: "Bordelau and Prevost 1994")[(openurl: "https://link.springer.com/article/10.1007/BF02183092")]). High salt concentrations reduce nodulation by impeding the root hair colonization process required for rhizobial invasion. Major differences in salt tolerance exist across legume species and among different cultivars of the same species. Alfalfa, yellow-lupin, faba bean, and mesquite are particularly salt-tolerant and could be options for farmers facing saline conditions ((link: "Zahran 1991")[(openurl: "https://www.researchgate.net/profile/Hamdi_Zahran/publication/226940249_Conditions_for_successful_Rhizobium-legume_symbiosis_in_saline_environments/links/57178cd608ae09ceb264b048/Conditions-for-successful-Rhizobium-legume-symbiosis-in-saline-environments.pdf")]). Variety trials in chickpea, for instance, have detected genotypes that can successfully form nodules and fix nitrogen at double the salinity levels that impede more salt-vulnerable chickpeas ((link: "Rao et al. 2002")[(openurl: "https://academic.oup.com/aob/article/89/5/563/205764")]). [[Next|conclusion]]

**Toxic Substances for Rhizobia** Nodulation failure can occur if rhizobia are killed through exposure to toxic substances before they are able to colonize the plant root. Known toxins include: * fungicides * solvents * alcohols * heavy metals (lead, cadmium, nickel, chromium, copper, zinc) * antibiotic seed coat exudates Rhizobial inoculants should not be exposed to any of these substances before or during inoculation or planting. This can be particularly tricky for fungicides which are often applied as seed coat treatments. However, where legumes face serious fungal threats and fungicidal seed coatings are desired, [[granular]] inoculants may help protect rhizobia from direct contact with fungicides and improve nodulation success ((link: "Graham 1981")[(openurl: "https://www.sciencedirect.com/science/article/pii/0378429081900605")]). Some legume seed coats produce exudates that are toxic to rhizobia. The toxicity and concentration of seed coat exudates vary across species leading to low success rates of seed preinoculation for those with higher concentrations and more toxic exudates ((link: "Deaker et al. 2004")[(openurl: "http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.584.2666&rep=rep1&type=pdf")]). These antibiotic seed coat effects can be mitigated by [[waiting to inoculate|pre-inoculated seeds]] until just before seeding ((link: "O'Hara et al. 2014")[(openurl: "https://grdc.com.au/__data/assets/pdf_file/0020/82082/grdc-booklet-inoculating-legumes.pdf.pdf")]). [[Next|conclusion]]

**Environmental Impediments to Nodulation** Even with attentive inoculation using high-quality rhizobia, nitrogen fixation success is no guarantee. Nitrogen fixation relies on a delicate partnership between the plant and bacteria. Each partner has a its own set of environmental requirements and tolerance ranges. Conditions outside the tolerance range for one or both partners will limit and sometimes elimimate nodulation and nitrogen fixation. Explore the links for more details about each of the important environmental factors: [[Soil pH]] [[Excess Nitrogen]] [[Soil moisture]] [[Temperature]] [[Nutrient Deficiencies]] [[Salinity]] [[Rhizobial Toxicity]]

**Pathways to Better Nitrogen Fixation** Legumes are a powerful tool for sustainable agriculture, but they require some thought and attention to maximize their nitrogen fixation potential and receive the best return on your cover crop investment. </style> <img src=https://imgur.com/WwKcGgC.jpg, style="max-width: 80%;"> </div> Here are 6 steps for better nodule farming: 1. Choose a legume that is a good match for your season, soil, and climate. 2. Inoculate well with live rhizobia of the correct [[type|inoculant type]]. 3. Do what you can to encourage plant health (appropriate moisture, nutrients, sunlight, etc). Healthier plants have higher potential for nitrogen fixation. 4. Check roots for nodule formation starting 30 days after planting. 5. If nodules have formed, try estimating the nitrogen availability for the subsequent crop with this (link: "online calculator")[(openurl: "http://aesl.ces.uga.edu/mineralization/")]. 6. If no nodules have formed, [[head|Start]] back to the beginning of this guide to learn more about potential causes and interventions to improve legume performance. [[credits]]

**A Guide to Troubleshooting Nodulation Failure in Leguminous Cover Crops** by: *Stephanie Kasper* </style> <img src=https://i.imgur.com/gpwLM4w.jpg, alt="guide to nodulation failure" style="max-width: 100%;"> </div> [[Click here to start!|Introduction]] *To navigate, click any blue link or use the grey forward and backward arrows.*

**Acknowledgments** This research was funded by a Conservation Innovation Grant from the U.S. Department of Agriculture, Natural Resources Conversation Service, grant number 69-3A75-17-281 and by a Graduate Student Grant from Southern Sustainable Agriculture Research and Education, grant number GS18-193. Stephanie Kasper was supervised by Dr. Alex Racelis, Dr. Bradley Christoffersen, and Dr. Pushpa Soti in her thesis work on legumes including the creation of this nodulation troubleshooting tool. Thanks to our farm collaborators at Hilltop Gardens, Andy Cruz and Joseph Kim, and to Mylen Arias, Alyssa Cano, Diana Cantu, Suzanne El-Haj, Allison Kaika, Matthew Kutugata, Katie Lavallee, Habraham Lopez, Lindsey Richards, Thalia Torres and Joy Youwakim for all their help with data collection. </style> <img src=https://i.imgur.com/MaeYT1f.png, style="max-width: 100%;"> </div>

</style> <img src=https://imgur.com/IXCP7JX.jpg, style="max-width: 100%;"> </div> In winter 2017, a research project on cover crops in south Texas experienced a 14-acre nodulation failure. Although we had acres of healthy-seeming crimson clover, vetch, and peas, they failed to form nodules and make the nitrogen contributions we expected. We were perplexed and spent the next year talking to farmers and advisors, reading the scientific literature, and running our own experiments trying to figure out what went wrong and we might improve legume performance. This guide aims to gather what we have learned into a helpful tool for other farmers facing similar nodulation failures or for any grower interested in maximizing the nitrogen returns and cost-effectiveness of legumes as cover crops. [[Begin|Start]]

Agroecology and Resilient Food Systems School of Earth, Environmental, and Marine Sciences

A Guide to Troubleshooting Nodulation Failure in Leguminous Cover Crops