A program of the National Center for Appropriate Technology • 800-346-9140 • www.attra.ncat.org Microbial Inoculants The symbiotic relationship By Barbara Bellows, A growing number of companies between plants and Texas Institute for Applied are selling or bios- soil . Environmental Research; timulants: microbial inoculants Source: Meena et al., 2019 Mike Morris, NCAT; and intended to enhance soil health. Colin Mitchell, NCAT In laboratory and greenhouse Published Sept. 2020 studies, the microbes con- ©NCAT tained in these products have IP604 proven eff ective in enhancing nutrient availability, decreasing Contents pest infestations, stimulating Introduction ...... 1 plant growth, protecting plants against stress conditions, and Overview of enhancing plant competition Plant-Benefi cial Microorganisms ...... 2 against invasive species. These benefi ts have not always been Reasons Why seen by farmers in “real world” Microbial Inoculants conditions, however—making May Not Work ...... 5 the value and cost-eff ective- Overcoming Microbial ness of these products a topic Inoculant Limitations .....7 of considerable uncertainty. Summary, Future To help you reach your own Directions, and conclusions, this publication Recommendations ...... 8 explains how microbial inoculants work, reviews current (in 2020) scientifi c literature and studies, and References ...... 10 off ers practical recommendations: factors to consider before buying and using these products. Further Resources ...... 12 Introduction

vast number of soil microorganisms infl u- contain benefi cial microorganisms that are ence nutrient uptake, water access, disease intended to help plants access their own nutrients A resistance, and many other factors related from the soil. Some of these products contain a to plant growth and health. For example, some single organism, others include a mixture of organ- bacteria, fungi, and viruses have been shown to isms, and some include proteins, enzymes, or other This material is based upon work increase competition against weeds, while others supported by the Southern Sustain- compounds intended to serve as food sources for able Agriculture Research and Edu- can remediate or immobilize soil contaminants, the microorganisms. cation program through Research and Education grant award number including heavy metals (Mishra and Sarma, 2017) LS14-264: Indicators and Soil Conser- Interest in these products is growing rapidly. vation Practices for Soil Health and and hydrocarbons (Janczak et al., 2018). Carbon Sequestration. According to Fortune Business Insights, the global Intense land use, tillage, and synthetic agricultural microbial inoculant market was valued at $1.34 inputs can greatly diminish the number, diversity, ATTRA (www.attra.ncat.org) billion in 2018 and is projected to reach $3.15 is a program of the National and benefi ts provided by soil microorganisms. Center for Appropriate Technology billion by the end of 2026. Seed treatments of (NCAT). The program is funded Many farmers and ranchers who want to restore through a cooperative agreement rhizobia and other nitrogen-fi xing microorganisms with the United States Department plant-microbial interactions are exploring microbial of Agriculture’s Rural Business- accounted for 82.7% of this market. Cooperative Service. Visit the inoculants: powders or solutions containing live NCAT website (www.ncat.org) Th e use of rhizobia bacteria to enhance nitrogen for more information on or dormant microorganisms. Unlike chemical fer- our other sustainable tilizers that contain plant nutrients (like nitrogen, fi xation by legumes (the Fabacae family, including agriculture and energy projects. phosphorus, and potassium), microbial inoculants peas, beans, alfalfa, clovers, and vetch) has been www.attra.ncat.org Page 1 thoroughly studied by researchers since 1896, their food from plants, and directly involved in and rhizobia inoculum has been widely used to plant growth processes. Still other microorganisms, enhance nitrogen fi xation since the 1950s. In con- like mycorrhizae, live partially within plant cells trast, there has been far less research on other and partially within the . microbial inoculants, especially under fi eld con- ditions. Controlled studies in greenhouses have Rhizobia shown benefi ts such as enhanced nutrient uptake, Rhizobia are a genus of : bacteria resistance to drought and saline conditions, and that live in the rhizosphere. Th ey live in large colo- protection against pest and disease infestations. nies in nodules on the roots of legumes, forming a Unfortunately, these benefi ts are not always seen Related ATTRA symbiotic (mutually benefi cial) relationship with in the fi eld, under real-world conditions. Th is Publications their plant hosts. Rhizobia take nitrogen from the publication explains how microbial inoculants air (N2) and transform it into ammonia (NH4), www.attra.ncat.org work, reviews current (in 2020) scientifi c litera- which plants can use to form proteins and other ture and studies, explains several possible factors Alternative Soil biomolecules needed for growth. In return, the that can limit their eff ectiveness, and off ers practi- Amendments plant provides a place to live on its roots, as well cal advice about buying and using these products. Nutrient Cycling as carbohydrates that the rhizobia use as energy. in Pastures Overview of Plant-Benefi cial To promote the formation of nodules and also Sustainable Soil nitrogen fi xation, rhizobia inoculum in a powder Management Microorganisms form is mixed with peat or biochar, wetted, and Plant roots provide an excellent habitat for soil ATTRA Soil Resources applied to legume seeds immediately before plant- organisms. Osmosis and root pressure pull soil ing. Th ere are eight diff erent rhizobia-legume nutrients towards plant roots, making the root cross inoculation groups, and it’s important to zone a nutrient-rich area. At the same time, plant use the correct type of rhizobia for the legume roots exude many organic chemicals that serve as being planted. (See the Further Resources section food sources for microorganisms. for guides to successful inoculation.) Plant-benefi cial microorganisms living in the rhizosphere (the area close to plant roots) include Mycorrhizae decomposers and bacteria involved in nitrogen Mycorrhizae are fungi that form symbiotic rela- transformations. Other microorganisms, called tionships with almost 80% of all land plants. , live inside or between plant cells: Th e mycorrhizae most important for agriculture protected from predators, getting most or all of are the arbuscular mycorrhizae (sometimes seen abbreviated as “AM”). Mycor- rhizae begin their life within the cells of plant roots, where they extract carbohydrate energy and produce hyphae, thread-like structures that grow through the soil. Since these hyphae are considerably thinner than plant roots, they can grow more easily through soil, obtaining hard-to- reach phosphorus and water that are transported to plant roots. As hyphae grow through and around soil particles, they bind soil particles into aggregates: clumps of soil mineral and organic matter that are key to soil nutrient and water-holding capacity and also enhance soil tilth or friabil- Plant-benefi cial activities of endophytes. Source: Brader et al., 2014 ity, decreasing soil compaction.

Page 2 Microbial Inoculants Rhizobium bacteria. Source: McMahon, 2016

Common mycorrhizal networks. Source: Jansa et al., 2013

Plant Growth-Promoting Rhizobacteria (PGPR) Plant growth-promoting rhizobacteria (PGPR) are rhizosphere bacteria that (as the name implies) stimulate plant growth. Some PGPR protect against pathogens, stimulate production of growth regulators, or enhance a plant’s ability to detoxify poisons. Others enhance the availability of nutrients by fi xing nitrogen, solubilizing phos- phorus and potassium from soil minerals (Khan et al., 2007), or facilitating uptake of iron (Dob- belaere and Okon, 2007). PGPR can also work with mycorrhizae during droughts to enhance nodule formation on legume roots and nitrogen Mycorrhizae. Source: Karas, no date fi xation by rhizobia (Pandey et al., 2016).

Among their other benefi ts, mycorrhizae can pro- duce chemicals that make iron and other micro- nutrients more available (Saha et al., 2015), make plants more resistant to saline conditions (Vuru- konda et al., 2016), or produce phytohormones that signal the plant to create chemicals hindering the growth of pests and diseases (Hohmann and Messmer, 2017). Mycorrhizae can also connect with the roots of other nearby plants, including plants of diff erent species. Th ese common mycor- rhizal networks can transfer nutrients, protective substances, and signals among plants—helping neighboring plants resist infestations from pests, diseases, or invasive plants. Some studies have shown that these mycorrhizal networks may be partially responsible for enhanced yields of multi- cropping systems (Walder et al., 2012). Plant growth-promoting rhizobacteria. Source: Kumar and Dubey, 2012 www.attra.ncat.org Page 3 Like mycorrhizae, PGPR can produce phyto- growth of pathogens or their ability to enter hormones, improving tolerance for salinity, and damage plant cells. PGPR can also stimulate drought, and extreme temperatures (Vurukonda protective responses to pest or disease infestation, et al., 2016). One group of PGPR produces such as exuding exopolysaccharides (Vurukonda antibiotics and other chemicals that inhibit the et al., 2016).

Table 1. Plant-Benefi cial Rhizobacteria Species and Their Benefi ts

Salinity Plant Nitrogen Enhance Phosphorus Disease Enhance > K uptake Drought & Root Fixation Nodules Solubilization Supression Stress Growth

Agrobacterium xx x

Arthrobacter x

Aspergillus x

Azoarcus x

Azotobacter xxxx

Azospirillum xx x

Bacillus xxx x xxxx

Burkholderia xxx

Diazotrophicus x

Enterobacter xx x x

Herbaspirillum x

Klebsiella xx x

Pseudomonas xx x xxxx

Rhizobium x x xxx

Rhizopus

Serratia xx x

Trichoderma xx Sources: Mabrouk et al., 2018; Gouda et al. 2018; Tabassum et al., 2017; Bhardwaj et al., 2014

Page 4 Microbial Inoculants Endophytes inadequate or inappropriate diversity, unfavor- able environmental conditions, unfavorable land As noted earlier, endophytes live inside or between management practices, and production factors plant cells—entering plant tissues through leaves, that limit an inoculant’s eff ectiveness. Let’s look stems or roots. Some PGPR are able to move from at each of these in more detail. the rhizosphere into root tissue and become endo- phytes. Like PGPR, endophytes can stimulate plant stem and root growth. Th ey can also protect In-Field Competition plants against growth inhibition due to salinity, Recommended application rates for most micro- drought, fl ooding, pest and disease attack, pres- bial inoculant products are extremely small. If the ence of heavy metal or organic contaminants, added microorganisms are to provide any benefi t, or temperature (Glick, 2015). Endophytes have they need to grow, reproduce, and outcompete the also been shown to enhance plant competitiveness vastly greater numbers of microorganisms already against weeds (Moura et al., 2020) and to form phytochemicals in medicinal plants. While rhi- zobia are an example of endophytes, we discussed them separately above because of their widespread use and importance, as well as because these ben- efi cial bacteria were known and promoted for use much earlier than other endophytes. Plant-Benefi cial Interactions Interactions among PGPR, mycorrhizae, and endophytes drive many important plant growth processes. For example, mycorrhizae hyphae, which typically only live fi ve or six days, pro- vide a source of food for PGPR (Staddon et al., 2003). In turn, substances produced by PGPR can aff ect root cell growth to facilitate the formation of mycorrhizal (Azcón et al., 2010). PGPR, mycorrhizae, and endophytes often work together to enhance plant uptake of mineral nutri- ents, stimulate plant root growth, reduce drought stress, or produce substances that protect plants from pest and disease infestations. Th ese interac- tions also enhance the nitrogen-fi xing function of rhizobia by providing the rhizobia with phos- Benefi ts provided by endophytes. Source: Feng et al., 2017 phorous and protecting the nodules from stress conditions (Staudinger et al., 2016).

Reasons Why Microbial Inoculants May Not Work Soil is home to a vast ecosystem of soil organisms. Attempting to change that eco- system by adding a relatively small amount of powder or solution containing new organisms—while sometimes dramati- cally eff ective—is also inherently problem- atic and unpredictable. Many factors can prevent microbial inoculants from work- ing. Th ese include competition with exist- ing organisms, compatibility problems, Soil microorganisms work together to provide plant benefi ts. Source: Quiza et al., 2015 www.attra.ncat.org Page 5 in the fi eld for access to plant roots and other bacteria involved in nitrogen fi xation, phosphorus nutrient resources. For the introduced microor- solubilization, organic matter decomposition, ganisms to persist into the next growing season, and drought protection can all work together they also need to benefi t the plant suffi ciently to provide nutrient cycling and continued plant to preferentially obtain nutrients and habitat growth under stress conditions. (Verbruggen et al., 2013). Environmental Conditions Microorganism/Plant Climate conditions, especially temperature, can Compatibility limit the growth and benefi cial properties of A microorganism may be parasitic or pathogenic to plant-benefi cial microbes. For example, high tem- a particular plant species (Jones and Smith, 2004), peratures can alter the release of metabolites from extracting lots of plant carbohydrates while provid- plant roots, favoring some microorganism strains ing few, if any, benefi ts. In order to be eff ective, a over others. Drought and soil salinity inhibit plant benefi cial microorganism must be compatible with growth and the formation of rhizobium nodules. the plant population, soil conditions, and existing However, certain PGPR can reduce these negative soil microbial populations: providing substantial impacts. Soil conditions, such as aggregation, benefi ts while effi ciently using (and not draining) aeration, and moisture, will also favor some the plant’s carbohydrates. microorganisms over others. Lack of Diversity Land Management Practices A given soil may lack the necessary plant and Modern agriculture creates conditions that are microbial biodiversity to respond to a microbial very diff erent from those under which most plant- inoculant. Diff erent plant species exude diff erent microorganism relationships evolved, sometimes chemicals, release diff erent nutrients back to the creating an unhospitable environment. Most crop soil upon decomposition, and provide diff erent plants are not native to the location where they types of microbial habitats within their rhizo- are now grown and, due to plant breeding, most sphere. Th is diversity of physical and chemical crops have characteristics that are diff erent from conditions provided by plant diversity supports native or heirloom varieties. a diversity of microorganisms. Working together, Benefi cial microorganisms are also aff ected by a diversity of microorganisms provides many farm inputs—especially manures, fertilizers, and functions or services back to plants. For example, pesticides. Symbiotic relationships between soil microbes and plants evolved under stress condi- tions, with limited plant access to soil nutrients. When nutrients are abundantly available from manure or synthetic fertilizers, a plant obtains limited benefi ts from developing associations with microorganisms and may not produce exudates to support the growth of these now-unnecessary microorganisms (Djuuna, 2014). Chemicals in pesticides can also kill or disrupt the growth of microorganisms. Microbial Inoculant Production Method Not all varieties of mycorrhizae or PGPR are read- ily made under laboratory or large-scale produc- tion conditions. For example, the mycorrhizae and PGPR that provide the greatest benefi ts to plants growing under stress conditions are typi- cally slower-growing varieties, whereas rapidly- Environmental factors aff ecting plant benefi ts from soil microorganisms. Source: growing varieties are appealing to manufacturers Compant et al., 2019 because they are more cost-eff ective to produce.

Page 6 Microbial Inoculants Growing conditions in commercial production are also usually very diff erent from fi eld condi- tions. In the greenhouse or in soilless culture, mycorrhizae are produced on a single host plant, while bacteria are produced in aerated vats of nutrients and minerals. Such favorable grow- ing conditions—better than typical fi eld con- ditions—favor organisms that provide fewer benefi ts than those grown under more stressed conditions (Hohmann and Messmer, 2017). Commercial microbial inoculants need microorganisms, a material to carry and nur- Overcoming Microbial ture them, and packing to enhance their viability. Source: Lobo et al., 2019 Inoculant Limitations Research has shown the following factors to be necessary for microbial inoculants to be eff ective: • Th e diversity of organisms in microbial inoculants, and the nutrients added to them, should be consistent with natural ecological processes. • Microbial inoculants should be produced under soil, crop production, and environ- mental conditions similar to those where these products will be used. • Processing, packaging, customer han- dling, and use of microbial inoculants need to protect the viability of the micro- organisms. • Microbial inoculants must be applied to seeds or plant roots in a way that favors Microbial inoculants may contain nutrients and hormones. Source: Agricen, no date competition of the added microorganisms with resident soil organisms. the product will be used. Most products commer- Let’s consider these one at a time: cially available in the United States are developed for a mass market. In developing countries, when Microbial and Nutrient Diversity products are developed for specifi c, localized con- No microbial inoculant is eff ective in all ecosys- ditions, inocula have proven eff ective (Gouda et tems. Some products include a small number of al., 2018). While currently cost-prohibitive for microbial species and are designed to provide a commercial production, genetic sequencing can specifi c function. Other products contain diverse be used to select microorganisms with specifi c species and are designed to provide multiple benefi cial or environmental traits (Paterson et benefi ts. Moreover, some products also contain al., 2017) and to identify processes infl uencing enzymes and micronutrients, intended to stimu- establishment success and persistence of inocu- late initial growth of the microorganisms. Before lants in the soil under fi eld conditions (Kokkoris buying, study the label to determine the type and et al., 2019). diversity of microorganisms in the product and Th e Rodale Institute guide How to Inoculate Arbus- whether any nutrients are provided. cular Mycorrhizal Fungi on the Farm, describes a method for creating a “diverse group of locally- Production Conditions adapted mycorrhizal fungi that can be used to Ideally, a microbial inoculant should be produced boost a farm’s native population” (Lohman et al., under conditions closely resembling those where 2010). Steps include: www.attra.ncat.org Page 7 1. Select a host plant and grow it to seedling approach is simply to dig up plants from local stage in vermiculite or seed starter. (Don’t healthy ecosystems (assumed to have plenty of use Brassicaceae species as host plants, since mycorrhizae), and harvest mycorrhizal inoculum most plants in that family don’t form mycor- by shaking soil loose from the roots and cutting rhizal associations.) the roots up into small pieces. 2. Transplant the seedlings into pots fi lled with 1:3 soil:sand mixture, using sterilized fi eld Packaging, Handling, and soil or soil from a natural area or fi eld that Application has not been used within two years to grow Mycorrhizal and PGPR inoculants are sold the crop you are planning to inoculate. either as powders or liquid solutions. Liquid for- 3. Make a dilute compost mix by combining mulations are less expensive to make but have compost (yard waste or dairy manure) with shorter storage times, losing viability after only a nutrient-poor substrate such as vermicu- about six months. Solid formulations are mixed lite, perlite, or peat in 7-gallon bags. For with clay, peat, biochar, and various agricultural yard-clipping compost, the article suggests by-products such as cassava starch, sugarcane a ratio of 1:4 (compost:vermiculate) on a bagasse, or talc. Encasing microorganisms in volume basis. microcapsules made of plant-based polymers or resins has been shown to enhance shelf life, 4. Collect soil from the top fi ve inches of soil at while allowing slow release of microorganisms various locations throughout a natural area. and providing protection from predation dur- Add about a quart of this local soil to each ing the establishment stage (Hernández-Montiel of the 7-gallon bags and mix. Transfer about et al., 2017). Another method that has shown fi ve seedlings into each 7-gallon bag. Weed eff ectiveness is to mix microbial inoculants with and water. Mycorrhizhae will proliferate as biofi lms and apply them to plant seeds. (Biofi lms the plants grow. are substances exuded by soil organisms onto 5. To harvest mycorrhizal inoculum after the plant root or leaf surfaces.) plants have grown, shake loose compost/ In general, seed application has proven to be the vermiculite mix from the root ball. Th en most eff ective method of applying mycorrhizal cut the root segments into short pieces (less and PGPR inoculants to fi eld crops, allowing than ½ inch) and add these to the compost/ vermiculite mix that you shook loose. benefi cial microorganisms to grow with the ger- minating seed. Seed treatment involves mixing a Th is method requires that you have access to nat- solution or moist powder containing spores of the ural soil with mycorrhizal fungi present. Another microorganisms with seeds just prior to planting. Transplanted vegetables are best inoculated by dipping the roots into a solution or paste of the inoculum. Inoculum can also be added to the transplant soil hole when transplanting trees or potted plants. Microbial applications have been very eff ective in horticulture and tree nurseries, primarily due to the direct application of the inoc- ulum to potted plants or tree seedlings.

Summary, Future Directions, and Recommendations More than 30 years of greenhouse-based controlled experiments have demonstrated highly benefi cial relationships among plants, mycorrhizae, PGPR, and endophytes. However, these benefi ts have not been consistently reported for the use of commer- The microbial inoculant production process. Source: Ahmad et al., 2018 cial products under fi eld conditions.

Page 8 Microbial Inoculants Standard sustainable agricultural practices will likely enhance the ben- efi ts provided by microbial inoculants. Th ese practices include • Eliminating pesticides that hin- der microbial growth • Avoiding tillage and other soil disturbance • Not over-applying fertilizers or manure • Enhancing plant biodiversity • Ensuring that a crop is grow- ing or has a root in the ground throughout the year An eff ective microbial inoculant needs the right microorganisms, production conditions, Inoculants can be produced on-farm packaging, and substances to enhance longevity. Source: Berniger et al., 2018 using soil from organic or limited input fi elds as the inoculum source. As this publication is going to press (in 2020), Alternatively, commercial inoculums can be the European Union is about to establish a combined with local soil to produce locally Fertilizing Products Regulation: providing adapted inoculum. a claims-based defi nition of “biostimulants” or microbial inoculants. To gain access to Researchers are currently (in 2020) studying the European markets, a manufacturer will need genetics of particular microorganism species and to produce data showing that the product isolating those organisms to produce inoculants reliably provides the benefi ts claimed (Ricci (Agnolucci et al., 2019). Recent research (Rou- et al., 2019). While similar legislation has phael and Colla, 2018) demonstrates synergis- not yet been proposed in the United States, tic benefi ts when microbial inoculants include a these EU standards may eventually infl uence combination of PGPR, mycorrhizae, and humic U.S. legislation. acids (a complex form of organic matter). Wild plants that are able to survive harsh environmen- Before using any commercial inoculant, read the tal conditions are also being explored as a source label carefully to determine the type of organ- for endophytes (Afzal et al., 2019). isms and supplementary nutrients or enzymes it contains. Also, check the production date to Since the most eff ective inoculants are developed ensure that the inoculum is still viable. Keep it for specifi c plants and environmental conditions, in a cool, dry location to slow loss of viability. eff ective commercial inoculants will likely be When using the product, make sure it adheres to developed sooner for high-value crops (such as the seeds and that the seeds are planted imme- ornamentals and vegetables) than for fi eld crops. diately, so the inoculant doesn’t dry out. To In the meantime, land management practices that enhance the benefi ts of any added inoculum, promote soil health and microbial populations are follow sustainable agricultural practices such as likely the most cost-eff ective method for enhanc- minimizing input use, planting a diverse mix of ing rhizosphere microbial communities. cover crops, and avoiding tillage.

www.attra.ncat.org Page 9 References

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www.attra.ncat.org Page 11 Further Resources A Guide to Troubleshooting Nodulation Failure in Leguminous Cover Crops. 2019. By University of Texas Rio Legume Seed Inoculation. USDA Natural Resource Grande Valley. www.utrgv.edu/agroecology/research/ Conservation Service (NRCS). Technical Note TX-PM-15-01. subtropical-soil-health/nodulation-guide/index.htm www.nrcs.usda.gov/Internet/FSE_PLANTMATERIALS/ publications/etpmctn12525.pdf Th is guide walks a producer through a decision tree to assist in determining the cause of nodulation failure in a legume Th is document from NRCS guides producers through the cover crop. process of selecting and inoculating legume cover crops. It also discusses considerations for storage of inoculant and checking for eff ective inoculation. How to Inoculate Arbuscular Mycorrhizal Fungi on the Farm, Part 1. 2010. By Rodale Institute, Kutztown, PA. https://rodaleinstitute.org/science/articles/how-to-innoculate- arbuscular-mycorrhizal-fungi-on-the-farm-part-1/ Th is resource provides an overview of mycorrhizal fungi’s role in a sustainable agroecosystem and then goes into detail on how to produce native mycorrhizal inoculant on-farm.

Microbial Inoculants By Barbara Bellows, Texas Institute for Applied Environmental Research; Mike Morris, NCAT; and Colin Mitchell, NCAT Published September 2020 © NCAT Cathy Svejkovsky, Editor • Amy Smith, Production This publication is available on the Web at: www.attra.ncat.org IP604 Slot 630 Version 092820

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