Marama Bean Microbiome Function Exploration and Future Development Design

MARAMA BEAN MICROBIOME FUNCTION EXPLORATION

AND FUTURE DEVELOPMENT DESIGN

YIWEN DENG

Submitted in partial fulfillment of the requirements for the degree of

Master of Science

Department of Biology

CASE WESTERN RESERVE UNIVERSITY

August, 2020

CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES

We hereby approve the thesis of Yiwen Deng

candidate for the degree of Master of Science

Committee Chair Karen Abbot

Committee Member Christopher Cullis

Committee Member Jean Moriuchi

Committee Member Leena Chakravarty

Date of Defense July 8, 2020

*We also certify that written approval has been obtained for any proprietary material contained therein.

Contents

Abstract ...... 4

2.Background...... 6

2.1 The Marama Bean ...... 6

2.2 Constraints on soybean production and current chemical fertilizer ...... 9

2.3 Soil microbes and Microbial fertilizer...... 11

2.4 Support for identifying and using microbiome supplements ...... 14

2.5 Current microbial fertilizer companies...... 15

2.6 Characterization of Marama Soil Microbiome ...... 22

3. Methods ...... 27

3.2 Isolation of bacteria ...... 28

3.3 Molecular characterization ...... 29

3.3 Experiment details ...... 30

4. Conclusion ...... 41

5. References ...... 43

Marama Bean Microbiome Function Exploration And Future Development Design

Abstract

YIWEN DENG

2018). Although it probably cannot become a major crop, it is likely to function mainly as a rescue crop to ensure some produce irrespective of how bad the year is. However, as an underserved crop, the lack of understanding of marama’s genetic variation and microbiome composition and their interactions present barriers to the study and cultivation of the plant for large-scale food production or even the use of its specialized characters. Since marama has the ability to grow in these harsh environments, including the ability to extract nutrients from the relatively poor soil, the mechanisms by which it does this nutrient extraction is of interest and may have applicability for mainline agricultural crops. Furthermore, the characterization of the identities and functions of the soil microbiome can not only benefit the marama bean itself, but also make contributions to other crops. This thesis provides a possible pathway to identify the associated soil microbes and their functions with the view to their application to other crops to

increase their fitness and yield. More generally, a possible outcome is identifying the best combination of microbes that would allow the development of an alternative microbial fertilizer.

By analyzing the growth status of marama beans in different microbial environments, the most important functional microbes would be identified. The population structure using DNA sequencing on these microorganisms will identify their species and functions. The possible utility of the marama soil microbiome for mainstream agriculture will be evaluated using soybean as a model system. From these experiments, it is expected that several microbial combinations will be identified as possible biological fertilizers. In the future, the plan is to determine the optimal composition for this novel biofertilizer. Although the initial test system is soybean, the same methodology will be used to determine the best microbe combinations, and their optimal conditions, for use as biofertilizers for other mainstream crops. The outcome of these experiments, will be to develop a new type of microbial fertilizer which is expected to help plants absorb nutrients, become more drought tolerant and to increase yield.

2.Background

2.1 The Marama Bean

The marama bean is a leguminous plant that grows in southern Africa. This plant is not grown at all as a conventional crop, but it does have important potential. The region where it grows in

Southern Africa is known for its drought and high temperatures. Marama beans are one of the few plants that can survive and thrive under these extreme conditions and have a useful agricultural product. Marama bean grows mainly on the western fringes of the Kalahari. The annual rainfall here is 250–500 mm and the rainfall is often sporadic, which means marama bean suffers from heavy storms, that can cause flooding, and extended dry periods. Years with almost no rain are relatively frequent. Marama bean grows on very sandy soils with extremely little organic matter, nitrogen and phosphorus (Cullis et al. 2018). Mean maximum temperatures during daylight in the main period of growth are 32°C (range 28–37°C). Solar radiation during the 3 hours either side of midday is considerable, ca 2,000µmol m-2 s-1(Cullis et al., 2019). Thus, the plant is adapted to an extreme environment to which current high yield food crops are not adapted, even when irrigated, suggesting that marama would have a particular, even unique role, in the agroecology of the region (Cullis et al., 2019). Nevertheless, marama beans are very nutritious (Cullis et al., 2019). The nutritional value is even comparable to one of the world’s mainstream crops, soybeans. It has long been proposed as a potential research direction to study the drought resistance and growth mechanism of marama bean and turn it into a crop targeted to dry areas (Cullis et al., 2018). However, the value of marama beans may not be just in the plants themselves, but also in the composition of the rhizosphere. The root systems are vital for plant growth and especially characteristically specialized under low moisture and nutrient conditions.

Although the arid environment can inhibit the growth of plant roots, plants growing in arid areas still try to expand their root as much as possible, utilizing drought avoidance mechanisms by increasing root depth and spread. However, as shown in Figure 1, the marama bean is different.

It does not have an extensive root system, mainly consisting of a long tap root (to drill for water), which can also develop into a huge tuber.

Marama bean is a perennial, tuberous geophyte, with two potential units of economic yield, the seed and tuber. The underground tuber, which when old (probably many decades) may be ca. 250 kg, becomes dormant in winter when the above ground vegetative foliage senesces. In spring, just before or at the start of the rainy season, several shoots emerge from the apex of the tuber, rapidly developing into vines (up to 6 m in length) which run along the soil surface.

Numerous bilobed leaves are produced alternately along the length of the vines. The tuber contains more than 80% of its fresh weight as water in seasons when the rainfall is sufficient.

This water is an important weapon of marama bean in its ability to withstand drought.

Stem Foliage Tuber Broken off tap root

Figure1. Middle sized tuber about 100lbs

In rainy or relatively humid weather, the tuber absorbs water from the soil and swells. Under drought conditions, the tuber uses stored water to maintain its own life. Under very dry conditions the tuber loses water and shrinks and the soil around the plant collapses. However, compared to other plants with well-developed root system, how does a marama bean with primarily a vertical main root absorb such a huge amount of water? An associated question is how does marama access the nutrients to grow sufficient foliage during the growing season to develop seeds and also lay storage in the tuber? In 1997, scientists conducted experiments on soybean cultivation under conditions of sufficient water and drought. The results show that the number of soybean seeds and the lushness of the foliage under drought conditions are far less than those with sufficient water (Agboma, Sinclair, Jokinen, Peltonen-Sainio, & Pehu, 1997). As shown in Figure2, under the same or even a poorer environment, the marama bean's foliage grows very lush, while other plants are much sparse. It is well known that the microbes in the soil play an important role in supporting the plant’s growth and development (Egamberdiyeva,

2007). The soil microbes associated with the marama bean may play an important role both in providing/mobilizing nutrients and in combating drought (Yang et al. 2019).

Figure2. Lush marama foliage. Under the same drought conditions, marama bean has more vegetative growth than on any of the other surrounding vegetation.

2.2 Constraints on soybean production and current chemical fertilizer

Soybean (Glycine max (L.) Merr.) has been classified among the most important commercial oilseed crops worldwide(Mózner, Tabi, & Csutora, 2012).It can substantially provide oils, micronutrients, minerals, and vegetable proteins suitable for livestock feed and human consumption. In addition, soybean has supplied materials for industrial uses, such as biodiesel, plastics, lubricants, and hydraulic fluids. Currently, world production of soybean is greater than any other oilseed crop. Globally, it accounts for approximately 68% of global crop legume production and 57% of world oilseed production. Collectively, soybean production occupies around 6% of the world’s available land (Hong, MacGuidwin, & Gratton, 2011).

People in many parts of Asia rely on soybeans as their main source of food. However, drought has a tremendous effect on soybean growth and development, thus negatively affecting the projected expansion of crop production. In recent years, drought has occurred more and more commonly as a result of climate change (Fan et al., 2013).

Nowadays, the high yield of soybeans in today's society is mainly due to the application of pesticides and phosphorus (P) potassium (K) fertilizers. Nodules are present on soybean root systems and fixing N by the V2 to V3 growth stage, and therefore the addition of N fertilizer to soybeans is not a recommended practice. Fertilizers provide important elements, phosphorus (P), and potassium (K)-PK for growth so that the yield of soybeans can be maintained. But at the same time, the use of fertilizers has also had a huge adverse impact on the natural environment.

The addition of chemical fertilizer destroys the material balance of the original soil (Carter,

Zhong, & Zhu, 2012) and the efficiency of chemical fertilizer use has decreased because of fertilizer saturation. The use of chemical fertilizers can also cause excessive phosphorus to be a component of the groundwater. Studies have shown that crops can take up only 30–50% of the applied chemical fertilizers, thus a great amount of the applied components is lost in the soil where it pollutes groundwater, resulting in eutrophication of the water (Mózner et al., 2012).

How to reduce the use of chemical fertilizers is an active current research area (Wang, Zhu,

Zhang, & Wang, 2018). Another important factor affecting soybean production is drought. Some

Asian land cannot sustain soybean growth due to low precipitation, and the land is left unused.

There is no doubt that this is an important waste of resources. These are some of the problems that need to be solved in the field of soybean cultivation.

2.3 Soil microbes and Microbial fertilizer

The rhizosphere hosts large and diverse communities of microorganisms that compete and interact with each other, and with plant roots(Lance, Burke, Hausman, & Burns, 2019). Within these communities, mycorrhizal fungi are almost ubiquitous. Mycorrhizal fungi colonize the roots of many plants and they don’t harm the plant; on the contrary, they develop a "symbiotic" relationship that helps the plant be more efficient at obtaining nutrients and water. In return, the plant provides energy to the fungus in the form of sugars. A mycorrhiza is a symbiotic association which is mutualism between a fungus and a plant. Symbiosis is any type of a close and long-term biological interaction between two different biological organisms, be it mutualistic (mutualism describes the ecological interaction between two or more species where each species has a net benefit), commensalistic (an association between two organisms in which one benefits and the other derives neither benefit nor harm), or parasitic (parasitism is a symbiotic relationship between species, where one organism, the parasite, lives on or in another organism, the host, causing it some harm, and is adapted structurally to this way of life). Mycorrhiza associations allow most terrestrial plants to colonize and grow efficiently in suboptimal and marginal soil environments (Buée, De Boer, Martin, Van Overbeek, &

Jurkevitch, 2009). Plant nutrient acquisition strategies and resistance to soil-borne pathogens strongly depend on mycorrhizal associations.

Fungal partners belonging to different mycorrhizal types modify local soil conditions and generate habitat patches of differential quality, which affects offspring establishment of plants

(Johnson, Clay, & Phillips, 2018). Ectomycorrhiza (EcM) which is a form of symbiotic relationship that occurs between a fungal symbiont, or mycobiont, and the roots of

various plant species and ericoid mycorrhiza (ErM) which is a mutualistic relationship formed between members of the plant family Ericaceae and several lineages of mycorrhizal fungi associations acidify soil by degrading recalcitrant litter, deplete available nutrients, and produce allelochemicals (Cipollini, Rigsby, & Barto, 2012). Allelopathy is a biological phenomenon by which an organism produces one or more biochemicals that influence the germination, growth, survival, and reproduction of other organisms. These biochemicals are known as allelochemicals and can have beneficial or detrimental effects on the target organisms and the community. At the same time, the arbuscular mycorrhizal (AM) which is a type of mycorrhiza in which the symbiont fungus penetrates the cortical cells of the roots of a vascular plant forming arbuscules can cause pathogens to accumulate in the soil to affect the plants growth (Kadowaki et al.,

2018). On the other hand, competitive interactions between plant individuals may be shaped by interactions with symbiotic fungi. For instance, EcM fungi reduce mycorrhizal root colonization of neighboring AM herbs and enhance the competitive dominance of EcM trees over AM herbs by promoting litter accumulation and limiting access to nutrients (Booth & Hoeksema, 2010).

Similarly, AM fungi were found to exacerbate iron deficiency in Eucalyptus seedlings and inhibit their establishment in Australian AM-dominated rainforest (Janos, Scott, Aristizabal, &

Bowman, 2013). In conclusion, Mycorrhizal fungi can regulate the composition of the plant population and the growth state of the plant by producing various substances to influence nutrient uptake (Tedersoo, Bahram, & Zobel, 2020).

Besides mycorrhizal fungi, plant symbiotic microorganisms such as rhizobium can directly supply nutrients, organic acids, amino acids and vitamins of nitrogen, phosphorus and other mineral elements. The most well-known of the soil microbes are rhizobia, which interact with plants by forming a symbiotic association in the root nodules with legumes. Nitrogen accounts

for 4/5 of the air composition, but it cannot be directly used by plants. Some microorganisms can convert nitrogen in the air into fixed-state nitrides that plants can use, either through the close association in the root nodule or through uptake from the soil. For example, some soil rhizobial bacteria (i.e., Rhizobium, Bradyrhizobium, Mesorhizobium, Sinorhizobium, and Allorhizobium) can use root nodules to sequester atmospheric nitrogen as ammonia, a form of N that can then be incorporated into organic components including proteins and nucleic acids (Pankievicz et al.,

2015). By virtue of their association with such nitrogen fixation soil microbes, it is equivalent to having your own nitrogen fertilizer production plant (King, 2018).

Other microorganisms can mobilize nutrients, such as phosphorus, that are otherwise unavailable to the plants. For instance, Duarah reported that application of both NPK fertilizer and a consortium of seven PSB (Phosphate solubilizing bacteria) strains selected for their high P solubilization properties (e.g., Staphylococcus epidermidis, P. aeruginosa, Bacillus subtilis, and Erwinia tasmaniensis) improved plants biomass and enhanced germination index in rice and cowpea owing to stimulation of specific enzyme biosynthesis such as amylase in seeds (Duarah,

Deka, Saikia, & Boruah, 2011). Moreover, a recent study evaluated the maize growth in response to impregnated DAP (Diammonium phosphate) fertilizer with Pseudomonas putida (prepared by coating DAP (20 g/kg) with a mixture of organic material containing compost, molasses, and the P. putida bacterial strain). This study demonstrated the benefit of the combined DAP and

PSB co-application as it improved maize dry matter (12%) yield and P uptake (33%) in addition to significant agronomic efficiency in terms of produced biomass that increased by 62% compared to unfertilized soil (Noor, Yaseen, Naveed, & Ahmad, 2017).

Microbial fertilizer, containing several kinds of highly active and beneficial microbial bacteria, suitable for use in various crops, can improve nutrient utilization, and has broad ubiquity,

breaking the specificity limitation of common chemical fertilizers which is that not only improve soil nutrients, crop stress and disease resistance and promote plant growth, but also shows no pollution to the environment. In general, any land with plant growth can be a site for the addition of microbial fertilizer to improve the soil and reduce the use of chemical fertilizer to promote crop growth. It helps to return the soil to its natural state and balance the pH of the soil to the extent needed by the crop. All of these effects improve soil fertility and help eliminate pollution in soil and water. It is a low-carbon, pure natural, non-toxic, harmless and non-polluting organic microbial agent, which can improve soil fertility, increase the number and activity of beneficial microorganisms in the soil and prevent soil compaction. In 2017, the microbial fertilizer containing the phosphorus-dissolving strain Pseudomonas plecoglossicida and potassium- dissolving strain Bacillus Aryabhata was developed to stimulate the growth of reed, to improve the soil nutrient and promote the restoration of plants in degraded wetland. It turned out that the microbial fertilizer significantly promoted the growth of reeds, increase the contents of total N, P and K in soil and the activities of soil sucrase and urease (Luo & Xing, 2010).

2.4 Support for identifying and using microbiome supplements

In 2015, ten bacterial strains were isolated from Yongin forest soil for which in vitro plant- growth promoting bacteria (PGPB) trait screenings, such as indole acetic acid (IAA) production, a phosphate solubilization test, and a siderophore (siderophores are small, high-affinity iron- chelating compounds that are secreted by microorganisms such as bacteria and fungi and serve primarily to transport iron across cell membranes) production tests were used to select one PGPB candidate—P. yonginensis DCY84T. Salt stress, drought stress and heavy metal (aluminum) stress challenges indicated that P. yonginensis DCY84T-inoculated plants were more resistant

than control plants (Sukweenadhi et al., 2015). In 2016, a tobacco study showed that microbial water-retention fertilizer (MWF) can help tobacco grow in extreme drought environments in

GuiZhou, China. Under different drought conditions, the largest leaf area was obtained in normal conditions combined with 45 g MWF. The LAI (leaf area index which is a dimensionless quantity that characterizes plant canopies is defined as the one-sided green leaf area per unit ground surface area in broadleaf canopies) significantly increased by 32.0% when compared to normal conditions without adding MWF (Yang et al., 2019). Besides, bacterial inoculation can increase crop nutritional value especially in nutrient deficient soil. These investigations were carried out in 2017 in pot experiments with calcareous calcisol soil which is soil that has calcium

carbonate (CaCO3) in abundance and causes nutrient deficiencies for many plants taken from

Sirdarya, Uzbekistan and control loamy sand from Muencheberg, Germany. The bacterial strains

Pseudomonas alcaligenes PsA15, Bacillus polymyxa BcP26 and Mycobacterium phlei MbP18, which were obtained from the National Collection of Agricultural Microorganisms, Centre of

Agroecology, Tashkent State University of Agriculture of Uzbekistan, were previously selected for their ability to stimulate plant growth in plate bioassay experiments had a great effect on plant growth and nitrogen (N), phosphorus (P) and potassium (K) uptake of maize in nutrient deficient calcisol soil (Egamberdiyeva, 2007).

2.5 Current microbial fertilizer companies

Plant microbiota has been explored in the last decades, particularly those colonizing the rhizosphere, but the awareness of their diversity and relevance has exploded in the last few years.

At the same time, agricultural production has had to face severe challenges due to the demographic development and climate change with extreme weather events and emerging

pathogens. Furthermore, our society demands more sustainable production systems, a number of chemicals (e.g. pesticides) will be taken from the market in the coming years and several countries do not support the use of genetic modification to improve crop traits. All these factors have led to an increasing awareness of the functions mediated by plant microbiota by academia as well as by the industry(Yin et al., 2015).

Indigo Ag, a plant microbiome company, was founded in 2013. The company has some impressive fundamentals, including US$650 million in funding, with a reported value of over

US$3.5 billion and 750 employees worldwide. Indigo was originally developed as a microbial company selling microbial-treated seeds. Since then, Indigo has completed multiple supply chains and joint ventures around the world, and has invested in warehousing, logistics and other fields. The idea now is not only to sell profitable alternatives in existing systems, but also to fundamentally change agriculture as we know it. Their focus is to identify which of these soil microbes are most vital to a plant’s health. The program starts with the identification of the microbes that have evolved in conjunction with plants over millions of years in order to optimize their health and maximize their productivity, and to construct a database of these microbes and their effects. Then algorithms and machine learning are applied to this database to predict which microbes are most beneficial to the plant’s health. The resulting products complement a plant’s natural processes to improve health and development across each phase of life, while boosting crop yields. Their first product, Indigo Cotton, which was a seed treatment containing bacteria isolated from cotton plants that is intended to improve yields under drought conditions, was launched in July 2016. By 2018 the company had also launched similar seed treatments to improve drought resistance in wheat, corn, soybeans, rice, and barley.

Indigo's business development and business model are unique and novel. Indigo’s central thesis is that commodity crops are not all created equal. One farmer's corn may contain more starch than most corn. The other may require less water or less pesticides to grow. The company hopes to find ways to compensate farmers for these attributes so that they are no longer forced to sell goods to anonymous grain markets. The price of the same crop on the market is the same, even if they have different characteristics. This is a waste of resources for farmers, producers, and society as a whole. Taking bread as an example, the production of bread is more inclined to use high-protein wheat. But the bread factory can only buy the same wheat from the standardized market, which is a loss for farmers and factories that plant high-protein wheat. Even some farmers do not know the characteristics of the crops they grow. Indigo wanted to change this situation, so they established a data collection apparatus called Indigo Research Partners. It's a global network of sensors, drones, and satellites that send one trillion data points per day back to

Indigo's Boston headquarters for analysis. 125 farm businesses averaging 8,000 acres in size, spread across the largest commodity hotbeds in the world, allow Indigo to collect data and run tests on new technology in the field. Indigo launched its own online “Marketplace” service to shepherd carefully-monitored grain—loaded with dozens of accompanying data points—to precisely the buyer who ordered it. In this way, indigo obtains massive amounts of data and connects with downstream manufacturers to make farmers and factories mutually beneficial and win-win. It also provides farmers with zero-interest loans to install grain bags. This new long- term storage method can last up to 11 months and is drier and cooler than traditional storage methods. In addition, if they allow Indigo to monitor the grain and harvest the resulting data, these farmers will receive monthly "rewards" directly from the company. For indigo, this is obviously necessary. Once these grains leave the farm and go elsewhere, all traceable data will

disappear. In addition, the huge amount of data gives it the ability to research and innovate. By exploring the different characteristics of the same crops, the reasons for variation can be investigated. Indigo can then continue to develop higher quality seeds or microbial fertilizers.

Using the data obtained from farmers to conduct scientific research, and then feedback the scientific research results to farmers, this smart business model makes Indigo develop better and better.

Founded in 2012, Agbiome is another plant microbial company. However, compared to Indigo

Ag, they mainly focus on the research of plant microorganisms. Their first product was a biological product that controlled major soil-borne diseases in greenhouses and major crops.

AgBiome will also apply the most advanced genomics and screening techniques to identify plant-related microorganisms, thereby enhancing plant health, resistance to diseases and insect pests and yield. They use the new knowledge of the microbiome associated with plants to develop innovative and cutting-edge solutions to create novel products. Through their extraordinary and expanding collection of microorganisms, they discovered microorganisms and proteins that kill pests, fungal pathogens and weeds. They first isolated microorganisms from global environmental samples and then sequenced the genome of each microorganism completely. Now they have discovered more than 3,500 new insect control genes, sequenced more than 70,000 genomes from the microbial strain collection, and possessed more than 200 active strains.

Compared to Indigo, Agbiome's business model is relatively simple. Focus on product development one by one. Each product goes through several phases of Discovery, Early

Research Proof of Concept, Proof of Concept in Target Crop, Event Selection, Product

Development, Regulatory & Market Development, and finally enters the market.

Although this business model is simple, it is a most suitable place from which a new start-up can learn.

As an African crop that has not been fully understood, Marama bean and related microorganisms have significant potential to be the basis for the development of a similar company and benefit mankind furthermore. It is now difficult for us to judge how much of the ability of marama beans to survive in harsh environments comes from its neighbors in the soil-microorganisms. But this uncertainty is also a huge market. Through the identification of these microorganisms and functional studies, we may find previously unknown variants. The function of these variants could make a huge contribution to the existing agricultural market. These contributions may include but are not limited to helping plants absorb nutrients, survive drought, resist disease and increase yields.

An important example is the worldwide food shortage and humans' application of nitrogen-fixing bacteria to the soil to increase crop yields. The global population is constantly rising and expected to reach 9.8 billion in 2050 and 11.2 in 2100. Currently, there are great expectations in the application of microbial inoculants as promising results have been reported and so far this approach has been hardly applied in crop production with the exception of N2-fixing rhizobial inoculants for legume production. Although is quite abundant in the Earth, nitrogen (N) is the most limiting nutrient for plants, which require it for the formation of amino acids and subsequently, proteins. Some prokaryotes have the ability to manage the process of combination or conversion of atmospheric nitrogen into organic forms, which can be finally assimilated by plants (Raymond, Siefert, Staples, & Blankenship, 2004). Amongst free-living rhizobacteria, members of the genera Azospirillum, Azotobacter, Beijerinckia, Bacillus, Paenibacillus,

Burkholderia, Gluconoacetobacter and Herbaspirillum were reported as nitrogen-fixing microorganisms. The genus Azospirillum is commonly associated with cereals in temperate zones, increasing crop yields in most of them, as well as in some legumes and sugarcane.

Members of the genus Azotobacter are able to fix nitrogen in rice crops (Sahoo, Ansari, Dangar,

Mohanty, & Tuteja, 2014). If we can find applicable microorganisms from the soil of Marama bean, and it is likely to solve other problems in plant growth like nitrogen-fixing bacteria and solve the shortage of human food.

Intrinsyx Bio is a good example of resolving the problem above. Intrinsyx Bio is a developer of plant biofertilizer intended to increase crop yield, reduce excess fertilizer and improve soil and water conditions. To achieve these intentions, the company uses microbiome to help crop plants, grasses, ornamental plants and trees to fix Nitrogen (N2) directly from the atmosphere, enabling farmers to increase crop yields both in optimum soil nutrition and in nitrogen deficient soils.

Their microbes were first discovered in trees growing in nutrient-poor conditions in the forests of the Pacific Northwest. It was determined that conventional nitrogen fertilization is only 30-50% efficient, in terms of the plant utilization of the added nitrogen. The rest is lost to the environment and may result in significant pollution. Another consequence of these fertilization practices is negative impacts on plant beneficial microbes. Their research process is of great help to the microbiome research of marama bean. The content of N in Pacific Northwest soil is relatively small, so it is very likely to find microorganisms in the forest soil that help trees absorb N. This is basically consistent with our research ideas. Marama bean grows in soil under arid conditions and nutrient deficiency, and there is a high probability that there are microorganisms in the soil that help the growth of marama bean. So the success of Intrinsyx Bio has further strengthened our confidence in potential of the marama soil microbiome.

Compared to the previous company, Biome Makers chose a different path. Also based on the microbiome as the foundation of the company, Biome Makers' goal is not crops, but the soil which carry crops. Through the detection of nutrient contents of the soil, combined with the crop planted in the soil, Biome Makers provides a detailed soil report for the farmers, from which farmers can know the nutrients that are lacking in their soil. After that Biome Makers provides farmers with corresponding microbial products through their huge database and a lot of microbiome, helping them to obtain higher yields. The company was founded in San Francisco in 2015, and achieved great success in the European market the following year, then won various entrepreneurial awards in the next three years. After getting a series of investments in 2019, it launched its own platform and app. This also provides new ideas for our entrepreneurial planning. As a rare potential crop that can grow under conditions of extreme drought and nutritional deficiencies, marama bean's soil microbiome can effectively fill the blanks in the current microbial product market. If the future experiment proves that the marama bean soil microbes really have the effect of helping crops to resist drought, we can also cooperate with similar platforms to license the marama bean soil microbiome patents to them. This model can enrich our business model, can increase the richness of the world microbial product market and contribute to the world's agriculture at the same time.

As noted above, the market for bio-fertilizers is huge. This means that establishing a company in this field, as long as the product is effective, would not be competing in an already saturated field. The global microbial biopesticide market accounted in 2014 for more than $800 million. A more recent analysis reported a global biocontrol market of $2.8 billion today rising to an expected over $11 billion in 2025 with about 60% microbial products. A CAGR (compound annual growth rate) of 17% is expected for the years 2015–2020. Similarly, the biostimulant

market is constantly increasing with an expected CAGR of 10.9% until 2022 (Sessitsch, Brader,

Pfaffenbichler, Gusenbauer, & Mitter, 2018).

However, compared to biopesticides and biostimulants, products for resisting drought and nutrients absorption have not been fully developed. The soil microbiome influence on the combination of these two functions apparent in Marama bean, is likely to reveal the new insights into the solution of these two aspects for improved growth under stressful environmental conditions. Therefore, the study of marama bean soil microorganisms has both a great scientific significance and market potential.

2.6 Characterization of Marama Soil Microbiome

Tylosema esculentum, the marama bean, is an orphan legume native to Southern Africa. Marama is a prostrate, tuber-producing, non-nodulating and trailing plant capable of growing in inhospitable arid, low fertility and low-water conditions while maintaining a high nutritive content comparable to peanuts and soybeans. Because of these qualities, Marama is an excellent candidate for microbiome study relating to how the plant can accesses scarce soil nutrients under water limitation and undergo rapid extensive vegetative growth. Under these less than optimal conditions, marama still gets high vegetative growth, large tuber formation and seed production.

Therefore, the plants must be able to access appropriate nutrients. Since they do not appear to have a high density root system, a reasonable assumption, which is proposed to be investigated, is that the soil microbiome is an active partner in providing the required nutrition.

The basic method of soil microbe discovery is to isolate soil microbes (and/or their DNA) and evaluate their potential as bioinoculants. The first step is constructing inventories of PGPB (Plant growth-promoting bacteria) from plants’ natural environments including soil, roots, or some

internal tissues. In general, these samples need to go through both nonselective and selective culture media to be tested whether they are able to grow in a specific artificial condition. Many microbes have been cultivated using an enrichment media method whereby a soil sample is mixed with water and the suspension serially diluted onto a nonselective medium such as nutrient agar or any generalized medium that contains a carbon source, amino acids, and salts.

Next steps often require the use of a culture medium that reveals a particular plant growth promoting (PGP) trait (Menendez & Garcia-Fraile, 2017). Due to the variety of PGP characteristics, the trait methods also vary according to different experimental needs. For example, in 2015, 100 PGPR strains isolated from different varieties of ginger (Zingiber officinale Rosc.) were first characterized for their morphological, biochemical, and nutrient mobilization traits in vitro. The PGPB were also screened in vitro for inhibition of Pythium myriotylum causing soft rot in ginger. Results revealed that only five PGPB showed >70% suppression of P. myriotylum. These 5 PGPB viz GRB (Ginger rhizobacteria) 25 – Burkholderia cepacia, GRB35 – Bacillus amyloliquefaciens; GRB58 – Serratia marcescens; GRB68 – S. marcescens; GRB91 and Pseudomonas aeruginosa were used for further growth promotion and biocontrol studies in the green house and field (Dinesh et al., 2015). Once a single species is isolated, it is usually identified by 16S ribosomal RNA (rRNA) gene sequence analysis.

However, as the number of microbes in plant soil is massive, it is hard to extract and sequence all the DNA at the same time. Moreover, even if the molecular research in the lab is relatively easy to accomplish, cultivation dependent experiments are always problematic because not all bacterial isolates can be grown in vitro. Indeed, it has been estimated, based on the discrepancy between the numbers of cells directly counted in an environmental sample versus the number growing in culture medium, that only approximately 1% of environmental microbes are

cultivable. Since the product target is for large-scale soil planting, it is not worth the effort to spend a lot of time and energy to conduct experiments in the laboratory that cannot be applied to the actual soil.(Katz, Hover, & Brady, 2016).

On the other hand, the interaction between different microbes in microbial community affects expression of function significantly. Single microbe inoculation ignores this to some extent (King,

2018). Soil inoculation is a new method to do the microbial community inoculation. Compared to original methods, this method retains the habitat in which microbes live so that makes it easier to establish in the new environment.

Recently, scientists found a reduced number of microbes closer to the roots of plants compared with soil in general, and these microbes which can establish near or even inside the plant root play more important roles in plant growth. Consequently, this is a great way to narrow down the scope of functional microbes.

For this reason, seven sets of controlled trials have been designed. These experimental conditions are aimed at determining three different responses:

a. Firstly, does the soil microbiome associated with marama promote plant growth on both

marama and soybean?

b. Secondly, is the effect on soybean an improvement on the current microbiome associated

with soybean cultivation?

c. Thirdly, does the presence of the marama soil microbiome (or subsets of it) improve the

drought tolerance of both marama and soybean?

1. In the first set of experiments, the sterilized marma bean seeds will be planted in

sterilized and non-sterilized marama bean soil to determine whether the microorganisms

in the soil play a positive role in the growth of marama bean.

2. In the second set of experiments, the sterilized soybean seeds will be planted in sterilized

and non-sterilized soybean soil as a control to confirm that the microorganisms in the soil

play a positive role in the growth of soybean.

3. In the third set of experiments, sterilized and non-sterilized marama bean seeds will be

planted in sterilized marama bean soil to investigate whether the microorganisms

attached to the seeds play an important role in the growth of marama bean.

4. The fourth group of experiments soybean seeds will be planted in sterilized marama bean

soil, non-sterilized marama bean soil and non-sterilized soybean soil. These will

determine whether the microorganisms in the marama soil have a growth promoting

effect on soybeans and if the marama bean soil microbiome is even better than current

soybean soil microbiome for promoting plant growth.

5. The fifth set of experiments is to plant marama bean and soybean seeds in sterilized soil.

After a period of time, the microorganism species in the soil are tested and identified to

determine whether the plant has a regulatory effect on the types of soil microorganisms.

6. The sixth set of experiment is to plant three groups of sterilized soybean seeds to

sterilized soil. In the first group adding marama microbiome (adding Namibian soil from

around well growing marama plants). Adding no microbiome to the second group and

adding normal soybean microbiome dressing to the last group. Drought will be invoked

by withdrawing watering at different growth stages and the extent and timing of wilting

of the plants, their recovery and yield after watering was resumed will be determined.

For all of the treatments, the microbiome composition will be determined using both basic microbe isolation and 16S ribosomal RNA (rRNA) sequencing for the bacterial composition and the internal transcribed spacer (ITS) region for the profiling of the fungal population. After the identification of the microorganisms is completed, it is hoped that one or several new functional microorganisms would have been discovered. Based on these data, new combinations of microorganisms can be developed into PGP products.

On the other hand, if the microbiome on the surface of the seeds and in the soil proves to have a positive effect on plant growth, then further exploration into the best conditions for this microbiota to be used directly as a microbial biofertilizer.

3. Methods

3.1 Soil sample collection and inoculation

Soil samples have been collected at four different sites of Namibia (Fig3) from different plants at each site with a range of growth habits, from large well growing to small relatively unproductive plants.

Figure3. Sample collecting sites

Selected marama bean plants at the four locations have also had their seeds collected. The

seeds at the four locations are recorded as seeds 1, 2, 3, and 4. The soil samples

surrounding marama bean has been collected from under the corresponding plants and

from a distance from the plants. Each soil location was sampled in a vertical cline from

below the tuber to the surface. This sampling strategy will include as many

microorganisms as possible as well as identifying any stratification of the microbiome.

The soil in the four locations is identified as soil 1, 2, 3, 4 along with the positing

underground for subsequent records.

Four sets of initial control experiments will be conducted on the seed and soil samples at

each location(Peacher & Meiners, 2020). For these initial tests, a mixture of the soil

across the depth profile will be used(Reinhart & Rinella, 2016). The following summary

of the protocol uses Seed sample 1 and Soil sample 1 (from the first location) as an

example(Cahill et al., 2017)(Rinella & Reinhart, 2017).

Sterilization regime:

Seeds: Sterilize the seeds with 95% ethanol, and then wash with ultrapure water. After

washing, put the seeds in 5% bleach for 20 minutes and rinse with ultrapure water several

times.

Soil sterilization: Autoclave at 121°C for 15 minutes

Growth of plants

All the individual plants will be grown individually in 8” pots with the appropriate soil

composition.

Seed yield (kg) ha-1 = Seed yield pot-1 (kg)/ Area of pot (m2) ×10000m2

3.2 Isolation of bacteria

After determining the best performing soil sample, bacteria and fungi will be isolated from it.

Roots and tuber will be separated from the rest of the marama bean plant (Döbereiner, 1989), and rhizospheric bacteria isolated from soil adhered to the roots and tuber. Root bacteria are isolated after surface disinfection, which is performed by washing the roots in running tap water, and then by immersion in 70% ethanol for 1 min and in sodium hypochlorite solution (4%, v/v) for

2 min, followed by five serial rinses in sterilized distilled water. Immediately after disinfection, the roots are sliced with a sterile scalpel. A total of 10 g of soil (for rhizospheric bacteria) and

10 g of root segments (for root bacteria) are placed in individual sterile 500 ml Erlenmeyer flasks containing 90 ml of sterile saline solution (0.85% NaCl). Sample is incubated at 28°C under agitation (200 rpm) for 16 h. Aliquots of 0.1 ml of three-fold serial dilutions were inoculated, in

triplicate, into vials containing 4 ml of semi-solid N-free medium (0.18% agar-agar). Five days after incubation at 28°C, those vials showing a veil-like pellicle near the surface of the medium will be considered to be positive for bacterial growth, and will be used for reinoculation into others vials containing the same semi-solid N-free medium previously utilized. The cultures from the positive vials are subjected to further purification steps by streaking them onto specific agar plates of the same medium as was used in the semi-solid vials, but containing 20 mg l−1 of yeast extract, and incubated at 28°C for 2 d. After incubation, distinct colonies are grown in liquid LB medium (LB broth contains, per ml, 10 mg tryptone (a mixture of peptides formed by the digestion of casein with the pancreatic enzyme, trypsin), 5 mg yeast extract (an autolysate of yeast cells), and 5 or 10 mg NaCl) at 28°C under agitation (200 rpm). After that, Gram testing will be done and then each pure culture is suspended in 50% sterilized glycerol solution and stored at −18°C.

3.3 Molecular characterization

DNA will be extracted from bacteria using the following protocol: a 1.5 ml sample of a bacterial culture grown in LB is centrifuged for 5 min at 12,000 g. The bacterial pellet is rinsed with

700 μl of TES buffer (10 mM Tris pH 8.0, 25 mM EDTA, 150 mM NaCl), recentrifuged and resuspended in 500 μl of TE buffer (10 mM Tris pH 8.0, 25 mM EDTA) plus 25 μl of lysozyme

(20 mg ml−1) with incubation at 37°C for 30 min. A 108 μl volume of sodium dodecyl sulfate

(20% SDS) and a 5 μl volume of proteinase K (20 mg ml−1) are added. The samples are homogenized for 30 s in a vortex and incubated at 56°C for 15 min. A 600 μl volume of ammonium acetate (8 M pH 8.0) is added and the samples are kept on ice for 30 min and subsequently centrifuged for 20 min at 12,000 g for precipitation of cellular waste. The pellet is

washed with 70% ethanol and subsequently air dried then resuspended in 50 μl of TE buffer. The

DNA quality and integrity were checked by electrophoresis in 0.8% agarose gels in 1 X Tris- borate-EDTA buffer with ethidium bromide and visualized by UV light (Molecular Cloning: A

Laboratory Manual, 3rd ed).

The selected universal primers for the bacterial 16S rRNA 27F (5'-

AGAGTTTGATCCTGGCTCAG-3') and 1392R (5'-GGTTACCTTGTTACGACTT-3') and the universal primers used for fungal amplification were ITS1 (5′TCC GTA GGT GAA CCT

GCG G 3′), and ITS4 (5′TCC TCC GCT TAT TGA TAT GC 3’) will be initially used for characterizing the complete soil microbiome (Felske, Rheims, Wolterink, Stackebrandt, &

Akkermans, 1997).. The amplifications and next generation sequencing will be done by BGI. A dendrogram showing the genetic relationships of isolates (roots and rhizopheric bacteria were summed up) was constructed for each sampled site. Sequence analyses will be performed using the Cyverse platform. DNA sequences will be compared with sequences from EzTaxon Server version 2.1 and GenBank database using BLASTN software(Rocha et al., 2020).

3.3 Experiment details

Experiment 1: Exploring the growth effect to marama bean of marama bean soil microbiome

branch, the number of flowers and pods (not in the first year) of the longest branch, the average weight of pods (not in the first year) on this branch and the weight of tuber as evaluation indicators. The plant can also be knocked out of the pot and the tuber measured and photographed. The experimental measurement table is shown below. After overwintering the plants are again gown and the progression to flowering noted. These measurements are repeated annually to determine if the soil has a measurable effect on any of the parameters. Since the marama bean cannot produce any pods at the first year, for the first round experiment the measurements about pots will not be used as evaluation indicators.

Table. 1 Experiment 1 measurement form seed1.1, 1.2, 1.3 respectively represent three repeated experiments. The comparison index of the experiment is the growth of marama bean in the sterilized soil

the the length of the number average The Seed number the of flowers weight of pods weight yield (kg) of longest and pods of on the longest of ha-1 branches branch(cm) the longest branch(g) tuber(kg) branch

Seed 1.1- sterilized Soil 1

Seed 1.2- sterilized Soil 1

Seed 1.3- sterilized Soil 1

Seed 1.1- unsterilized Soil 1

Seed 1.2- unsterilized Soil 1

Seed 1.3- unsterilized Soil 1

For the samples of the other three locations, the same set of experiments will also be carried out and recorded.

Possible observation and further study:

By comparing the experimental results of the two groups, I hope to see that in the presence of marama bean soil microbiome, the indicators such as pod number, tuber size and yield are much higher than the other group. This shows that marama bean soil microbiome plays an important positive role in the growth of marama bean itself. Next, you can extract the microorganisms in the soil for separation and identification, explore their functions, and lay the foundation for the future development of microbial fertilizers. If there is no significant difference between the two sets of experiments, it means that the role of miciobiome in the soil is not critical. Then conduct experiments to explore the microorganisms attached to the seeds.

Experiment 2: Exploring the growth effect to soybean of soybean soil microbiome

The seeds 1 and a part of the soil 1 were sterilized. The sterilized seeds are planted in the soil 1 in pot after sterilization, and the other part is planted in the soil 1 without sterilization. Three replicates are carried out on the seeds in each soil in three different pots. The two groups of seeds are grown under the exact same experimental conditions, ensuring that the only difference is the microorganisms in the soil. After 90 days when soybeans are ripe, the two groups of plants are measured. Take the number of branches of each plant, the length of the longest branch, the number of flowers and pods of the longest branch, the average weight of pods on this branch as evaluation indicators. The plant can also be knocked out of the pot and photographed. The presence and abundance of nodules will be recorded. The experimental measurement table is shown below.

Table. 2 Experiment 2 measurement form seed1.1, 1.2, 1.3 respectively represent three repeated experiments. The comparison index of the experiment is the growth of soybean in the sterilized soil

the the length of the number average Seed yield number the longest of flowers weight of pods (kg) ha-1 of branch(cm) and pods of on the longest branches the longest branch(g) branch

Seed 1.1- sterilized Soil 1

Seed 1.2- sterilized Soil 1

Seed 1.3- sterilized Soil 1

Seed 1.1- unsterilized Soil 1

Seed 1.2- unsterilized Soil 1

Seed 1.3- unsterilized Soil 1

For the samples of the other three locations, the same set of experiments will also be carried out and recorded.

Possible observation and further study:

By comparing the experimental results of the two groups, I hope to see that in the presence of marama bean soil microbiome, the indicators such as pod number, tuber size, nodule number and yield are much higher than the other group. This shows that soybean soil microbiome plays an important positive role in the growth of soybean itself.

Experiment 3: Explore the effect of the microorganisms attached to the seeds of marama bean on the growth of Marama bean

A part of seeds 1 is sterilized, and the other part is not sterilized. Inoculate sterilized and non- sterilized seeds in the sterilized soil 1 respectively in pot. Three repeated experiments are carried out on the seeds in soil in three different pots. The two groups of seeds are grown under the exact same experimental conditions, ensuring that the only difference is the microorganisms attach seeds Ten weeks later at the end of the first growing season, the two groups of plants are measured. Take the number of branches of each plant, the length of the longest branch, the number of flowers and pods (not in the first year) of the longest branch, the average weight of pods (not in the first year) on this branch and the weight of tuber as evaluation indicators. The plant can also be knocked out of the pot and the tuber measured and photographed. The experimental measurement table is shown below. After overwintering the plants are again gown and the progression to flowering noted. These measurements are repeated annually to determine if the soil has a measurable effect on any of the parameters. Since the marama bean cannot produce any pods at the first year, for the first round experiment the measurements about pots will not be used as evaluation indicators.

Table. 3 Experiment 3 measurement form seed1.1, 1.2, 1.3 respectively represent three repeated experiments. The comparison index of the experiment is the growth of marama bean in the unsterilized soil

the the length of the number average Seed The number the longest of flowers weight of pods yield (kg) weight of of branch(cm) and pods of on the longest ha-1 tuber(kg) branches the longest branch(g) branch

Sterilized Seed 1.1-unsterilized Soil 1

Sterilized Seed 1.2-unsterilized Soil 1

Sterilized Seed 1.3-unsterilized Soil 1

Unsterilized Seed 1.1- unsterilized Soil 1

Unsterilized Seed 1.2- unsterilized Soil 1

Unsterilized Seed 1.3- unsterilized Soil 1

For the samples of the other three locations, the same set of experiments will also be carried out and recorded

Possible observation and further study:

By comparing the experimental results of the two groups, I hope to see that in the presence of marama bean seeds attached microbiome, the indicators such as pod number, tuber size and yield are much higher than the other group. This shows that seed attached microbiome plays an important positive role in the growth of marama bean itself. Next, you can extract the microorganisms attached to the seed for separation and identification, explore their functions, and lay the foundation for the future development of microbial fertilizers. If there is no significant difference between the two sets of experiments, it means that the role of miciobiome attached to the seeds is not critical. Then conduct experiments to explore the microorganisms in

the soil. If neither of them shows positive result, it means microbiome of marama bean is not an important factor for its growth.

Experiment 4: Investigate whether marama bean soil microbiome can play a positive role on soybean growth

The first group of sterilized soybean seeds are planted in the marama bean soil 1 after sterilization, the second group of sterilized soybean is planted in the marama bean soil 1 without sterilization and the last group of sterilized soybean is sown in the unsterilized soybean soil.

Three repeated experiments are carried out on the seeds in each soil. The three groups of seeds are grown under the exact same experimental conditions, ensuring that the only difference is the microorganisms in the soil. After 90 days, soybeans are ripe, the two groups of plants are measured. Take the number of branches of each plant, the length of the longest branch, the number of flowers and pods of the longest branch, the average weight of pods on this branch as evaluation indicators. The plant can also be knocked out of the pot, the root system photographed and characterized for nodules and size. The experimental measurement table is shown below.

Table. 4 Experiment 4 measurement form. seed1.1, 1.2, 1.3 respectively represent three repeated experiments.

the number of the length of the the number of average weight Seed yield branches longest flowers and of pods on the (kg) ha-1 branch(cm) pods of the longest longest branch branch(g)

Soybean Seed 1.1-sterilized Soil 1

Soybean Seed 1.2-sterilized Soil 1

Soybean Seed 1.3-sterilized Soil 1

Soybean Seed 1.1-unsterilized Soil 1

Soybean Seed 1.2-unsterilized Soil 1

Soybean Seed 1.3-unsterilized Soil 1

Soybean Seed 1.1-soybean Soil 1

Soybean Seed 1.2-soybean Soil 1

Soybean Seed 1.3-soybean Soil 1

For the samples of the other three locations, the same experiment is also carried out and recorded.

Possible observation and further study:

I expect soybeans planted in marama bean soil to achieve the best results, followed by soybeans in their own soil, with the lowest yield and other indicators grown in sterilized soil. If the experimental results are as above, it shows that marama bean soil microorganisms have great potential. Not only can it help itself to grow, but it also has a very strong positive effect on the

growth of other plants. It also shows that marama bean soil microbiome can be successfully developed as future products and attract investment. The next step is to plant different crops in marama bean soil, determine the impact of Marama bean soil on different crops, and gradually explore the formulation of future products. At the same time, you can also isolate, purify and identify the marama bean soil microorganisms as in experiment 1.

Experiment 5: Explore the effects of soybean and marama bean on soil microbial composition

The seeds of Marama bean and soybean are planted in sterile soil. Each seed was subjected to three parallel experiments. One year later, the microorganisms in the soil were isolated and identified. Compare differences in microbial composition. Find out the different microorganisms attracted by the two plants. Since the marama bean and soybean experimental samples grow in the exact same environment, in theory, there should be no difference in soil microbiome. But if different microorganisms are detected in different plant soils. It is very likely that these microorganisms have a unique effect on the growth of this plant. Therefore, it will be meaningful to continue further study about this/these microbes.

Experiment 6: Exploring marama bean soil microbiome’s effect of helping soybean tolerate drought.

Divide sterilized soybean seeds to three parts. First plant all the seeds to sterilized soil. Adding

Namibia soil from around well growing marama plant for inoculating marama bean microbiome to the first part. Adding soybean soil to the second part for inoculating soybean microbiome and adding nothing to the last part. The three groups of seeds are grown under the exact same

experimental conditions, ensuring that the only difference is the microorganisms in the soil.

After 50 days, when the canopy is fully developed, stop watering seeds. Every late morning observe plants and record data. Data were recorded for leaf wilting using a 1–5 scale (1=no wilting, 2=few top leaves showed wilting, 3=half of the leaves showed wilting, 4=severe wilting,

~75% of the leaves showed wilting, and 5=severely wilted). Record the time every group to to reach and stay at each level. After they reach the level 5, start to re-watering them and record how long time it can fully recover. 40 days later after the plants recover (if thety recover), check their yields. The experimental measurement table is shown below.

Table. 5 Experiment 6 measurement form. seed1.1, 1.2, 1.3 respectively represent three repeated experiments.

Days to Days to Days to Days to Days to Days to Seed yield reach reach reach reach reach recover (kg) ha-1 wilting wilting wilting wilting wilting after level 1 level 2 level 3 level 4 level 5 restart after stop after stop after stop after stop after stop watering watering watering watering watering watering

Soybean Seed 1.1-marama bean microbiome

Soybean Seed 1.2- marama bean microbiome

Soybean Seed 1.3- marama bean microbiome

Soybean Seed 1.1-no adding

Soybean Seed 1.2- no adding

Soybean Seed 1.3- no adding

Soybean Seed 1.1-soybean microbiome

Soybean Seed 1.2- soybean microbiome

Soybean Seed 1.3- soybean microbiome

For the samples of the other three locations, the same set of experiments will also be carried out and recorded.

Possible observation and future study:

I expect that the group inoculated with the marama bean microbiome will have the longest time to reach each wilt level, the shortest recovery time, and the highest yield. If the result is as I expected, it means that marama bean soil microbes have the effect of helping plants resist drought. The next plan is the same as above, isolate and identify microorganisms, explore functional functional microorganisms, and further develop these microorganisms, hoping to get a new microorganism product.

4. Conclusion

As one of a few plants capable of tolerating extreme drought and nutritional deficiencies, marama bean itself has huge development potential. At the same time, its microbiome also has great value. After a series of experiments, I’m looking forward to confirming the growth promotion effect of Marama bean microbiome on the growth of marama bean and prove that this microbiome also has a positive effect on the growth of other crops. After the separation and identification of microorganisms, I hope to find one or several new types of microorganisms, and understand their role in plant growth and development. If the above goals can be achieved, we can learn from the business model of companies in the microbiome field to create our own company. The research on the effect of marama bean microbiome on soybean drought resistance research is a fast and crucial research. It will take about half a year to know whether the microbiome of marama bean has anti-drought effect. If it proves successful, the microbiome can be directly made into a microbial product to help legumes resist drought. If one or several new anti-drought microorganisms can be identified, a patent can be applied for. At the same time as applying for a patent, we can start writing an SBIR (Small Business Innovation Research) and plan for future commercialization. Then explore the functional principles of the discovered microorganisms, as a basis for the production of more products. The product is not limited to agriculture. It is also possible to grant patents for this microorganism to other companies to help them improve their databases.

The rest of the experiments on the growth of marama beans, due to the long growth cycle of marama beans, and the need to be measured every year, can continue to carry out scientific

research after the establishment of the company to provide more vitality for the company in the future.

5. References

Agboma, P. C., Sinclair, T. R., Jokinen, K., Peltonen-Sainio, P., & Pehu, E. (1997). An evaluation of the effect of exogenous glycinebetaine on the growth and yield of soybean: timing of application, watering regimes and cultivars. Field Crops Research, 54(1), 51–64. Booth, M. G., & Hoeksema, J. D. (2010). Mycorrhizal networks counteract competitive effects of canopy trees on seedling survival. Ecology, 91(8), 2294–2302. Buée, M., De Boer, W., Martin, F., Van Overbeek, L., & Jurkevitch, E. (2009). The rhizosphere zoo: an overview of plant-associated communities of microorganisms, including phages, bacteria, archaea, and fungi, and of some of their structuring factors. Plant and Soil, 321(1– 2), 189–212. Cahill, J. F., Cale, J. A., Karst, J., Bao, T., Pec, G. J., & Erbilgin, N. (2017). No silver bullet: different soil handling techniques are useful for different research questions, exhibit differential type I and II error rates, and are sensitive to sampling intensity. The New Phytologist, 216(1), 11–14. https://doi.org/10.1111/nph.14141 Carter, C. A., Zhong, F., & Zhu, J. (2012). Advances in Chinese agriculture and its global implications. Applied Economic Perspectives and Policy, 34(1), 1–36. Cipollini, D., Rigsby, C. M., & Barto, E. K. (2012). Microbes as targets and mediators of allelopathy in plants. Journal of Chemical Ecology, 38(6), 714–727. Cullis, C., Chimwamurombe, P., Barker, N., Kunert, K., & Vorster, J. (2018). Orphan legumes growing in dry environments: marama bean as a case study. Frontiers in Plant Science. https://doi.org/10.3389/fpls.2018.01199 Cullis, C., Lawlor, D. W., Chimwamurombe, P., Bbebe, N., Kunert, K., & Vorster, J. (2019). Development of marama bean, an orphan legume, as a crop. Food and Energy Security, 8(3), e00164. https://doi.org/10.1002/fes3.164 Dinesh, R., Anandaraj, M., Kumar, A., Bini, Y. K., Subila, K. P., & Aravind, R. (2015). Isolation, characterization, and evaluation of multi-trait plant growth promoting rhizobacteria for their growth promoting and disease suppressing effects on ginger. Microbiological Research, 173, 34–43. https://doi.org/https://doi.org/10.1016/j.micres.2015.01.014 Döbereiner, J. (1989). Isolation and identification of root associated diazotrophs. In Nitrogen fixation with non-legumes (pp. 103–108). Springer. Duarah, I., Deka, M., Saikia, N., & Boruah, H. P. D. (2011). Phosphate solubilizers enhance NPK fertilizer use efficiency in rice and legume cultivation. 3 Biotech, 1(4), 227–238. Egamberdiyeva, D. (2007). The effect of plant growth promoting bacteria on growth and nutrient uptake of maize in two different soils. Applied Soil Ecology, 36(2), 184–189. https://doi.org/https://doi.org/10.1016/j.apsoil.2007.02.005 Fan, X.-D., Wang, J.-Q., Yang, N., Dong, Y.-Y., Liu, L., Wang, F.-W., … Li, H.-Y. (2013). Gene expression profiling of soybean leaves and roots under salt, saline–alkali and drought stress by high-throughput Illumina sequencing. Gene, 512(2), 392–402. https://doi.org/https://doi.org/10.1016/j.gene.2012.09.100 Felske, A., Rheims, H., Wolterink, A., Stackebrandt, E., & Akkermans, A. D. L. (1997). Ribosome analysis reveals prominent activity of an uncultured member of the class Actinobacteria in grassland soils. Microbiology, 143(9), 2983–2989. Hong, S. C., MacGuidwin, A., & Gratton, C. (2011). Soybean Aphid and Soybean Cyst

Nematode Interactions in the Field and Effects on Soybean Yiel. Journal of Economic Entomology, 104(5), 1568–1574. https://doi.org/10.1603/EC11084 Janos, D. P., Scott, J., Aristizabal, C., & Bowman, D. M. J. S. (2013). Arbuscular-mycorrhizal networks inhibit Eucalyptus tetrodonta seedlings in rain forest soil microcosms. PLoS One, 8(2). Johnson, D. J., Clay, K., & Phillips, R. P. (2018). Mycorrhizal associations and the spatial structure of an old-growth forest community. Oecologia, 186(1), 195–204. Kadowaki, K., Yamamoto, S., Sato, H., Tanabe, A. S., Hidaka, A., & Toju, H. (2018). Mycorrhizal fungi mediate the direction and strength of plant–soil feedbacks differently between arbuscular mycorrhizal and ectomycorrhizal communities. Communications Biology, 1(1), 1–11. Katz, M., Hover, B. M., & Brady, S. F. (2016). Culture-independent discovery of natural products from soil metagenomes. Journal of Industrial Microbiology & Biotechnology, 43(2), 129–141. https://doi.org/10.1007/s10295-015-1706-6 King, A. (2018). Plants’ bacterial zoos. Chemistry World. Lance, A. C., Burke, D. J., Hausman, C. E., & Burns, J. H. (2019). Microbial inoculation influences arbuscular mycorrhizal fungi community structure and nutrient dynamics in temperate tree restoration. Restoration Ecology, 27(5), 1084–1093. https://doi.org/10.1111/rec.12962 Luo, X. X., & Xing, Z. Q. (2010). Comparative study on characteristics and influencing factors of soil respiration of reed wetlands in Yellow River Estuary and Liaohe River Estuary[J]. Procedia Environmental Sciences, 2, 888–895. Mózner, Z., Tabi, A., & Csutora, M. (2012). Modifying the yield factor based on more efficient use of fertilizer—The environmental impacts of intensive and extensive agricultural practices. Ecological Indicators, 16, 58–66. Noor, S., Yaseen, M., Naveed, M., & Ahmad, R. (2017). Effectiveness of diammonium phosphate impregnated with Pseudomonas putida for improving maize growth and phosphorus use efficiency. Plant Sci, 27, 1–8. Pankievicz, V. C. S., do Amaral, F. P., Santos, K. F. D. N., Agtuca, B., Xu, Y., Schueller, M. J., … Ferrieri, R. A. (2015). Robust biological nitrogen fixation in a model grass–bacterial association. The Plant Journal, 81(6), 907–919. https://doi.org/10.1111/tpj.12777 Peacher, M. D., & Meiners, S. J. (2020). Inoculum handling alters the strength and direction of plant–microbe interactions. Ecology, 101(4), e02994. https://doi.org/10.1002/ecy.2994 Raymond, J., Siefert, J. L., Staples, C. R., & Blankenship, R. E. (2004). The natural history of nitrogen fixation. Molecular Biology and Evolution, 21(3), 541–554. Reinhart, K. O., & Rinella, M. J. (2016). A common soil handling technique can generate incorrect estimates of soil biota effects on plants. New Phytologist, 210(3), 786–789. https://doi.org/10.1111/nph.13822 Rinella, M. J., & Reinhart, K. O. (2017). Mixing soil samples across experimental units ignores uncertainty and generates incorrect estimates of soil biota effects on plants. New Phytologist, 216(1), 15–17. https://doi.org/10.1111/nph.14432 Rocha, S. M. B., Mendes, L. W., de Souza Oliveira, L. M., Melo, V. M. M., Antunes, J. E. L., Araujo, F. F., … Araujo, A. S. F. (2020). Nodule microbiome from cowpea and lima bean grown in composted tannery sludge-treated soil. Applied Soil Ecology, 151, 103542. Sahoo, R. K., Ansari, M. W., Dangar, T. K., Mohanty, S., & Tuteja, N. (2014). Phenotypic and molecular characterisation of efficient nitrogen-fixing Azotobacter strains from rice fields

for crop improvement. Protoplasma, 251(3), 511–523. Sessitsch, A., Brader, G., Pfaffenbichler, N., Gusenbauer, D., & Mitter, B. (2018). The contribution of plant microbiota to economy growth. Microbial Biotechnology, 11(5), 801– 805. https://doi.org/10.1111/1751-7915.13290 Sukweenadhi, J., Kim, Y.-J., Choi, E.-S., Koh, S.-C., Lee, S.-W., Kim, Y.-J., & Yang, D. C. (2015). Paenibacillus yonginensis DCY84T induces changes in Arabidopsis thaliana gene expression against aluminum, drought, and salt stress. Microbiological Research, 172, 7– 15. Tedersoo, L., Bahram, M., & Zobel, M. (2020). How mycorrhizal associations drive plant population and community biology. Science, 367(6480). Wang, Y., Zhu, Y., Zhang, S., & Wang, Y. (2018). What could promote farmers to replace chemical fertilizers with organic fertilizers? Journal of Cleaner Production, 199, 882–890. Yang, X., Shao, X., Mao, X., Li, M., Zhao, T., Wang, F., … Guang, J. (2019). Influences of Drought and Microbial Water-Retention Fertilizer on Leaf Area Index and Photosynthetic Characteristics of Flue-Cured Tobacco. Irrigation and Drainage, 68(4), 729–739. Yin, C., Fan, F. L., Song, A. L., Cui, P. Y., Li, T. Q., & Liang, Y. C. (2015). Denitrification potential under different fertilization regimes is closely coupled with changes in the denitrifying community in a black soil[J]. Applied Microbiology and Biotechnology, 99(13), 5719–5729.