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Marama Bean Microbiome Function Exploration and Future Development Design

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Marama Bean Microbiome Function Exploration and Future Development Design MARAMA BEAN MICROBIOME FUNCTION EXPLORATION AND FUTURE DEVELOPMENT DESIGN by 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 by YIWEN DENG 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 and low-water conditions while maintaining a high nutritive content comparable to peanuts and soybeans (Cullis, Chimwamurombe, Barker, Kunert, & Vorster, 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
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