"The Effects of B. Cereus, Rhizobacteria, Mycorrhizal Fungi, and Oyster Mushrooms on Plant Health and Growth" by Sara Hemaid

The Effects of B. Cereus, Rhizobacteria, Mycorrhizal Fungi, and Oyster Mushrooms on Plant Health and Growth

Sara Hemaid, Farleigh Dickinson University

Abstract: Although fertilizers are the most widely used and effective plant supplements, utilizing microbes to enhance plant health and growth is an environmentally sustainable alternative. The overall objective of this experimental study was to test whether different microbes, including oyster mushrooms, mycorrhizal fungi, Bacillus cereus bacteria, and rhizobacteria, promote plant growth. This experimental study tested the effects of the microbes on Wisconsin Fast Plants. It was conducted in four trials with a sample size of 15 plants for each of the four microbes plus a control group. Each trial lasted two weeks, during which the plants were watered, their height was measured, and their health was recorded on a scale of 0 to 3. Three indicates the highest level of health, with green and leaf growth on the plants, while zero indicates an unhealthy plant and no growth. The data was recorded for each plant after the four trials were completed. The results from each trial showed there were positive effects of the treatment on plant growth and health. When the data was analyzed, the control group exhibited the lowest average height and health of the plants. Overall, the plants that were treated with rhizobacteria had the highest average height, and those that were treated with mycorrhizal fungi had the highest average health compared to the other treatments. In conclusion, the numerical data collected were statistically significant, and there was supporting evidence that with the addition of microbes, there was increased growth and health of the Wisconsin Fast Plants. 

Agriculture associations have often relied on the use of chemical fertilizers to help maintain the health and growth of various plants and crops. The use of microbes such as bacteria and fungi is a healthier alternative and plays a role in the crossroads of plant health and the environment. These microbes are important for their antimicrobial effects, promoting growth, and forming symbiotic relationships with the plants. In addition to the plant-enhancing qualities the microbes offer, they also contribute to the soil quality, enhancing its fertility and ecological processes. Ultimately, the use of microbes will help improve the future of sustainable agriculture while simultaneously maintaining the environment. 

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Literature Review

This experiment was conducted to observe the different effects of bacteria and fungi on plant growth. The experiment involved manipulating B. cereus bacteria, rhizobacteria, oyster mushrooms, and mycorrhizal fungi to observe their effects on plant health and growth. The effects of the treatments were demonstrated by monitoring the plant's height, leaf size, and color by comparing them to the untreated control group. Agriculture associations in America rely on chemical fertilizers and rarely use natural resources in their crops. Research suggests that using bacteria to ensure plant health is more effective than chemical fertilizers and produces less damage to the environment. B. Cereus is a type of actinobacteria that is beneficial and more efficient than commercial chemical fertilizers and pesticides. These types of bacteria are prolific antibiotic producers, which are desirable in the agricultural setting because they can aid in suppressing disease and diverse pathogenic microbes in the soil and rhizosphere (Yang et al., 2023). Renuka et al. (2023) conducted a study in India using native actinobacteria to cure a fruit root pathogen affecting their chili population. Renuka et al. (2023)  concluded that the native actinobacteria were an eco-friendly alternative to synthetic fungicides at pre- and post-harvest pathosystems, effective at suppressing plant diseases, promoting plant growth, increasing crop yield, and enhancing soil fertility (Renuka et al., 2023). The use of B. cereus and other actinobacteria offers an environmentally sustainable approach to agriculture by effectively enhancing soil health and crop productivity. 

In addition to the B. Cereus bacteria, plant growth-promoting rhizobacteria contribute to plant health in many ways. This includes support during climatic conditions and severe stresses on crop productivity. Rhizobacteria also influence the soil root, safeguarding the root from soil-borne diseases and improving the resilience of plants to climatic variability (Bhat et al., 2023). The rhizosphere is a hotspot of microbial interactions, as exudates released by plant roots serve as the primary food source for microorganisms (Raaijmakers et al., 2009). Ultimately, the plant-associated and soil microorganisms can act as biofertilizers and increase the availability of nutrients in the rhizosphere.

While B. cereus and rhizobacteria play a significant role in improving plant health and growth, fungi such as mycorrhizal and oyster mushrooms also offer numerous advantages. Oyster mushrooms in the rhizosphere carry out vital ecosystem roles, particularly in carbon cycling and as symbiotic partners with various other organisms (Hamed et al., 2021). Oyster mushrooms contain many important minerals, but they also absorb many minerals. These minerals can include Calcium, Copper, Iron, Potassium, Magnesium, Manganese, Phosphorus, and Zinc (Boadu et al., 2023). Furthermore, mycorrhizal fungi form a symbiotic relationship with plant roots to create a mutually beneficial association known as mycorrhiza. This relationship is crucial in promoting plant growth and enhancing overall plant health. Through improved nutrient and water absorption, enhanced stress resistance, and contributions to soil structure, these fungi contribute significantly to the overall health and vitality of plants (van der Heijden & Scheublin, 2007). Ultimately, each microbe plays a unique role in enhancing plant health and can serve as an environmentally sustainable alternative to the chemical fertilizers widely used in agriculture.

 Methods & Materials

The experiment used three packs of Wisconsin Fast Plant seeds and three packs of oyster mushroom kits, each of which can produce mushrooms in 10 days. 150 soil pots were utilized to account for the sample size. For identification of the treatment applied to the plants, one roll of labeling tape was used. The five soil beds were used to group the plant pots, so one treatment was applied to the plants in the designated soil bed. Biological agents, including mycorrhizal fungi, rhizobacteria, and biofilms from B.cereus and Bacillus subtilis, were provided in amounts adequate to treat 90 plants each, supporting plant growth and health. Lastly, rulers helped with recording observations and measuring plant growth. 

Procedure

There were four trials with five soil beds and 15 soil pots in each bed, with three seeds in each pot. Four of the five soil beds were designated for the specific bacteria or fungi treatment. The following were placed into their own soil bed: B. cereus, mycorrhizal fungi, rhizobacteria (Pseudomonas sp.), and oyster mushrooms. B. cereus and rhizobacteria were grown in nutrient broth in a water bath for one day, and 10 microliters of the solution were then incubated on a plate to determine the colony-forming unit factor. Bacterial solutions were diluted, and the selected concentration of 1:100 was used. This concentration was prepped with nutrient broth and stored overnight in a shaking water bath. Then, 1 mL was added to each pot in the designated soil bed. Lastly, 1 gram of the mycorrhizal pellets and mushroom pieces was incorporated into pots of the designated soil bed, while the fifth soil bed served as the control and remained untreated, receiving only water and light throughout the 4 trials. The soil beds were constantly exposed to light and watered with 30-40 mL two times a week, and were measured in centimeters twice every week. 

Once each trial was completed, new soil was added to the mycorrhizal and oyster mushroom experiment groups, while the soil for the rhizobacteria and B. Cereus was reused by boiling the used soil in a beaker with water in the microwave for 3 minutes, or until the soil was dry. This was done to kill the bacteria so a new sample of bacteria can be added to the soil. For the data collection, the wilting condition and color of the plant were considered, in addition to the quantitative data. The null and alternative hypotheses were formulated to guide the experiment. The null hypothesis states that there will be no difference in the growth and health of the plants due to the additives. On the other hand, the alternative hypothesis states that the additives of bacteria and fungi increase growth and health. The two A-Nova T-tests were used to analyze the data between the control and experimental groups for the height and health of the plants. These tests helped determine whether the alternative hypothesis was supported or rejected.

Results

The average health of the plants across four trials for each additive or microbe was determined on a scale from 0 to 3, as previously mentioned, and was assigned based on the plants’ physical characteristics. This included color, height, whether or not they wilted, and the number of leaves on the plant. Figure 3 illustrates how these metrics correspond to the scoring system. The treatment applied to the plant was abbreviated as shown in the tables and graphs. The abbreviations for the treatments are as follows: control (CNL), B. cereus (BC), rhizobacteria (RZ), mycorrhizal (MY), and oyster mushrooms (OM). The results for plant health across the four trials are illustrated in Table 2. The graph in Figure 2 shows that the plants containing mycorrhizal fungi were the healthiest overall, with an average of 2.7875, closely followed by the plants grown with rhizobacteria, which had an average of 2.5919. The plants grown under the influence of the B. Cereus bacteria had the least healthy plants overall, with an average of 2.1372. Additionally, Figure 1 displays a graph of the average height of the plants. The plants grown with rhizobacteria clearly had the most significant average height of 7.4764 cm compared to the height of the control plants, along with the ones grown under the influence of the other additives. The control had the lowest average height of 4.1323 cm.

An ANOVA single-factor test was performed to support the data statistically through an extension software on Google Sheets. An ANOVA test determines differences between research results from three or more unrelated samples or groups. This experiment compares multiple bacteria and fungi, such as rhizobacteria, B. cereus bacteria, mycorrhizal fungi, and oyster mushrooms. From the test, it was concluded that the results are statistically significant because the p-value for health was <0.01, as shown in Table 4, and the p-value for height was 0, as shown in Table 3. Since these values are less than the alpha value of 0.05, the experiment supports the alternative hypothesis that the additives of bacteria and fungi increase growth and health. The overall results indicated that the bacteria, specifically the rhizobacteria, had the greatest influence on the health and height of the plants. 

Conclusion

In conclusion, the data were statistically significant, and there is supporting evidence that with the addition of microbes, B. cereus, mycorrhizal fungi, rhizobacteria (Pseudomonas sp.), and oyster mushrooms, there was increased growth and health of the Wisconsin Fast Plants. The data for both height and health showed a p-value less than 0.05, supporting the relationship between the treatments and their effects. Although the data were statistically significant, some experimental errors occurred. The variables supposed to remain constant during the experiment were not controlled appropriately. For example, in the first trial, the amount of water given to the plants, the number of seeds planted in each pot, and the time the plants were supposed to grow were not constant. The plants were not all grown at the same time, as some started growing before others, so the time the data was recorded was not the same. However, the plants were set to grow for two weeks straight with four check-ins for each trial, regardless of which plant it was and when it started to grow. 

In the future, the experiment should have more constant variables to ensure no confounding variables skew the data. For instance, regulating how many seeds are in each pot and how much water is given. Furthermore, to expand on this hypothesis, testing these microbes on more complex organisms would help determine if these natural fertilizers can be used in agriculture. Also, since most of the microbes have disease-fighting tendencies, it would be interesting to test how the bacteria and fungi resist different types of antagonistic bacteria. 

References

Bhat, M. A., Mishra, A. K., Jan, S., Bhat, M. A., Kamal, M. A., Rahman, S., Shah, A. A., & Jan, A. T. (2023). Plant Growth Promoting Rhizobacteria in Plant Health: A Perspective Study of the Underground Interaction. Plants, 12(3). http://dx.doi.org/10.3390/plants12030629

Boadu, K. B., Nsiah-Asante, R., Antwi, R. T., Obirikorang, K. A., Anokye, R., & Ansong, M. (2023). Influence of the chemical content of sawdust on the levels of important macronutrients and ash composition in Pearl Oyster Mushroom (pleurotus ostreatus). PLOS ONE. https://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0287532

Hamed, H. A., Mohamed, M. F., Hosseny, M. H., & El-Shaikh, K. A. A. (2021, March 1). IOPscience. IOP Conference Series: Earth and Environmental Science. https://iopscience.iop.org/article/10.1088/1755-1315/690/1/012028 

Raaijmakers, J. M., Paulitz, T. C., Steinberg, C., Alabouvette, C., & Moënne-Loccoz, Y. (2009). The rhizosphere: A playground and battlefield for soilborne pathogens and beneficial microorganisms. Plant & Soil, 321(1/2), 341-361. https://doi.org/10.1007/s11104-008-9568-6

Renuka, R., Prabakar, K., Anandham, R., Pugalendhi, L., Rajendran, L., Raguchander, T., & Karthikeyan, G. (2023). Exploring the potentiality of native actinobacteria to combat the chilli fruit rot pathogens under post-harvest pathosystem. Life, 13(2), 426. https://doi.org/10.3390/life13020426

Van Der Heijden, M. G., & Scheublin, T. R. (2007). Functional traits in mycorrhizal ecology: Their use for predicting the impact of arbuscular mycorrhizal fungal communities on plant growth and ecosystem functioning. New Phytologist, 174(2), 244-250. https://doi.org/10.1111/j.1469-8137.2007.02041.x

Yang, B., Zheng, M., Dong, W., Xu, P., Zheng, Y., Yang, W., Luo, Y., Guo, J., Niu, D., Yu, Y., & Jiang, C. (2023). Plant disease resistance-related pathways recruit beneficial bacteria by remodeling root exudates upon Bacillus cereus AR156 treatment. Microbiology Spectrum, 11(2), e0361122. Advance online publication. https://doi.org/10.1128/spectrum.03611-22