The Study of the Genomic Diversity Regarding pH, Soil Moisture, and Biodiversity between soil samples from root (carrot) and vine (squash) plants.

Alyssa Remenius, Runxin Wang, and Wybie Loper

Purdue University

Rationale

The goal of this study is to research whether the soil of root plants vs vine plants has different qualities. It is important to know these things to be able to understand what growing conditions are best for each species of plant. It has been found that different soil pH has a significant impact on the nutrients available in the soil, as well as how the nutrients are absorbed by the plant (Ferrarezi et al.). It has also been demonstrated that at a lower, more acidic pH certain elements of soil can become toxic to plants (Ferrarezi et. al.). Another factor that needs to be taken into consideration is soil moisture. If moisture concentration is too high there is a lack of oxygen supply to the plants, causing them to be unable to absorb nutrients and moisture. Too little moisture can cause the leaves of the plant to shrivel and seed production to lower (Hsia and Jackson). Carrots have been shown to prefer a neutral to slightly acidic pH, as well as draining soil with a medium moisture content (Rohwer). Squash tends to prefer a slightly acidic soil pH, as well as a well-draining soil (Ezzo et al.). These plants prefer similar conditions; however, the squash was struggling to grow while the carrots were looking healthy and growing well. These experiments can be beneficial to urban gardening and regular gardening, as they will provide insight into how different conditions of soil affect carrots and squash. Urban gardening communities can use this information to monitor or change the growing conditions of their soil to best fit the conditions of each plant.

Eerie Street Garden West Lafayette, In 47906 September 10, 2023

The carrots are on the left side of the garden and the squash are on the right side of the garden. Samples collection. To collect the samples we used a soil core.

Study Aim

The aim of the study is to compare the levels of biodiveristy, soil mositure, and pH levels in soil. We will be comparing soil samples from root plants (carrots) and vine plants (squash). We are concentrating on if the carrots being physically surrounded by the soil creates a difference in these factors, allowing them to grow healthier than the suqash, as the squash are currently dying. To collect data, we will be performing a series of tests including soil concentration, richness, eveness, shannon diversity index, culturing bacteria, and pH levels will be examined. This study can enrich the garden and lives of those living in the Lafayette community. Our findings from the research conducted will be relayed to the volunteers of the Eerie Street Garden. Based off our data the volunteers will be able to make large or small changes to their gardening methods. These changes will allow the community members to have access to fresh produce. Not only will be be helping curate a healthier garden, but we will also be fulfilling our community.

Squash Plant

These images depict the squash struggling to grow. This is also where the squash soil samples were obtained.

Wybie takes their squash soil sample.

Runxin takes her squash soil sample.

Alyssa takes her squash soil sample.

As a group we hold our squash soil samples! The soil is very thick.

Carrot Plant

These images depict the healthiness of the carrots, and their ability to thrive. This is where the soil samples were taken.

Alyssa takes her carrot soil sample.

Wybie takes their carrot soil sample.

Runxin takes her carrot soil sample.

As a group we hold our carrot soil samples. Soil is drier and crumbly.

Experimental Plan

It has been shown that diversity is a function of nutrients (Cowling et al. 1996). Based on the observations, we found that the plant states of carrots and squash are completely different. Carrots are growing well and squash are struggling to survive. Therefore, the study was conducted to figure out how the different soil properties of the squash and carrots cause a different biodiversity that provides different nutrients. The properties of soil can be measured by pH values and water concentration. In these tests, the independent variables are the pH values and water concentration of the squash and carrot soil. Based on the cultural dishes, we can clearly observe how the bacteria cluster together, the size of the colonies, and their positioning on the dish. The dependent variable in this scenario is the biodiversity. Bacteria interactions and the size of the soil can be different based on different spatial scales of soil (Vos et al. 2013). This can be measured by the concentration of bacteria in the study. Culturing bacteria tests will be used to measure the concentration of the bacteria in the soil. Evenness and richness are two measurable indexes that give us hints about the biodiversity of the soil samples. According to Salem (2017), a low evenness is associated with the community structure which is dominated by one or more species. Richness is the basic descriptor of different species in a community. Therefore, evenness and richness can be used when concluding the biodiversity of the soil samples. The Shannon diversity index will be calculated, which gives the number of varying substrates utilized by the microbial communities, this is a way to measure functional diversity (Habig and Swanepoel, 2015). Shannon diversity index depends on evenness and richness, representing the probability that two species will randomly be chosen, this index increases as diversity decreases (Kim et al., 2017). The 16S rRNA gene sequence analysis will be used to identify the poorly described or uncultured bacteria (Clarridge, 2004). Finally, the absorbance value from the EcoPlate test, Shannon, and evenness indexes with pH values and water concentration of the squash and carrot soil samples will be compared. The relations between pH values, water concentration, and biodiversity will be explored. The results of this experiment will indicate the properties of the soils, telling us what needs to change to aid the plants in their growth.

Carrot Petri Dish

Squash Petri Dish

Figure 1: pH

Figure 1: pH Concentration of Squash VS Carrots. In the graph the average mean pH value of squash a vine plant and carrots a root plant. These results were obtained by adding water to the soil samples and then the samples were vortexed to create a solution. The samples were then incubated, and a pH meter was used to find the pH values of each sample. The graph depicts the average mean of 8 soil samples pH values along with their pH values. There was also a T-test performed to determine if there was a significant difference between the squash and carrot soil samples. The p-value found was 0.031. This means there was a significant difference between the two samples because the p-value is less than 0.05.

The method used was adding water to the samples. After water was added the solution was vortexed and then a pH meter was inserted into the mixture and the pH was found.

Per the evidence the average pH value of squash is 7.726 with a standard deviation of 0.121. The average pH value of carrots is 7.848 with a standard deviation of 0.0718. These values do have a significant difference with a p-value of 0.031. This tells us that the pH of the squash and carrots could be affecting their health. The pH values have a ten percent difference between, with the pH of the carrots having a higher average. This means the carrots are more basic than the squash. Neither the squash nor carrots are in their optimum pH range. Carrots tend to thrive in a more acidic soil and squash in a more alkaline soil. This means that lime should be added to the carrot soil to lower the pH levels. Sulfur should be added to raise the pH of the squash.

The carrot and the squash do have unique pH values. This is supported by the statistically significant difference between their pH values per the results of the T-test. I am certain of this analysis because the p-value that resulted was 0.031, confirming that there is a statistical difference between the pH of the carrot and squash soil samples. I am confident in this analysis because the data demonstrated a p-value lower than 0.05. The squash being in an acidic soil is contributing to their struggle to thrive. The carrots are also in an incorrect pH range; however, they are a root plant. This leads me to believe that the surrounding plants and soil are providing carrots with the nutrients they need to grow and prosper. Since the carrots are emersed in the soil they are protected from outside factors. Factors such as air pollutants in the air could be directly affecting the squash and raising their pH values.

In conclusion, the pH of the squash is contributing to the lack of survival of the squash. Squash is in a category of cucurbits. Thus, the pH of squash should be more alkaline (Lerner and Dana, 1). The pH of the squash is too acidic. The squash needs lime added to the soil to the soil to raise the pH. On the other hand, carrots thrive in an acidic environment (Schuh and Mackenzie, 1). The carrots are surrounded by the soil and can be pulling other nutrients allowing them to prosper. There is a statistical difference between the pH of the squash and carrots with a p-value of 0.031 which is less than 0.05. This leads to the conclusion that pH is playing a role in the survival of the squash and carrots.

p-value: <0.05

Figure 2: Moisture Concentration

To find these results, we weighed our soil samples, dried them, and weighed them after to find the percent difference based on the weights. The graph demonstrates the average percent moisture content for carrots and squash, along with error bars for their standard deviations. We also ran an unpaired t-test assuming unequal variance and generated a p-value of 0.0159, indicating a significant statistical difference across results.

The method used to gather this data included weighing our soil samples, dehydrating them, and weighting them to calculate the percentage of weight lost in water.

The average percent moisture content of carrots and squash are significantly different based on the averages, as well as the T-Test performed on the data that was collected. The average moisture content of the carrot samples collected was 21.78, while the average moisture content of squash was about 20.7% higher, at 27.43. The generated p-value from our T-Test was 0.016, which is <0.05, indicating a significant statistical difference between the moisture content of the two conditions.

Based on the collected evidence, it can be concluded that the soil that the squash was being grown in had a significantly higher moisture content than the soil that the carrots were being grown in. Based on the values and data obtained, we can say with confidence that this conclusion is accurate. Across all of the samples, squash had a higher moisture content than carrots.

These results can be caused by a multitude of things; however, it could explain certain things that were observed about the actual plants that were growing from the soil samples that we obtained. The carrots were thriving, while the squash was struggling. This could have been caused by the squash soil’s higher moisture content.

Moisture content of soil can be affected by many different things, including soil temperature, organic matter present, and soil texture. (Rasheed et. al.) Compaction of soil can also affect permeability, which can change the moisture content as well (Kuzucu et al.). Compaction and soil texture is something that can be used to explain the higher moisture content observed in the squash plants. We observed that the squash soil was much more dense than the carrot soil. The compaction of the soil can explain why the squash soil did not drain as quickly as the carrot soil, making it have a higher moisture content.

Figure 3.1 bar graph of Shannon(H), Richness(S) and Evenness (E) averages in two conditions

Data Analysis:

Based on the data above, carrot (condition 2) has an average of 29.125 and squash (condition 1) has 30, which shows that squash has 2.92% more average richness values than carrot. Also, carrot ‘s average values are more separated than squash. Although squash has more richness index than carrot, based on their P-value, which is 0.684, there is no big statistical difference between them. For Shannon diversity index, based on p value, these two conditions’ Shannon diversity index indicates a huge difference, which can be found in 0.0232 P value (less than 0.05). In comparison, squash has 1.36 % more Shannon diversity index than carrot ‘s with a larger fluctuation around the average. However, both carrot and squash soil have large evenness although the evenness value in squash is 0.50% higher than carrots. The 0.0783(more than 0.05) P value indicates there is almost no huge difference between the two conditions’ evenness values.

Conclusion:

In conclusion, we can confidently conclude that squash has more Shannon diversity index than carrot based on P value 0.0232 less than 0.05, which means there is a significant difference between carrot and squash’s H. Also, there is a high certainty that H between two conditions is significantly different with P value 0.0232 which is less than 0.05. Shannon diversity index indicates the variety and abundance of different land cover types (Shannon, 1948; Turner et al., 2001; McGarigal et al., 2012). Therefore, the larger Shannon diversity index, the more diverse and abundant the soil is. Based on the data we have; we can speculate that squash soil has more diverse and abundant. However, we cannot confidently conclude that there is any difference of richness and evenness between these two conditions. The P values of S and E are 0.0684 and 0.0783, which both are more than 0.05. Also, we cannot be certain about the comparison between carrot and squash based on p values (0.0684 and 0.0783) more than 0.05, even though squash has more richness and evenness values than carrot.

Figure 3: Shannon, richness, and evennness of the ecoplate

Figure Legend 1:

The Figure 3.1 represent the average values of Shannon diversity index, Richness and Evenness of two conditions (squash and carrot) in eight samples total. The richness is calculated by summing the positive number responses which are the wells that met the change in absorbance threshold 0.25 with the analysis of Eco plate. The Shannon diversity index can be obtained by −1 X Σ [pi × ln(pi)], pi is the ratio of color development in one well to well color developments in all the wells ln is the natural log. Dividing Shannon diversity index by ln (Richness), we can get the values of evenness. Then we calculated the average values of H, S and E in these eight samples by considering the ratio of sum number given number to the sum of total values. The bar graph can be presented based on average values of H, S and E and standard deviation to indicate how spread the data are around mean values. The P value of H, S and E also be considered to show how different two conditions based on one specific value. If P values is less than 0.05, that means there is a significant different between two conditions.

Method:

Richenss is calculated by the sum of positive number of the well from Eco plate met the absorbance 0.25. The Shannon diversity index equals to −1 X Σ [pi × ln(pi)]. Evennness is E = H / ln(S). The average of H, S and E is the ratio of sum number given number to the sum of total sample numbers. If P value is less than 0.05, there is a significant different between two conditons.

Figure 3.2, group data of relative utilization efficiency graphs

Figure Legend 2:

Figure 3.2 indicates the Relative Utilization Sum which can be get by summing of all the carbon sources of our group that meet the threshold of 0.25. The Carbon Relative Utilization Efficiency which is the ratio of growth over carbon uptake (Manzoni et al., 2012) also be shown on the figure.

Method:

Relative Utilization Sum is the sume of all our group carbon sources that meet the threshold 0.25. The Carbon Relative Utilization Efficientcy is the ratio of grwoth over carbon uptake.

Data Analysis:

Based on our group relative utilization sum, the utilization of carbon in squash is one larger than carrot in overall. However, carrot has slightly more relative utilization efficiency in carboxylic 27.6%, amino acids 20.7%, polymers 13.8% and Amine 6.90%, which means bacteria in carrot soil is more efficient in utilization these carbon sources than squash. Squash bacteria is more expert in consuming carbohydrates with 33.3% efficiency than 31.0% in carrot. However, the total efficiency two conditions utilized is the same, both up to one hundred percent.

Conclusion:

More Carbon Utilization Efficiency indicates more carbon sources are converted to biomass and biological products relative to the amount consumed (Allison et al., 2010; Manzoni et al., 2012; Hessen et al., 2004; Jiao et al., 2014; Sterner and Elser, 2002). As result, we can conlude that both carrot and squash soil have a high biomass and biological products based on their same Carbon Utilization Efficiency.

Figure 4: Shannon, richness, and evenness from extracted bacterial DNA

The results of our Shannon (H), richness (S), and evenness (E) tests were obtained through DNA extraction from bacteria. The extracted DNA samples were sent to a lab to be sequenced and Nephele was used to analyze the data collected from the sequencing and analysis of the extracted DNA. The graphs in figure 4.3 demonstrate the average Shannon, richness, and evenness values of the two conditions with standard deviation shown.

There was an unpaired t-test assuming unequal variance ran for the H, S, and E values. The Shannon and richness values generated a p-value of >0.05 (Shannon Value: 0.432)(Richness Value 0.856), indicating no significant statistical differences between the two conditions analyzed. However, the evenness values generated a p-value of <0.05 (0.00649, indicating a significant statistical difference. This indicates that the biodiversity of the bacteria (H) and number of organisms (S) are statistically similar between the two conditions. The number of different bacteria and their concentration (E) can be seen as statistically different.

The values obtained demonstrates that each factor plays a different role in the soil samples. The large biodiversity could be contributing to the death of the squash. Too many factors could be in play causing the squash to struggle because excess factors could be present. The squash could also be intaking too many carbon sources, causing them to die. In all, there are many factors that play into the death of the squash, these are just a small possibility that are intermixed in the bigger picture.

In conclusion, based on evidence above, squash has more evenly distributed bacteria in a sample than carrot. This statement can be confidently proved by the P value (0.00649) less than 0.05, which means there is a significant difference between the evenness of these two conditions. Also, there is a high certainty that they have some differences in evenness based on P value 0.00649 less than 0.05, which indicates that there is a huge difference between the evenness of squash and carrot. Therefore, we can be confident and certain to conclude that squash has more even bacteria distribution than carrot. On the contrary, we cannot confidently conclude that there is any obvious difference between Shannon diversity index and richness of these two conditions due to 0.432 and 0.856 P values that are more than 0.05, which means that there is no significant difference of Shannon diversity index and richness between these two conditions. In addition, the certainty cannot be considered based on p values (0.432 and 0.856), which represents there is no significant difference between the Shannon diversity index and richness. As result, with the consideration of Shannon diversity index, richness and evenness, it is only possible for us to conclude that squash has more evenly distributed bacteria in a sample than carrot due to different evenness between two conditions with P value 0.00649.

Discussion

What makes our research project interesting is that we are comparing soil samples from a root plant and a non-root plant to determine the differences in soil pH, water concentration, their microbiomes, and biodiversity. This is intriguing because it allows not only us as researchers, but also the community to expand thier knowledge. By the end of the project we have relayed our findings to the volunteers of the community garden; helping them make changes to the garden. These small or large changes will allow for more produce to be harvested, giving more families the opportunity to have a meal. Having fresh vegetables and fruits is vital to the families in the community because it lowers the risk of stroke, heart disease, blood pressure, eye, and digestive problems (Harvard). The projects has an immense impact on us researchers as well, because we are studying something that we once viewed as mundane; soil.

Acknowledgments

Thank you to Purdue University for funding this research project and for encouraging our ideas. Thank you Proffessor Jacob Adler for pushing us to explore our ideas. For encouraging our plans and for supporting us through the project. Thank you to our teach assistand Tyler. You allowed us to bounce ideas off of you and you helped curate our skills in biology. Thank you to our teaching interns as well for always being there for us. We appreciate all of you.

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