Veronica's Research on Aeroponics

Dr. Mackey is going to start recording this session, so for anybody, if you'd like to ask questions, you can type them into the chat and we'll get to those at the end. Otherwise, please stay muted and I'll get started. Thank you so much everyone who took time out of your day to attend today's session. This is my presentation for my Master's of Science Research Project. The effects of plant growth promoting microorganisms on microbial community dynamics and plant growth responses of aeroponically cultivated spinach. Again, my advisor is Dr. Mackey, who introduced me, and then Drs. Alan Robertis and Josh Sharp are here as well, and they are on my committee. So conventional soil-based agriculture has been able to keep up with our growing population, but it comes at a cost. This system uses... 37% of the earth's land, while only 12.21% of that is truly arable. Conventional systems also involve soil degrading techniques such as over-fertilization, pesticide application, and then monoculture crops. These result in reduced yields, lower biodiversity, reduced soil quality, and reduced crop variety. These practices also contribute to agrochemical pollution and public health problems. And they also influence pathogen distribution and the growth and then nutrient availability to the plants and the release of biologically active substances. Current large-scale systems also don't take advantage of resident microbial populations that can help suppress those pathogens and improve crop productivity. Conventional farming also faces additional challenges in the Upper Peninsula of Michigan as they are not possible year round due to our cold climates and short growing seasons. This is going to create a lack of access to quality locally sourced and affordable foods year round, resulting in regional food insecurity. Food insecurity is a household level economic and social condition of limited access to affordable foods. And according to Feed in America Organization 2022, at 14... or sorry, 15.2% of children and 14.8% of the total population of the UP face food insecurity. So it's really important that we find ways to increase the quality and quantity of foods that we're producing. One option is to transition to organic systems as these systems can increase soil carbon levels, improve soil quality and decrease soil erosion while using little to no synthetic pesticides. But one of the most significant criticisms of organic agriculture are its lower yields, which present a challenge as populations continue to grow, and they also don't address the climate issues faced by the Upper Peninsula. Indoor growing systems such as hydroponics could help provide a potential solution for food production in cold climates like the Upper Peninsula. Hydroponics are an indoor system that uses water-based nutrient solution rather than soil. And as shown here, there are a wide variety of hydroponic systems that you can choose from. And by shifting from a soil-based conventional system to a modified hydroponic system, yields could improve as these systems do not require fertile land and can be operated indoors year-round in all sorts of spaces like converted abandoned warehouses or your own home kitchen or garage. And this technology can offer growers more control over production with the ability to increase yields and improve sustainability. Our focus was on aeroponics. Aeroponics are a modified subgroup of hydroponics that use no growth medium and induce growth by misting a nutrient rich solution onto the roots at specified intervals. This photo on the right here shows the inside of an aeroponic system. where the roots are suspended and water and nutrients are sprayed onto the roots. These systems can reduce the amount of space used per unit of yields by utilizing vertical space. And then the indoor location means that production relies less on the external conditions, such as cold temperatures, lack of sunlight, meaning they can be operated year-round. They also can reduce water usage by up to 98 percent, fertilizer usage by up to 60 percent, and pesticide usage by up to 100 percent. And this is going to increase our overall efficiency and reduce the potential for pollution of ground and surface water. Aeroponic systems might also allow plants to take up more vitamins and minerals, increasing their nutritional value, as well as the increased aeration of the nutrient supply as it's being sprayed onto the roots. And the oxygen that's being provided by that, it may prevent plant diseases and reduce water and nutrient usage. So this is what our aeroponic system looked like. This image on the left here is kind of like almost exactly what it looked like. So we had a water reservoir, a water and nutrient reservoir, in which the water nutrients were pulled up by a pump and pumped into the system. And then this is what the inside of the system looked like. So they're pumped into these tubes and then sprayed onto the roots and then the water collected at the base and then drained back into the reservoir and was reused by the system. Aeroponics do have their disadvantages though. These systems have little room for nutrient application error as the system has almost no buffering capacity. They can be initially expensive to set up and are vulnerable to power outages as they rely on electricity. They're also vulnerable to user error. If you look at this image here, all these plants are dead. And that is because a tube slipped out of the reservoir overnight and when I came back in the morning... all the plants were dead. So I've made changes to prevent that, but you can have user error like that. They're also vulnerable to pathogen spread due to the high nutrient concentrations and infrequent cleanings. And the spread of pathogens can be devastating to food crops by reducing output and increasing per unit cost. So we propose that by incorporating organic nutrients and plant growth promoting microorganisms into these systems, it might mitigate some of these issues. So organic amendments are materials that can be added to improve the physical and chemical and biological properties of a system and their natural sources of macro and micronutrients. Common amendments, as Dr. Maggie mentioned before, biochar, and then manure and compost. The one that we chose to use is vermicompost. And vermicompost is a combination of the defecated products of worms and the microflora of the worm's gut. Vermicompost tea, which is shown here, is a process in which the vermicompost is brewed, much like how you brew your coffee, which is why I have kind of a coffee pour over there. You brew it and then you filter it. And then this process extracts the nutrients and microbes from the vermicompost and allows them these to be added as a spray, more or less, versus the typical vermicompost itself. And by providing additional nutrients and increased availability of potentially beneficial bacteria, this could be helpful to plant productivity. Then also Dominguez 2004 demonstrated that Burma compost has a low carbon to nitrogen ratio and organic nutrients with low carbon to nitrogen ratios of less than 10 can suppress plant pathogens, while increasing the availability of beneficial bacteria and fungi. The plant growth promoting microorganisms are bacteria and fungi found in the rhizosphere. capable of protecting plants from diseases and abiotic stressors while promoting growth. They can enhance plant growth by increasing seed emergence and weight and yield, and then inducing systemic resistance to pathogenic fungi and bacteria and viruses. The terms epiphyte and endophyte are used often throughout this presentation. Epiphytes are microbes that colonize the surface. of the plant root and are in contact with the outer environment. So they're exposed to temperatures and humidity in water. Whereas endophytes are the microbes that are capable of residing inside of healthy plant tissues without causing damage to their hosts. The bacteria and mycorrhizal fungi that we selected. are good candidates for inoculation because it can colonize both the internal and external plant tissues while creating a favorable growing environment without harming the plants. And then all of the organisms except for Klebsiella aerogenes are biosafety level one, which are suitable for use by healthy humans. which is important if these systems are going to be used by the public. These plant growth-promoting microorganisms provide direct and indirect benefits to the plants, such as synthesis of phytohormones, like indolecetic acid and gibberellins, the production of beneficial secondary metabolites, such as terpenoids and peroxidase, production of iron-scavenging siderophores, which can... take up iron that helps the plant and prevents pathogens from using the iron in the environment. They can solubilize organic and inorganic phosphates. Some of them can provide biological nitrogen fixation and the nitrogen fixed by these organisms is less prone to leaching and denitrification. Some of them can also produce fungicides to prevent wilt and rot diseases and some of them can help reduce the effects of salinity, which is important in an aeroponic system where those nutrients can build up and we can have salt build up. So our objective was to evaluate the impact of adding organic amendments and plant growth promoting microorganisms through a comparative study of aeroponic and soil-based systems. Our goals were to determine the effects of vermicompost tea and plant growth promoting microorganisms on the nutritional quality and yield of aeroponically grown spinach, to examine pathogen prevalence in aeroponically grown spinach following incorporation of vermicompost tea in plant growth promoting microorganisms, to assess the persistence of inoculated plant growth promoting microorganisms in soil and aeroponic systems, and to assess the composition of epiphytic and endophytic plant growth promoting microorganisms in root tissue. Okay. So we had three treatments shown here. Regular aeroponics, which is short coded by RA if you see that in future slides, that's what that's referring to. Modified aeroponics or MA and soil, which is sometimes referred to as S in some of the graphs. And the regular aeroponics and modified aeroponics were very similar setup wise. And then the soil were in pots of soil. These treatments varied in their plant growth promoting microorganism application. Our regular aeroponics was like our control. It had only a hydroponic solution and water applied to it, whereas the modified aeroponics had the hydroponic solution and water, as well as vermicompost tea and plant growth promoting microorganisms. And the soil had water in those plant growth promoting microorganisms and vermicompost tea as well. For all treatments, water is dechlorinated weekly. And then hydroponic solution concentration was increased weekly for the two aeroponic treatments. The mycorrhizal fungi and beneficial bacteria were regrown every week and added to the freshly dechlorinated water for the modified aeroponic and soil treatments. The mycorrhizal fungi were applied during germination for MA and soil. And then the plant growth-promoting microorganisms and vermicompost tea were applied, again, during those water changes. Fifteen plants were grown per treatment with four replications of these treatments. Mycorrhizal fungi, commercial mycorrhizal fungi, were inoculants, sorry, were mixed together and then applied as a thin layer at the base of the seed germination chamber for the modified aeroponic. and soil treatments. The seeds were then sown directly on top of these mycorrhizal inoculants, whereas the regular aeroponics were grown without these inoculants. Bacterial application. So to determine the quantity of bacteria to use, 100 microliters of frozen stock, cultures were added to five milliliters of nutrient broth. These were incubated until turbid. and then serially diluted. Then dilutions with colony forming units of 30 to 300 and the differences in the growth rates of each species were used to determine the quantity of each species to add to the mixture of the, to add to the mixture that went into the water. So a total of 15 mils of plant growth promoting microorganisms were added per four gallons of water. And these were replaced with the weekly water replacements. So after germination and then prior to transplanting into the systems, pre-measurements of germination percentage, initial root length from the base of the hevcodal to the tip of the radical in centimeters, and then initial. sorry, initial root length and then initial stem length from the base of the hippocotyl to the first node below the apical bud were measured. Then post-harvest, so after we pulled the plants from the system, the final stem length in centimeters, percent change in stem length, final root weight, final leaf weight, the total number of leaves, and then 16S rRNA gene amplicon sequencing of root tissue, both epiphytic and endophytic, and nutritional content analysis of leaf tissue were conducted. The bacterial 16S rRNA and fungal ITS1 gene amplicon sequencing was completed for two of the four replicates of each treatment, because this is a relatively cost-prohibitive process, especially for a grad student. But the process itself was we took 60 grams of roots from each treatment and then extracted the DNA using Kyogen's D&Easy Power Soil Kit. And then we sent these extractions to the University of Minnesota Genomics Center for Bioinformatic Analysis. The University of Minnesota uses Illumini MISEC high throughput sequencing to amplify the microbiome marker genes. And then... That microbiome profile is prepared using their dual indexing method that was developed in-house at University of Minnesota. And then bioinformatic analysis of those 16S and ITS1 regions were completed using CHIME 2 analysis software. Nutritional content analysis of the leaf tissue. All leaf tissue was bagged and then sent to Midwest Laboratories in Omaha, Nebraska. They reported back to us the percent of macronutrients and the parts per million of micronutrients in these leafy tissues. All right. So our results, all data were analyzed using Closet Cloud RStudio 2022. Plant traits and mineral content among the three treatment groups were compared using means. Normality and equality of variance were assessed. appropriate ANOVAs were run. If ANOVAs were significant, post hocs were performed. Then looking at these figures, the line in the box, well this one's easier to see, the line in the box plot indicates the median, while as the open circle, the open black circle indicates the mean. In these two figures, the application of mycorrhizal inoculants significantly increased germination rate for the soil and modified aeroponics. as well as significantly increased the initial stem length for those two treatments. Initial root length did not differ between the treatments. And then a study by Barron and Rigobello in 2021 demonstrated that mycorrhizal fungi have the ability to produce phytohormones, indolecetic acid, and gibberellic acid, which are essential to plant growth and have positive effects on germination. of the shoot and root development by division and cellular elongation and differentiation of vascular tissues. So that supports, aside from our initial roots not having different lengths, it does help support that the micro-risal fungi helped promote those benefits. In the following slides, we're going to discuss the effects of treatment and tissue type on bacterial and fungal community structure and richness using the data from UMGC. The term tissue type refers to whether the microorganisms are epiphytic or endophytic. While these organisms might look like they share a very similar habitat based off their proximity, they're actually vastly different. The epiphytes, which are the ones, again, on the outside of the root. are exposed to the temperature and the humidity and rainfall. While in contrast, the endophytes, the internal organisms, are a little bit more sheltered, but they have to deal with those plant defenses. And so these stress factors can affect community composition. To analyze the effects of treatment and tissue type on bacterial and fungal community structure, first we had to calculate the percentage of organisms identified at each taxonomic level to determine which level to use for analysis. After calculating that, we determined to use fungal species and genus and bacterial genus and family. The dark green indicates known and identified organisms, whereas the light green indicates unidentified organisms that were just marked as unidentified. After these were calculated, after these percentages were calculated, then a perminovo was used to compare the multivariate means of the bacteria and fungi among the treatment groups and to tissue types. For each taxonomic level, two perminovas were run because, or sorry, two perminovas were run. One was on the knowns only and the other was on the knowns plus the unknowns. So the entire group. analysis set. Following a significant interaction, post-hocs were run to determine whether the treatment groups or tissue types differed. For the following slides and for my paper, we chose to report the perminobular results only for correctly identified or known organisms because while using unknowns at all the taxonomic levels created a significant interaction term, UMGC did not give each unknown its own unique ID. So like species one unknown, species two unknown. But instead, they group all the unknowns into one ID. So it could not be said with confidence which of the unknown organisms may have created a significant interaction. And so since they didn't contain taxonomic information, they were removed. And this was guided by a study by Gundale et al. 2016. Results were then visualized using non-metric multidimensional scaling, or NMDS. The polygons here indicate the lines between the outer points of each treatment, encircling the other points within the group, and then these treatments have labeled regular aeroponics, modified aeroponics, and soil. The analysis of the effect of treatment and tissue type on fungal community structure shows that fungal genus, shown here, And fungal species shown on the next slide, community compositions were significantly influenced by treatment type, but not tissue type. And so then this is the fungal species showing that it differed by treatment type, but tissue type is not shown because it was not significant. I showed this two graphs here because this is the figure initially produced by NMDS. It was significant, but it's hard to visualize because number 11, sample 11 was an outlier. So I removed sample 11 just so you guys could see what was going on there, but this is the actual figure that it produced. And then while fungal samples differed by treatment only, bacterial was a little bit different. Bacterial genus community structure was significantly influenced by both treatment and tissue type. So we have treatment over here and then tissue type over here. And then... bacterial family was only significantly different between tissue types. And Nazarov et al. 2020 supports this, that these differences between fungal and bacterial community compositions could be because fungi can easily penetrate into or around epidermal cells without requiring unique structures or conditions. Whereas endophytic bacteria often require either damaged tissues or access to specialized structures such as stomata or a vector such as an insect burrowing into that tissue. Next, taxa richness among the three treatment groups and two tissue types were compared using means. Again, normality and equality of variances were assessed. They were not met. So a non-parametric two-way permutation was used to compare the means. of the measured tax of richness among treatment groups and tissue types. Following that significant interaction, a post-hoc was performed, and these are the results. So a colored dot indicates, like that colored dot right there, indicates the mean richness, and the cap bars indicate the standard error. And then the line here shows the interaction between treatments, so treatments across the x-axis, and tissue type. And the tissue types are indicated by color. So endophytic are the seafoam green and epiphytic are the darker blue color. Okay. So here we see that the richness or the results say that the richness of fungal species, fungal genre. So we got fungal species, fungal genera, bacterial genera, and bacterial family significantly differed among tissue type but not among treatment groups. So this demonstrates that healthy plants are selective in allowing microorganisms access into their internal structures. Next, we look at the 16S rRNA for bacteria and the fungal ITS1 data from UMGC, and we visualize it by relative abundance, as that's what these graphs are showing. And then across the x-axis, if you look down here, you see some shorthand for the samples. MA, again, stands for modified aeroponics. RA stands for regular aeroponics. And then soil, soil. Endo for endophytic tissue. epi for endo for endophytic microbes so from the inside of the roots and then epi for epiphytic microbes from the outside of the roots and then trial a or b like trial one or two that we selected from the send and then you will also see the number of species present in each sample So relative abundance of fungal taxa at the species level, there were 299,832 total known reads representing 81 unique species. These species were often shared between the samples, as you can see more based off the color in the figure. Then the relative abundance of fungal taxa at the genus level There were 63 unique genera. Again, a lot, a couple of these were shared among the samples. Then relative abundance of bacterial taxa at the genus level. There were 288 unique genera. Again, quite a few shared, as you can see. And then there were 177 unique bacterial families. And again, a lot of those were shared by the samples. But for both fungal and bacteria individual richness, the endophytic tissue contains significantly less individual reads than the epiphytic tissue, and this supports our taxa richness results. Next, we looked at the taxa with the highest abundance for fungi and bacteria. This table shows the top five fungal genera with each sample with their individual reads. Then each taxa had a unique dominant fungal genre, and it was shared by the trials and tissue types. So, olpidium was the dominant fungal genre in the modified aeroponics. This genera acts as a vector for multiple different plant viruses, and though we did, but we didn't observe any visual viral symptoms in the treatments. Then Fusarium was the dominant fungal genera for the regular aeroponic treatments. This is a pathogenic genus of great economic importance as it can produce mycotoxins that can decrease yields and often adversely affect consumer health. Sorry, my mouse was stuck. Rodriguez et al. 2008 found that some fusarium species can also confer salt tolerance to the host plant. So this would be beneficial to the aeroponic systems. And as the fusarium were not identified to species level, I can't say whether or not these were the beneficial ones or if they were pathogenic. Then the dominant... fungal genera in soil was Thermothylovoides, which is a thermophilic decomposer capable of growing at temperatures of 20 to 55 degrees Celsius, and that plays an important role in the carbon cycle by breaking down polysaccharides. This table shows the top five bacterial genera for each sample with their individual wreaths, but unlike the fungal genera, the dominant bacterial genera were not strictly shared by treatment. or tissue type. Some shared genera include Massilia. And Massilia is a widely distributed genus with species having unique ecological functions such as improving soil environment, degrading pollutants, and improving survival rate in harsh environments. Pseudomonas was another shared genera. or genus. And it's a multifaceted genus with multiple species having beneficial effects of disease and pathogen inhibition, bioremediation and growth promotion. One species can also be pathogenic to spinach, but the species were, no species were identified to the species level. It was all just to the genus level. So I can't again say whether or not it was pathogenic or beneficial. variety. And lastly, Escherichia shigella was also shared by some of the treatments, the SOIL Trial A and Modified Aeroponics Trial A endophytic tissues. Escherichia shigella is a human pathogen that uses plants as an alternative host due to their stable environment. It uses similar mechanisms to invade the plant cells as it does animal cells. and can inhibit the innate immune response of the infected plants, resulting in adverse growth effects. Endophytic tissues infected with the shriek yeshigala are still capable of transmitting the bacteria and could pose a serious food safety risk since spinach is often consumed without cooking. And although this was root tissue, bacteria can move throughout the plant, so there's potential that it was present in the leaf tissue. This bacteria, how could it have been transmitted? Well, potentially by the vermicompost, especially if the soil that the worms had ingested had previously had an infected manure applied to it. So this kind of poses an additional question is, is vermicompost even safe for use? And so a potential solution to that may be to pre-screen the vermicompost, but that's kind of expensive to like see what kind of species and genus are present. Or there's a study that examined autoclaving, which they showed increased the nutrient availability by lysing the cell components of the microbes in the vermicompost. And that could also potentially reduce the variability of microbes that are being added. And we could look more at the effects of our intended microorganisms. So looking back at these relative abundance graphs for fungal and bacterial genera, the analysis revealed that of the 11 plant growth-promoting microorganisms that we added to the soil and modified aeroponics treatments, UMGC was not able to identify any of them to the species level. And while some of these, the genera were detected, whether or not these genera contain the added species remains uncertain. Edmund Zettel, 2020, found that. selection pressure during early seedling development, so within those first 12 days by the root exudates, determines the residential microbial community. So the fact that our microorganisms were added after transplant, so after they had grown and emerged, germinated, this could have contributed to the lack of the uptake by the plants. And then Lobanov et al. 2022 adds that plants exert a strong selection pressure for their coexisting microbes to meet their particular needs regardless of microbial additions. So the plants just could have possibly not wanted to host the microbes that we had. And although our specific plant growth-promoting microorganisms that we had inoculated weren't detected, other species within these genera can provide plant growth-promoting properties. Also, some of the benefits that we've seen could have been due to horizontal gene transfer between inoculated plant growth-promoted microorganisms and the resident microorganisms. And that could have contributed to the increased growth and mineral acquisition that we'll see. Tagavi et al. 2005 demonstrated that the endophytes that they induced did not persist. However, the trait for toluene breakdown that they were interested in was present in the endophytic community. suggesting that the horizontal gene transfer had occurred. And then additionally, plant growth-promoting microorganisms could have affected the DNA methylation in the plant roots. Chen et al. 2022 demonstrated that although plant growth-promoting microorganisms did not persist that they inoculated, they were able to influence plant growth via epigenetic modification in the plant roots. So these are some things that could have contributed to the overall benefits that we see. Then of the 41 pathogens capable of infecting spinach and causing disease symptoms as listed by the American Phytopathological Society, only the fungal pathogen Alternaria alternata, which is Alternaria leaf spot fungus, was identified. RA, so regular aeroponics, had the highest infection rates, which we're seeing here, and then on these little guys. And then modified aeroponics in soil had low or non-existent rates of this pathogen. The reduced pathogen load by the addition of plant-worthful micro-organisms appears to be vital in these systems because they otherwise suffer from rapid pathogen growth, which we're seeing here. Then looking at all potential pathogens, Fusarium and Pseudomonas genera were also identified. These two genera could contain Fusarium oxysporum and Pseudomonas syringae. But again, UMGC couldn't identify these taxa to the species level. And so it can't be definitively said if these genera contain pathogens specific to spinach. And the colonization of epiphytic and endophytic tissues by... plant growth-formulating microorganisms in the modified aeroponics and soil systems could have contributed to the absence of pathogens by production of the siderophores, so for the iron chelating, antibiotic production, production of enzymes that can help lyse pathogenic cells, or the induction of non-specific systemic resistance. And these could have led to changes in the plant's metabolism that could have increased its overall general resistance to pathogens. Next, we looked at the effect of treatment type on mineral acquisition by the plants. We were interested in producing nutrient dense plants and thought that by adding the mycorrhizal fungi and the beneficial bacteria, that they would complement each other and improve nutrient acquisition in plant productivity. There was significant variation observed in the mineral composition of the three treatments. Mineral concentration of sodium and copper in leafy tissue significantly differed in all treatments with soil having the lowest concentration of each. While these concentrations of minerals were significantly different, Midwest laboratories had their own rating system and rated the sodium levels. as of modified aeroponics and regular aeroponics as excessive. So we're seeing that they're significantly different, but they're both excessive and soil as sufficient. And then in copper, the regular aeroponics were excessive and the modified aeroponics and soil were low and deficient. Calcium and boron. were also significantly lower in soil treatments and they were rated as deficient in these treatments by Midwest laboratories, while they were rated as high or excessive in the aeroponic systems. Concentrations of nitrogen, phosphorus, potassium, and sulfur were all significantly higher in the soil systems. The concentrations of nitrogen and phosphorus were excessive in soil and high in the aeroponic systems. while potassium was excessive in all three treatments, and sulfur was sufficient in all. Zinc concentration was significantly lower in modified aeroponic systems, and they were rated as excessively high in soil and regular aeroponics. And then the concentrations of magnesium, iron, and manganese did not significantly differ in any of the treatments. And magnesium was, they were all rated as excessive, iron low and deficient. and manganese as all high and excessive. So while soil had more nutrients at significantly high levels, the overall nutrient profile of each treatment from Midwest laboratories showed that the aeroponic systems had more overall nutrients at sufficient high or excessive levels. And this could be due to the aeroponic system's lack of nutrient diffusion limits. Water availability determines the diffusion limits of a system and aeroponics have continuous water availability, whereas soil systems are susceptible to periods of drought. So we're looking at post-harvest measurements here. So if you remember back to one of the first slides, regular aeroponics had a significantly shorter initial stem length. But then we have no significant difference in the average final stem lengths of the treatment groups, which results in regular aeroponics having a significantly higher percent change in stem length. Additional post-harvest measurements of leaf number, or sorry, leaf weight, leaf number, and root weight were all significantly lower in soil treatments. And root and shoot development are positively correlated. So roots uptake your minerals and water and leaves bring in your CO2 and the sugars and they combine and they produce cellular components necessary for growth. A study by Rona Wade et al. 2017 also investigated soilless systems and found that they outperform soil on plant yields. So why might soil plants and soil systems have produced less leaf and root tissues? These plants didn't bolt. Spinach are really good at telling you if they're experiencing heat stress, they will bolt. So it wasn't likely heat stress. The smaller root and shoot weights could be due to water stress. The soil treatment had a significantly lower calcium concentration than the aeroponic treatments. And the movement of calcium depends on transpiration through the xylem, which doesn't occur efficiently in a water stress plant as they close their stomata. And calcium is essential for cell division and elongation, and insufficient quantities can result in slower growth or stunted growth. And then the binding site for calcium is also in competition with the binding site for potassium, of which soil also had the highest amount or the most significant amount. And... This could have resulted in calcium deficiency symptoms, which would have been necrosis. But aside from being small, the plants didn't have necrotic symptoms. They were healthy in appearance. But the high levels of potassium during times of water stress could also have provided a temporary benefit of maintaining turgor pressure. So making the plants look like they're nice and happy and healthy. So to revisit our findings. The community composition of fungi, these little guys right here, varied between the treatments, but not the tissues, whereas bacterial communities vary between tissues. This could be attributed to the ability of fungal hyphae to, again, penetrate those internal structures easily, whereas bacteria... require a vector or a weakened immune system to access those internal structures. Fungal and bacterial richness significantly differ between tissue types, indicating that endophytic, so internal, and epiphytic environments provide those distinct habitats affecting colonization and efficacy. The application of mycorrhizal fungi during germination significantly improved germination rates and early stem growth, likely due to the production of essential phytohormones like indolecetic acid and dibrelin. Alternaria alternata was the only pathogen specific to spinach detected in the samples with the highest prevalence in the regular aeroponic treatments. Aeroponic treatments had significantly higher leaf and leaf number and leaf weight and root weight, and more leaves equals more food, more people fed. So it's a great thing. And then treatments varied in their mineral content with soil having the highest nitrogen, potassium, phosphorus and sulfur, but significantly lower sodium, copper, boron and calcium levels. While the aeroponic systems did not outperform the soil on certain minerals, they did produce an overall enhanced nutrient acquisition and the continuous water supply provided to the roots by the aeroponic systems could be responsible. making them less susceptible to drought, and then facilitating that nutrient uptake. So when selecting a growth system for a population such as the Upper Peninsula, it would be crucial to consider the nutritional needs of that population as well. And this project demonstrates the potential benefits of adding organic amendments and plant growth-promoting microorganisms to enhance yield, nutritional quality, pathogen resistance of aeroponically grown spinach and could play an important role in sustainable agriculture and regional food insecurity, especially in cold climates such as the Upper Peninsula. Are there questions? Thank you for listening. Okay, thank you, Veronica. I think I did see a couple of questions come through the chat, so why don't we start with those first. So one of the questions was how much would a system like this cost when you put together your aeroponic system? Do you have an estimate of what that cost? Yeah. So the system that I used that you saw in there costs approximately $425 to build. And a lot of that cost comes from the light itself. But you can purchase these systems from... like Amazon or a local growth store. And they range from $125 without lights. So again, lights is one of the factors that you need to consider up to $650 with lights, but it's a prebuilt system. So you could bring that cost down if you want to like build your own system. So you can buy one of those $125 systems, pay like $260 for some lights, and we're still saving money. It is, again, expensive. That is kind of expensive for people, though. Okay, another question was, what was the timing of the aeroponic, the misting timing? Okay, yeah, so they were on, so they were spraying the roots. for two seconds and then they would shut off for five seconds and just repetitive on for two off for five the entire time that they were growing okay um another question from carrie nice job she says i may have missed this but do you have any idea uh of the microbes that came from the vermicompost tea i do not That's an awesome question and definitely something that we need to look at or future studies need to look at because analyzing the microbes was very expensive. It was about $1,500 to analyze to send them to the University of Minnesota. And so I would have loved to analyze the vermicompost tea to see what microbes were getting added so that I could maybe... do a little bit more analysis with that because then I would have an idea of what I started with more and how maybe that out-competed what I was trying to add. So we never had the actual vermicompost itself analyzed initially. Correct. But then with your tea brewing, I mean, the study, the results that you were showing included the microbes coming from the tea. It wasn't because you inoculated, what, 12, 11 microbes that we knew. that we inoculated. Yes. Okay. I think that's it in the chat for now, but if other people want to type in that's I'm monitoring the chat. Are there any, any other questions you can either send it via chat or just unmute yourself and ask? I guess I have a question. So I know as part of your plant growth promoting microorganisms, the one potential human pathogen in there was the Klebsiella erogenes. Why was that one specifically added? I was just curious. Yeah, okay. So the Klebsiella... Thank you, Josh, for this question. uh clubziella which was when i did my study formally uh enterobacter orogenes so it's been a minute because it was recently reclassified um that one can help produce uh indolecetic acid and uh gibberellins and then can also catabolize excessive hormones like that and then they also help produce those iron chelating agents so they can sequester iron from the environment and they can solute all eyes inorganic phosphates and they can do some biological nitrogen fixation so they It was selected because of those attributes, but moving forward, it could be removed because there are a lot of other microbes in that selection that produce the same effects. So moving forward, that one would be excluded, especially if we want to look at making the system available to the public. Yeah, that was one of the reasons I was asking that because, you know, someone wanted to, you know, reconstruct these systems and it's going to be easier for... someone to get a hold of you know bsl1 you know organisms and two would be a potential problem yeah and where did you get your mycorrhizal fungi from uh so the uh fungi came from commercial inoculants uh so the trichoderma uh were together in i believe uh root shield plus and you can just order that on amazon or pick it up at the store i'd like a like a hydroponic store and the glomus species were i think dynamico um again just ordered that um there's there's a lot of um There's a lot of beneficial microorganisms available online, which we could use ones that only could be ordered and that would improve the study so that it could be actually usable by the public. And when I looked at it, you can get your vermicompost, your mycorrhizal fungi, so there's Trichoderma anglomus, the streptomyces, both bacillus strains, and Lactococcus from Amazon. all of those can come from Amazon. Then the erbis corillium and the pseudomonas must be, I could not find those as in any mixture or as individuals through any site that provided beneficial organisms. So like moving forward, it would be interesting to see if there's a difference if we took out those ones that people don't have. access to, and especially removing that Klebsiella immediately. And then the herbus beryllium and the pseudomonas, seeing what changes we might see without those versus with those. Okay. And then another question came in via chat. Was the compost tea a foliar spray or a reservoir additive? It was a reservoir additive, and then it was sprayed onto the roots. for the aeroponic system. And then for the soil, it was like mixed with the water and then watered on as needed. So it could have gotten on the leaves. It was meant as a soil like application though. Right. Okay. Okay. Well, it's getting close to the hour being finished. So I think we'll wrap up the questions for now. And I'm going to ask everyone who came in for the seminar, if you could please leave. Thank you for attending. Thank you. And Veronica will stick with her committee here and we'll continue on.

This is my presentation for my Master's of Science Research Project. The effects of plant growth promoting microorganisms on microbial community dynamics and plant growth responses of aeroponically cultivated spinach. Again, my advisor is Dr. Mackey, who introduced me, and then Drs.

Alan Robertis and Josh Sharp are here as well, and they are on my committee. So conventional soil-based agriculture has been able to keep up with our growing population, but it comes at a cost. This system uses... 37% of the earth's land, while only 12.21% of that is truly arable. Conventional systems also involve soil degrading techniques such as over-fertilization, pesticide application, and then monoculture crops.

These result in reduced yields, lower biodiversity, reduced soil quality, and reduced crop variety. These practices also contribute to agrochemical pollution and public health problems. And they also influence pathogen distribution and the growth and then nutrient availability to the plants and the release of biologically active substances. Current large-scale systems also don't take advantage of resident microbial populations that can help suppress those pathogens and improve crop productivity.

Conventional farming also faces additional challenges in the Upper Peninsula of Michigan as they are not possible year round due to our cold climates and short growing seasons. This is going to create a lack of access to quality locally sourced and affordable foods year round, resulting in regional food insecurity. Food insecurity is a household level economic and social condition of limited access to affordable foods.

And according to Feed in America Organization 2022, at 14... or sorry, 15.2% of children and 14.8% of the total population of the UP face food insecurity. So it's really important that we find ways to increase the quality and quantity of foods that we're producing. One option is to transition to organic systems as these systems can increase soil carbon levels, improve soil quality and decrease soil erosion while using little to no synthetic pesticides. But one of the most significant criticisms of organic agriculture are its lower yields, which present a challenge as populations continue to grow, and they also don't address the climate issues faced by the Upper Peninsula.

Indoor growing systems such as hydroponics could help provide a potential solution for food production in cold climates like the Upper Peninsula. Hydroponics are an indoor system that uses water-based nutrient solution rather than soil. And as shown here, there are a wide variety of hydroponic systems that you can choose from.

And by shifting from a soil-based conventional system to a modified hydroponic system, yields could improve as these systems do not require fertile land and can be operated indoors year-round in all sorts of spaces like converted abandoned warehouses or your own home kitchen or garage. And this technology can offer growers more control over production with the ability to increase yields and improve sustainability. Our focus was on aeroponics. Aeroponics are a modified subgroup of hydroponics that use no growth medium and induce growth by misting a nutrient rich solution onto the roots at specified intervals. This photo on the right here shows the inside of an aeroponic system.

where the roots are suspended and water and nutrients are sprayed onto the roots. These systems can reduce the amount of space used per unit of yields by utilizing vertical space. And then the indoor location means that production relies less on the external conditions, such as cold temperatures, lack of sunlight, meaning they can be operated year-round. They also can reduce water usage by up to 98 percent, fertilizer usage by up to 60 percent, and pesticide usage by up to 100 percent. And this is going to increase our overall efficiency and reduce the potential for pollution of ground and surface water.

Aeroponic systems might also allow plants to take up more vitamins and minerals, increasing their nutritional value, as well as the increased aeration of the nutrient supply as it's being sprayed onto the roots. And the oxygen that's being provided by that, it may prevent plant diseases and reduce water and nutrient usage. So this is what our aeroponic system looked like. This image on the left here is kind of like almost exactly what it looked like.

So we had a water reservoir, a water and nutrient reservoir, in which the water nutrients were pulled up by a pump and pumped into the system. And then this is what the inside of the system looked like. So they're pumped into these tubes and then sprayed onto the roots and then the water collected at the base and then drained back into the reservoir and was reused by the system. Aeroponics do have their disadvantages though. These systems have little room for nutrient application error as the system has almost no buffering capacity.

They can be initially expensive to set up and are vulnerable to power outages as they rely on electricity. They're also vulnerable to user error. If you look at this image here, all these plants are dead.

And that is because a tube slipped out of the reservoir overnight and when I came back in the morning... all the plants were dead. So I've made changes to prevent that, but you can have user error like that. They're also vulnerable to pathogen spread due to the high nutrient concentrations and infrequent cleanings. And the spread of pathogens can be devastating to food crops by reducing output and increasing per unit cost.

So we propose that by incorporating organic nutrients and plant growth promoting microorganisms into these systems, it might mitigate some of these issues. So organic amendments are materials that can be added to improve the physical and chemical and biological properties of a system and their natural sources of macro and micronutrients. Common amendments, as Dr. Maggie mentioned before, biochar, and then manure and compost. The one that we chose to use is vermicompost.

And vermicompost is a combination of the defecated products of worms and the microflora of the worm's gut. Vermicompost tea, which is shown here, is a process in which the vermicompost is brewed, much like how you brew your coffee, which is why I have kind of a coffee pour over there. You brew it and then you filter it.

And then this process extracts the nutrients and microbes from the vermicompost and allows them these to be added as a spray, more or less, versus the typical vermicompost itself. And by providing additional nutrients and increased availability of potentially beneficial bacteria, this could be helpful to plant productivity. Then also Dominguez 2004 demonstrated that Burma compost has a low carbon to nitrogen ratio and organic nutrients with low carbon to nitrogen ratios of less than 10 can suppress plant pathogens, while increasing the availability of beneficial bacteria and fungi. The plant growth promoting microorganisms are bacteria and fungi found in the rhizosphere. capable of protecting plants from diseases and abiotic stressors while promoting growth.

They can enhance plant growth by increasing seed emergence and weight and yield, and then inducing systemic resistance to pathogenic fungi and bacteria and viruses. The terms epiphyte and endophyte are used often throughout this presentation. Epiphytes are microbes that colonize the surface. of the plant root and are in contact with the outer environment.

So they're exposed to temperatures and humidity in water. Whereas endophytes are the microbes that are capable of residing inside of healthy plant tissues without causing damage to their hosts. The bacteria and mycorrhizal fungi that we selected.

are good candidates for inoculation because it can colonize both the internal and external plant tissues while creating a favorable growing environment without harming the plants. And then all of the organisms except for Klebsiella aerogenes are biosafety level one, which are suitable for use by healthy humans. which is important if these systems are going to be used by the public. These plant growth-promoting microorganisms provide direct and indirect benefits to the plants, such as synthesis of phytohormones, like indolecetic acid and gibberellins, the production of beneficial secondary metabolites, such as terpenoids and peroxidase, production of iron-scavenging siderophores, which can...

take up iron that helps the plant and prevents pathogens from using the iron in the environment. They can solubilize organic and inorganic phosphates. Some of them can provide biological nitrogen fixation and the nitrogen fixed by these organisms is less prone to leaching and denitrification.

Some of them can also produce fungicides to prevent wilt and rot diseases and some of them can help reduce the effects of salinity, which is important in an aeroponic system where those nutrients can build up and we can have salt build up. So our objective was to evaluate the impact of adding organic amendments and plant growth promoting microorganisms through a comparative study of aeroponic and soil-based systems. Our goals were to determine the effects of vermicompost tea and plant growth promoting microorganisms on the nutritional quality and yield of aeroponically grown spinach, to examine pathogen prevalence in aeroponically grown spinach following incorporation of vermicompost tea in plant growth promoting microorganisms, to assess the persistence of inoculated plant growth promoting microorganisms in soil and aeroponic systems, and to assess the composition of epiphytic and endophytic plant growth promoting microorganisms in root tissue. Okay. So we had three treatments shown here.

Regular aeroponics, which is short coded by RA if you see that in future slides, that's what that's referring to. Modified aeroponics or MA and soil, which is sometimes referred to as S in some of the graphs. And the regular aeroponics and modified aeroponics were very similar setup wise.

And then the soil were in pots of soil. These treatments varied in their plant growth promoting microorganism application. Our regular aeroponics was like our control. It had only a hydroponic solution and water applied to it, whereas the modified aeroponics had the hydroponic solution and water, as well as vermicompost tea and plant growth promoting microorganisms. And the soil had water in those plant growth promoting microorganisms and vermicompost tea as well.

For all treatments, water is dechlorinated weekly. And then hydroponic solution concentration was increased weekly for the two aeroponic treatments. The mycorrhizal fungi and beneficial bacteria were regrown every week and added to the freshly dechlorinated water for the modified aeroponic and soil treatments.

The mycorrhizal fungi were applied during germination for MA and soil. And then the plant growth-promoting microorganisms and vermicompost tea were applied, again, during those water changes. Fifteen plants were grown per treatment with four replications of these treatments. Mycorrhizal fungi, commercial mycorrhizal fungi, were inoculants, sorry, were mixed together and then applied as a thin layer at the base of the seed germination chamber for the modified aeroponic.

and soil treatments. The seeds were then sown directly on top of these mycorrhizal inoculants, whereas the regular aeroponics were grown without these inoculants. Bacterial application.

So to determine the quantity of bacteria to use, 100 microliters of frozen stock, cultures were added to five milliliters of nutrient broth. These were incubated until turbid. and then serially diluted. Then dilutions with colony forming units of 30 to 300 and the differences in the growth rates of each species were used to determine the quantity of each species to add to the mixture of the, to add to the mixture that went into the water.

So a total of 15 mils of plant growth promoting microorganisms were added per four gallons of water. And these were replaced with the weekly water replacements. So after germination and then prior to transplanting into the systems, pre-measurements of germination percentage, initial root length from the base of the hevcodal to the tip of the radical in centimeters, and then initial.

sorry, initial root length and then initial stem length from the base of the hippocotyl to the first node below the apical bud were measured. Then post-harvest, so after we pulled the plants from the system, the final stem length in centimeters, percent change in stem length, final root weight, final leaf weight, the total number of leaves, and then 16S rRNA gene amplicon sequencing of root tissue, both epiphytic and endophytic, and nutritional content analysis of leaf tissue were conducted. The bacterial 16S rRNA and fungal ITS1 gene amplicon sequencing was completed for two of the four replicates of each treatment, because this is a relatively cost-prohibitive process, especially for a grad student. But the process itself was we took 60 grams of roots from each treatment and then extracted the DNA using Kyogen's D&Easy Power Soil Kit. And then we sent these extractions to the University of Minnesota Genomics Center for Bioinformatic Analysis.

The University of Minnesota uses Illumini MISEC high throughput sequencing to amplify the microbiome marker genes. And then... That microbiome profile is prepared using their dual indexing method that was developed in-house at University of Minnesota. And then bioinformatic analysis of those 16S and ITS1 regions were completed using CHIME 2 analysis software.

Nutritional content analysis of the leaf tissue. All leaf tissue was bagged and then sent to Midwest Laboratories in Omaha, Nebraska. They reported back to us the percent of macronutrients and the parts per million of micronutrients in these leafy tissues. All right.

So our results, all data were analyzed using Closet Cloud RStudio 2022. Plant traits and mineral content among the three treatment groups were compared using means. Normality and equality of variance were assessed. appropriate ANOVAs were run. If ANOVAs were significant, post hocs were performed.

Then looking at these figures, the line in the box, well this one's easier to see, the line in the box plot indicates the median, while as the open circle, the open black circle indicates the mean. In these two figures, the application of mycorrhizal inoculants significantly increased germination rate for the soil and modified aeroponics. as well as significantly increased the initial stem length for those two treatments.

Initial root length did not differ between the treatments. And then a study by Barron and Rigobello in 2021 demonstrated that mycorrhizal fungi have the ability to produce phytohormones, indolecetic acid, and gibberellic acid, which are essential to plant growth and have positive effects on germination. of the shoot and root development by division and cellular elongation and differentiation of vascular tissues. So that supports, aside from our initial roots not having different lengths, it does help support that the micro-risal fungi helped promote those benefits. In the following slides, we're going to discuss the effects of treatment and tissue type on bacterial and fungal community structure and richness using the data from UMGC.

The term tissue type refers to whether the microorganisms are epiphytic or endophytic. While these organisms might look like they share a very similar habitat based off their proximity, they're actually vastly different. The epiphytes, which are the ones, again, on the outside of the root.

are exposed to the temperature and the humidity and rainfall. While in contrast, the endophytes, the internal organisms, are a little bit more sheltered, but they have to deal with those plant defenses. And so these stress factors can affect community composition. To analyze the effects of treatment and tissue type on bacterial and fungal community structure, first we had to calculate the percentage of organisms identified at each taxonomic level to determine which level to use for analysis. After calculating that, we determined to use fungal species and genus and bacterial genus and family.

The dark green indicates known and identified organisms, whereas the light green indicates unidentified organisms that were just marked as unidentified. After these were calculated, after these percentages were calculated, then a perminovo was used to compare the multivariate means of the bacteria and fungi among the treatment groups and to tissue types. For each taxonomic level, two perminovas were run because, or sorry, two perminovas were run. One was on the knowns only and the other was on the knowns plus the unknowns. So the entire group.

analysis set. Following a significant interaction, post-hocs were run to determine whether the treatment groups or tissue types differed. For the following slides and for my paper, we chose to report the perminobular results only for correctly identified or known organisms because while using unknowns at all the taxonomic levels created a significant interaction term, UMGC did not give each unknown its own unique ID.

So like species one unknown, species two unknown. But instead, they group all the unknowns into one ID. So it could not be said with confidence which of the unknown organisms may have created a significant interaction.

And so since they didn't contain taxonomic information, they were removed. And this was guided by a study by Gundale et al. 2016. Results were then visualized using non-metric multidimensional scaling, or NMDS. The polygons here indicate the lines between the outer points of each treatment, encircling the other points within the group, and then these treatments have labeled regular aeroponics, modified aeroponics, and soil.

The analysis of the effect of treatment and tissue type on fungal community structure shows that fungal genus, shown here, And fungal species shown on the next slide, community compositions were significantly influenced by treatment type, but not tissue type. And so then this is the fungal species showing that it differed by treatment type, but tissue type is not shown because it was not significant. I showed this two graphs here because this is the figure initially produced by NMDS. It was significant, but it's hard to visualize because number 11, sample 11 was an outlier.

So I removed sample 11 just so you guys could see what was going on there, but this is the actual figure that it produced. And then while fungal samples differed by treatment only, bacterial was a little bit different. Bacterial genus community structure was significantly influenced by both treatment and tissue type.

So we have treatment over here and then tissue type over here. And then... bacterial family was only significantly different between tissue types. And Nazarov et al. 2020 supports this, that these differences between fungal and bacterial community compositions could be because fungi can easily penetrate into or around epidermal cells without requiring unique structures or conditions. Whereas endophytic bacteria often require either damaged tissues or access to specialized structures such as stomata or a vector such as an insect burrowing into that tissue.

Next, taxa richness among the three treatment groups and two tissue types were compared using means. Again, normality and equality of variances were assessed. They were not met. So a non-parametric two-way permutation was used to compare the means.

of the measured tax of richness among treatment groups and tissue types. Following that significant interaction, a post-hoc was performed, and these are the results. So a colored dot indicates, like that colored dot right there, indicates the mean richness, and the cap bars indicate the standard error.

And then the line here shows the interaction between treatments, so treatments across the x-axis, and tissue type. And the tissue types are indicated by color. So endophytic are the seafoam green and epiphytic are the darker blue color.

Okay. So here we see that the richness or the results say that the richness of fungal species, fungal genre. So we got fungal species, fungal genera, bacterial genera, and bacterial family significantly differed among tissue type but not among treatment groups.

So this demonstrates that healthy plants are selective in allowing microorganisms access into their internal structures. Next, we look at the 16S rRNA for bacteria and the fungal ITS1 data from UMGC, and we visualize it by relative abundance, as that's what these graphs are showing. And then across the x-axis, if you look down here, you see some shorthand for the samples.

MA, again, stands for modified aeroponics. RA stands for regular aeroponics. And then soil, soil. Endo for endophytic tissue.

epi for endo for endophytic microbes so from the inside of the roots and then epi for epiphytic microbes from the outside of the roots and then trial a or b like trial one or two that we selected from the send and then you will also see the number of species present in each sample So relative abundance of fungal taxa at the species level, there were 299,832 total known reads representing 81 unique species. These species were often shared between the samples, as you can see more based off the color in the figure. Then the relative abundance of fungal taxa at the genus level There were 63 unique genera. Again, a lot, a couple of these were shared among the samples.

Then relative abundance of bacterial taxa at the genus level. There were 288 unique genera. Again, quite a few shared, as you can see.

And then there were 177 unique bacterial families. And again, a lot of those were shared by the samples. But for both fungal and bacteria individual richness, the endophytic tissue contains significantly less individual reads than the epiphytic tissue, and this supports our taxa richness results.

Next, we looked at the taxa with the highest abundance for fungi and bacteria. This table shows the top five fungal genera with each sample with their individual reads. Then each taxa had a unique dominant fungal genre, and it was shared by the trials and tissue types.

So, olpidium was the dominant fungal genre in the modified aeroponics. This genera acts as a vector for multiple different plant viruses, and though we did, but we didn't observe any visual viral symptoms in the treatments. Then Fusarium was the dominant fungal genera for the regular aeroponic treatments.

This is a pathogenic genus of great economic importance as it can produce mycotoxins that can decrease yields and often adversely affect consumer health. Sorry, my mouse was stuck. Rodriguez et al. 2008 found that some fusarium species can also confer salt tolerance to the host plant. So this would be beneficial to the aeroponic systems.

And as the fusarium were not identified to species level, I can't say whether or not these were the beneficial ones or if they were pathogenic. Then the dominant... fungal genera in soil was Thermothylovoides, which is a thermophilic decomposer capable of growing at temperatures of 20 to 55 degrees Celsius, and that plays an important role in the carbon cycle by breaking down polysaccharides.

This table shows the top five bacterial genera for each sample with their individual wreaths, but unlike the fungal genera, the dominant bacterial genera were not strictly shared by treatment. or tissue type. Some shared genera include Massilia. And Massilia is a widely distributed genus with species having unique ecological functions such as improving soil environment, degrading pollutants, and improving survival rate in harsh environments.

Pseudomonas was another shared genera. or genus. And it's a multifaceted genus with multiple species having beneficial effects of disease and pathogen inhibition, bioremediation and growth promotion.

One species can also be pathogenic to spinach, but the species were, no species were identified to the species level. It was all just to the genus level. So I can't again say whether or not it was pathogenic or beneficial. variety. And lastly, Escherichia shigella was also shared by some of the treatments, the SOIL Trial A and Modified Aeroponics Trial A endophytic tissues.

Escherichia shigella is a human pathogen that uses plants as an alternative host due to their stable environment. It uses similar mechanisms to invade the plant cells as it does animal cells. and can inhibit the innate immune response of the infected plants, resulting in adverse growth effects.

Endophytic tissues infected with the shriek yeshigala are still capable of transmitting the bacteria and could pose a serious food safety risk since spinach is often consumed without cooking. And although this was root tissue, bacteria can move throughout the plant, so there's potential that it was present in the leaf tissue. This bacteria, how could it have been transmitted? Well, potentially by the vermicompost, especially if the soil that the worms had ingested had previously had an infected manure applied to it. So this kind of poses an additional question is, is vermicompost even safe for use?

And so a potential solution to that may be to pre-screen the vermicompost, but that's kind of expensive to like see what kind of species and genus are present. Or there's a study that examined autoclaving, which they showed increased the nutrient availability by lysing the cell components of the microbes in the vermicompost. And that could also potentially reduce the variability of microbes that are being added. And we could look more at the effects of our intended microorganisms. So looking back at these relative abundance graphs for fungal and bacterial genera, the analysis revealed that of the 11 plant growth-promoting microorganisms that we added to the soil and modified aeroponics treatments, UMGC was not able to identify any of them to the species level.

And while some of these, the genera were detected, whether or not these genera contain the added species remains uncertain. Edmund Zettel, 2020, found that. selection pressure during early seedling development, so within those first 12 days by the root exudates, determines the residential microbial community. So the fact that our microorganisms were added after transplant, so after they had grown and emerged, germinated, this could have contributed to the lack of the uptake by the plants. And then Lobanov et al. 2022 adds that plants exert a strong selection pressure for their coexisting microbes to meet their particular needs regardless of microbial additions.

So the plants just could have possibly not wanted to host the microbes that we had. And although our specific plant growth-promoting microorganisms that we had inoculated weren't detected, other species within these genera can provide plant growth-promoting properties. Also, some of the benefits that we've seen could have been due to horizontal gene transfer between inoculated plant growth-promoted microorganisms and the resident microorganisms.

And that could have contributed to the increased growth and mineral acquisition that we'll see. Tagavi et al. 2005 demonstrated that the endophytes that they induced did not persist. However, the trait for toluene breakdown that they were interested in was present in the endophytic community. suggesting that the horizontal gene transfer had occurred.

And then additionally, plant growth-promoting microorganisms could have affected the DNA methylation in the plant roots. Chen et al. 2022 demonstrated that although plant growth-promoting microorganisms did not persist that they inoculated, they were able to influence plant growth via epigenetic modification in the plant roots. So these are some things that could have contributed to the overall benefits that we see.

Then of the 41 pathogens capable of infecting spinach and causing disease symptoms as listed by the American Phytopathological Society, only the fungal pathogen Alternaria alternata, which is Alternaria leaf spot fungus, was identified. RA, so regular aeroponics, had the highest infection rates, which we're seeing here, and then on these little guys. And then modified aeroponics in soil had low or non-existent rates of this pathogen. The reduced pathogen load by the addition of plant-worthful micro-organisms appears to be vital in these systems because they otherwise suffer from rapid pathogen growth, which we're seeing here. Then looking at all potential pathogens, Fusarium and Pseudomonas genera were also identified.

These two genera could contain Fusarium oxysporum and Pseudomonas syringae. But again, UMGC couldn't identify these taxa to the species level. And so it can't be definitively said if these genera contain pathogens specific to spinach. And the colonization of epiphytic and endophytic tissues by...

plant growth-formulating microorganisms in the modified aeroponics and soil systems could have contributed to the absence of pathogens by production of the siderophores, so for the iron chelating, antibiotic production, production of enzymes that can help lyse pathogenic cells, or the induction of non-specific systemic resistance. And these could have led to changes in the plant's metabolism that could have increased its overall general resistance to pathogens. Next, we looked at the effect of treatment type on mineral acquisition by the plants. We were interested in producing nutrient dense plants and thought that by adding the mycorrhizal fungi and the beneficial bacteria, that they would complement each other and improve nutrient acquisition in plant productivity.

There was significant variation observed in the mineral composition of the three treatments. Mineral concentration of sodium and copper in leafy tissue significantly differed in all treatments with soil having the lowest concentration of each. While these concentrations of minerals were significantly different, Midwest laboratories had their own rating system and rated the sodium levels.

as of modified aeroponics and regular aeroponics as excessive. So we're seeing that they're significantly different, but they're both excessive and soil as sufficient. And then in copper, the regular aeroponics were excessive and the modified aeroponics and soil were low and deficient.

Calcium and boron. were also significantly lower in soil treatments and they were rated as deficient in these treatments by Midwest laboratories, while they were rated as high or excessive in the aeroponic systems. Concentrations of nitrogen, phosphorus, potassium, and sulfur were all significantly higher in the soil systems. The concentrations of nitrogen and phosphorus were excessive in soil and high in the aeroponic systems.

while potassium was excessive in all three treatments, and sulfur was sufficient in all. Zinc concentration was significantly lower in modified aeroponic systems, and they were rated as excessively high in soil and regular aeroponics. And then the concentrations of magnesium, iron, and manganese did not significantly differ in any of the treatments.

And magnesium was, they were all rated as excessive, iron low and deficient. and manganese as all high and excessive. So while soil had more nutrients at significantly high levels, the overall nutrient profile of each treatment from Midwest laboratories showed that the aeroponic systems had more overall nutrients at sufficient high or excessive levels.

And this could be due to the aeroponic system's lack of nutrient diffusion limits. Water availability determines the diffusion limits of a system and aeroponics have continuous water availability, whereas soil systems are susceptible to periods of drought. So we're looking at post-harvest measurements here.

So if you remember back to one of the first slides, regular aeroponics had a significantly shorter initial stem length. But then we have no significant difference in the average final stem lengths of the treatment groups, which results in regular aeroponics having a significantly higher percent change in stem length. Additional post-harvest measurements of leaf number, or sorry, leaf weight, leaf number, and root weight were all significantly lower in soil treatments.

And root and shoot development are positively correlated. So roots uptake your minerals and water and leaves bring in your CO2 and the sugars and they combine and they produce cellular components necessary for growth. A study by Rona Wade et al. 2017 also investigated soilless systems and found that they outperform soil on plant yields.

So why might soil plants and soil systems have produced less leaf and root tissues? These plants didn't bolt. Spinach are really good at telling you if they're experiencing heat stress, they will bolt.

So it wasn't likely heat stress. The smaller root and shoot weights could be due to water stress. The soil treatment had a significantly lower calcium concentration than the aeroponic treatments. And the movement of calcium depends on transpiration through the xylem, which doesn't occur efficiently in a water stress plant as they close their stomata.

And calcium is essential for cell division and elongation, and insufficient quantities can result in slower growth or stunted growth. And then the binding site for calcium is also in competition with the binding site for potassium, of which soil also had the highest amount or the most significant amount. And... This could have resulted in calcium deficiency symptoms, which would have been necrosis. But aside from being small, the plants didn't have necrotic symptoms.

They were healthy in appearance. But the high levels of potassium during times of water stress could also have provided a temporary benefit of maintaining turgor pressure. So making the plants look like they're nice and happy and healthy. So to revisit our findings.

The community composition of fungi, these little guys right here, varied between the treatments, but not the tissues, whereas bacterial communities vary between tissues. This could be attributed to the ability of fungal hyphae to, again, penetrate those internal structures easily, whereas bacteria... require a vector or a weakened immune system to access those internal structures.

Fungal and bacterial richness significantly differ between tissue types, indicating that endophytic, so internal, and epiphytic environments provide those distinct habitats affecting colonization and efficacy. The application of mycorrhizal fungi during germination significantly improved germination rates and early stem growth, likely due to the production of essential phytohormones like indolecetic acid and dibrelin. Alternaria alternata was the only pathogen specific to spinach detected in the samples with the highest prevalence in the regular aeroponic treatments. Aeroponic treatments had significantly higher leaf and leaf number and leaf weight and root weight, and more leaves equals more food, more people fed.

So it's a great thing. And then treatments varied in their mineral content with soil having the highest nitrogen, potassium, phosphorus and sulfur, but significantly lower sodium, copper, boron and calcium levels. While the aeroponic systems did not outperform the soil on certain minerals, they did produce an overall enhanced nutrient acquisition and the continuous water supply provided to the roots by the aeroponic systems could be responsible.

making them less susceptible to drought, and then facilitating that nutrient uptake. So when selecting a growth system for a population such as the Upper Peninsula, it would be crucial to consider the nutritional needs of that population as well. And this project demonstrates the potential benefits of adding organic amendments and plant growth-promoting microorganisms to enhance yield, nutritional quality, pathogen resistance of aeroponically grown spinach and could play an important role in sustainable agriculture and regional food insecurity, especially in cold climates such as the Upper Peninsula.

Are there questions? Thank you for listening. Okay, thank you, Veronica. I think I did see a couple of questions come through the chat, so why don't we start with those first.

So one of the questions was how much would a system like this cost when you put together your aeroponic system? Do you have an estimate of what that cost? Yeah.

So the system that I used that you saw in there costs approximately $425 to build. And a lot of that cost comes from the light itself. But you can purchase these systems from... like Amazon or a local growth store. And they range from $125 without lights.

So again, lights is one of the factors that you need to consider up to $650 with lights, but it's a prebuilt system. So you could bring that cost down if you want to like build your own system. So you can buy one of those $125 systems, pay like $260 for some lights, and we're still saving money. It is, again, expensive. That is kind of expensive for people, though.

Okay, another question was, what was the timing of the aeroponic, the misting timing? Okay, yeah, so they were on, so they were spraying the roots. for two seconds and then they would shut off for five seconds and just repetitive on for two off for five the entire time that they were growing okay um another question from carrie nice job she says i may have missed this but do you have any idea uh of the microbes that came from the vermicompost tea i do not That's an awesome question and definitely something that we need to look at or future studies need to look at because analyzing the microbes was very expensive.

It was about $1,500 to analyze to send them to the University of Minnesota. And so I would have loved to analyze the vermicompost tea to see what microbes were getting added so that I could maybe... do a little bit more analysis with that because then I would have an idea of what I started with more and how maybe that out-competed what I was trying to add. So we never had the actual vermicompost itself analyzed initially. Correct.

But then with your tea brewing, I mean, the study, the results that you were showing included the microbes coming from the tea. It wasn't because you inoculated, what, 12, 11 microbes that we knew. that we inoculated.

Yes. Okay. I think that's it in the chat for now, but if other people want to type in that's I'm monitoring the chat.

Are there any, any other questions you can either send it via chat or just unmute yourself and ask? I guess I have a question. So I know as part of your plant growth promoting microorganisms, the one potential human pathogen in there was the Klebsiella erogenes. Why was that one specifically added? I was just curious.

Yeah, okay. So the Klebsiella... Thank you, Josh, for this question. uh clubziella which was when i did my study formally uh enterobacter orogenes so it's been a minute because it was recently reclassified um that one can help produce uh indolecetic acid and uh gibberellins and then can also catabolize excessive hormones like that and then they also help produce those iron chelating agents so they can sequester iron from the environment and they can solute all eyes inorganic phosphates and they can do some biological nitrogen fixation so they It was selected because of those attributes, but moving forward, it could be removed because there are a lot of other microbes in that selection that produce the same effects.

So moving forward, that one would be excluded, especially if we want to look at making the system available to the public. Yeah, that was one of the reasons I was asking that because, you know, someone wanted to, you know, reconstruct these systems and it's going to be easier for... someone to get a hold of you know bsl1 you know organisms and two would be a potential problem yeah and where did you get your mycorrhizal fungi from uh so the uh fungi came from commercial inoculants uh so the trichoderma uh were together in i believe uh root shield plus and you can just order that on amazon or pick it up at the store i'd like a like a hydroponic store and the glomus species were i think dynamico um again just ordered that um there's there's a lot of um There's a lot of beneficial microorganisms available online, which we could use ones that only could be ordered and that would improve the study so that it could be actually usable by the public.

And when I looked at it, you can get your vermicompost, your mycorrhizal fungi, so there's Trichoderma anglomus, the streptomyces, both bacillus strains, and Lactococcus from Amazon. all of those can come from Amazon. Then the erbis corillium and the pseudomonas must be, I could not find those as in any mixture or as individuals through any site that provided beneficial organisms.

So like moving forward, it would be interesting to see if there's a difference if we took out those ones that people don't have. access to, and especially removing that Klebsiella immediately. And then the herbus beryllium and the pseudomonas, seeing what changes we might see without those versus with those. Okay. And then another question came in via chat.

Was the compost tea a foliar spray or a reservoir additive? It was a reservoir additive, and then it was sprayed onto the roots. for the aeroponic system. And then for the soil, it was like mixed with the water and then watered on as needed. So it could have gotten on the leaves.

It was meant as a soil like application though. Right. Okay.

Okay. Well, it's getting close to the hour being finished. So I think we'll wrap up the questions for now. And I'm going to ask everyone who came in for the seminar, if you could please leave. Thank you for attending.

Thank you. And Veronica will stick with her committee here and we'll continue on.

Transcript for:Veronica's Research on Aeroponics

Transcript for:
Veronica's Research on Aeroponics