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Effects of Soil Microbes on Soil Health and Plant Productivity

CHAPTER 1- Introduction

A.      Approx. 2-10 pages – explain the purpose of the thesis

B.      Bulk soil  rhizosphere  roots  endophytic microbes

Within the soil, the immediate regions surrounding the plant roots (rhizosphere), is a microbial “hot spot” which is considered to be one of the most dynamic borders on the earth (Philippot et al., 2013).  The microbial community which inhabits the rhizosphere partakes in a complex food web though acquisition and utilization of nutrients released by the plant and found within the surrounding soil (Mendes et al., 2013).  The rhizosphere is divided into three distinct zones, which include the endorhizosphere (root tissue), rhizoplane (external region of roots), and ectorhizosphere (soil surrounding the roots).  In many studies these three regions are consider one region –  the rhizosphere (Huang et al., 2014). The plant selection and soil type are major driving forces which regulate the microbial population, diversity, and activities within the immediate locality of the plant root system (Mendes et al., 2013).  The foremost source of species diversity and richness is received though bulk soil, the soil which is neighboring to the rhizosphere, through changes occurring in the bulk soil such as land-use changes which affects assembly and the final composition of the rhizospheric community (Mendes et al., 2014).  Plants and microbes have evolved symbiotic relations that enable them to coexist together and utilize symbiotic relations/interactions to survive (a)biotic stressors (Nihorimbere et al., 2011).  The interactions between a plant and its environment is a dynamic process. Previously researched spent minimal time focusing on the beneficial effects of plant-microbe interactions, while the majority of the research focused on the actual plant. However, in recent years, the plant microbiome has gained a significant amount of attention, as it has influence on both the plants health and productivity (Lakshmanan et al., 2014).  A soil microbiome is a unique collective term used to describe the microbes (fungi and bacteria) and small animals (amoebae, insect larvae, mites, nematodes, and protozoa) that reside in particular environment (Bronkowski et al., 2009).  A plant microbiome includes a diverse gene pool that has originated from viruses, prokaryotes, and eukaryotes which are associated with various habitats on, within, and neighboring the host plant (Lakshmanan et al., 2014).  The interaction that occurs between a host plant and neighboring microbes are dynamic processes, in which the plant monitors the adjoining environments and responds accordingly to the present of (a)biotic stressors (Fig. 1) (Chaparro et al., 2012).  While many previous experiments have attempted to simplify plant-microbe interactions these interactions are dynamic process which include a vast diverse assortment of microbes which produce synergistic effects which cannot be simplified (Mendes et al., 2011).  Earlier studies have shown that the soil characteristics play an important role in the determination of the arrangement and functions of the inhabiting bacterial and fungal communities (Lauber et al., 2008).  While former reports have shined light on these attributes, there still remains a great deal of scarcity in the comprehensive knowledge concerning specific aspects which have influence on the microbial communities (Liang et al., 2012). With the recent advancements in “omics” research, these techniques can help shed light on the taxonomy, function, and how these interactions relate and influence one another (Morales and Holben 2011). Developing a better understanding of these aspects will provide comprehensive information for the use in agroecosystem’s management including environmental impact assessments (Liang et al., 2012).
The interactions, both passive and active, which occurs between a plant and its surroundings is considered a dynamic process, where the plant monitors its surroundings and responds accordingly to the stressors.  Previously, it was believed that the root system only provided anchorage/stability and the uptake of water and nutrients, however it has now been determined that it is one of the crucial components in the interactions/communication between plants and microbes (Chaparro et al., 2012).  Plants and microbes communicate though chemical signaling, microbes release chemical signals which are received by the plants and then respond though the release of root exudates (Chaparro et al., 2012). Secretory compounds will vary depending on the plant species and requirements; as plants require unique sets of microbes which will vary depending on development stage or stressor.  A diverse range of exudates released by the roots assists in the development of a unique environment within the rhizosphere and within the plant system, through the release of sugars, amino acids, flavonoids, aliphatic acids, proteins, and fatty acids (Badri et al., 2009).  The chemicals which are released from the roots (root exudates) can interact with the surrounding microbes and initiate communication, symbiotic or pathogenic interaction with the microbial population in the surrounding soil (Bais et al., 2006).  The concentration and composition of root exudates will vary depending on the signals received by the rhizospheric microbiota, environment, soil type, (a)biotic, and age of the plant (De-la-Pena et al., 2010). Rhizospheric microbiota identification through “omic” techniques can provide a glimpse of the stressors which a plant is experiencing, through microbial identification.

Figure 1: Visual representation of the plant-microbe interactions and complexity surrounding these interactions. Microbes can confer beneficial effects to plants through symbiotic relations assisting the plants in obtaining nutrients and increasing stress tolerance (Coleman-Derr & Tringe, 2014).
The first objective of this research is aimed to evaluate the similarities and dissimilarities in the taxonomic and functional capability of bulk soils, ectorhizospheric, and endorhizospheric microbial profile within wild forest lowbush blueberry and field managed lowbush blueberry systems.  The second objective of the project is to isolate and identify potential nitrogen fixing, phosphate solubilizing, flavin secreting microbes from the endorhizosphere in managed fields and forest locations. I hypothesis the plant microbiome associated with wild forest blueberry systems will have a higher diversity and functionally capability of beneficial microbes in comparison to field managed blueberry systems.
This research is aimed to develop an alternative method that complements the improvement of the plant’s fitness and resistance to abiotic and biotic stresses.  In doing so, this research has the potential to increase wild blueberry production efficiency, thought the manipulation of the soil microbial communities into a more sustainable agricultural practice.

Chapter 2: background (related work)

1.       Approx. 20 pages – provide a summary of the subfield you are working in. To provide some context for the reader to put your work in.  Discuss related work done by other researchers, and explain how yours relates to it.

2.1 Wild (lowbush) Blueberry (Vaccinium angustifolium Ait.)
Wild (lowbush) blueberries (Vaccinium angustifolium Ait.) are a perennial, rhizomatous, cross-pollinating shrub, that is native to Atlantic Canada and one of Nova Scotia’s largest agricultural commodities (Debnath, 2009).  Farmers will rents bees to ensure cross pollination of the downward facing bell shaped flowers (McIsaac, 1997).  In 1980 3.8 million kg were produced and in 2015 29.5 million pounds were produced in Nova Scotia (Food Institute Report –January 21, 2016). The plants have adapted to handle the low fertile sandy acidic soils of northeastern North America.  The ideal conditions for growing blueberries are on sandy well-drained acidic soils, with sufficient organic matter (5-10%), with a pH range from 4-5 (Yarborough, 2012).  While these growing conditions tend to be less ideal for many other crops, lowbush blueberries cannot be planted like conventional crops (potatoes, maize, and soybean).  Thus, this can become one of the many challenges producers face as they are not capable of crop cycling (Drummond et al., 2009).  The use of abandoned farm land with natural wild blueberry plant is ideal for production, as farmers do not have to clear land of stumps, roots, trees, weeds, and other plant material allowing the wild blueberries rhizome (runners) to give rise to new roots and shoots (McIsaac, 1997). The greater the density of the initial wild unmanaged plant the less time it will require to produce new crops (McIsaac, 1997).
Blueberries have shallow fibrous root system with roots attached to rhizomes, mature plants require approximately 25mm of water during the growing and harvesting season and a porous soil.  Wild blueberries thrive in soils that are loose and ideally with no competing weeds.  Since mature plants only reach 0.5m high, it becomes important to increase plant density to ensure maximum yield (Yarborough, 2012).  The majority of the root system can be found within the first 10cm of the soil horizon, porous loose soil is required to allow roots to venture out, as they do not reach deep distances within the soil. (Rellan-Alvarez et al., 2016; Jeliazkova and Percival 2003b). Wild blueberries have adapted to landscape where natural disturbances such as forest fires and lightning strikes can occur (Drummond, et al., 2009).  This is made possible due to only 30% of the plant’s biomass being aboveground, while the remaining 70% of the biomass is found belowground, allowing quick regeneration of the aboveground shoots and leaves (Drummond et al., 2009).  Unmanaged plant’s rhizomes will grow roughly 8 cm per year, while in managed plant’s rhizomes will grow roughly 38 cm in a single year (McIsaac, 1997). The rhizomes provide carbohydrate reservoirs and lateral water transport for the plant, allowing blueberries to adapt to unfavorable conditions such as drought (Jeliazkova and Percival, 2003a).  Since the crops are derived from native wild plants, the farmers are capable of advertising as their crops as “wild” blueberries.
Blueberries are grow on a two-year production cycle (sprout and crop year) most fields are divided into both vegetative and harvesting so ensure yearly crop production. Mowing is performed in the fall post harvesting of the berries which is then followed by fertilizer application in the spring of the sprout year (Chang et al., 2016). Bare spots and weed coverage can vary from 30-50% within juvenile wild blueberry fields (Zaman et al., 2008).  The application of fertilizers and herbicides application occurs in spring of vegetative year (Percival and Sanderson 2004). The leaf and floral buds are produced during vegetative year (McIsaac, 1997).  On newly development shoots the ratio of floral buds to leaf buds is greater and more winter hardy in comparison to that of older shoots, thus farmers can drastically increase their yields (McIsaac, 1997).  After the initial pruning of the fields, applications of herbicides are normally required, spot treatment may be also required (McIsaac, 1997). Weeds have become a major limiting factor, the use of specialized herbicides has allowed for the increase of fertilization, pollination, and irrigation, which overall has increased production four-fold (Yarborough, 2004).  Previous studies have shown that the use of fertilization can improve the yield within conventional fields when N and P levels are below 1.6% and 1.3% respectively within the leaves (Drummond et al., 2009).  Fertilize application when N/P levels are adequate can stimulate the growth of weeds and will not improve the overall yield of the blueberries (Drummond et al., 2009).
2.2. The effects of host a plant on the soil microbiome

o        Signaling for microbes (root exudates)

Relationships created with the microbes

What benefits do the plants provide for these microbes?

The soil matrix is the main reservoir for potential microbes used to create each plant’s unique rhizospheric microflora.  Soil microbes compete for nutrients and other resources which are sparsely found within the soil system, due to these resource limitations bacterial proliferation in the soil tends to be slow (Doornbos et al., 2012).  However, the microbial activity within the soil is greatly influenced by the plants root system.  Plants use their root system to explore sub-terrestrial world, obtain nutrients to sustain growth and development, reservoir for carbohydrates, and communicate with their environmental partners and rivals through the release of exudates (Vacheron et al., 2013; Rellan-Alvarez et al., 2016). The roots play a significant role in nutrient and water uptake, anchorage, and nutrient storage from photosynthesis.  Many of the factors which affect root growth and physiology arise from the structure of the soil at the microscopic level (Rellan-Alvarez et al., 2016).  The soil type and texture can affect the roots ability to obtain nutrients and explore the sub-terrestrial world. Soil type will determine pore size as well as the ability for water to flow through the soil (Rellan-Alvarez et al., 2016). Root exudates have close ties with the surrounding microbial population (Chaparro et al., 2014).  The roots release carbon containing metabolites into the soil matrix resulting in rhizodeposition (Doornbos et al., 2012).  Rhizodeposition releases on average 5-21% of a plant’s photosynthetically fixed carbon however, in some cases they can release up to 40% of the photosynthetically fixed carbon this includes the shedding of root cells and exudates (the secretion and leakage of secondary metabolites sugars, organic acids, amino acids, and proteins) into the soil (Huang et al., 2014; Doornbos et al., 2012).
Microbes can utilize rhizodeposite compounds released by the roots resulting in an increase in microbial biomass within the rhizospheric regions in comparison to bulk soil region. This allows for the generation of microbial communities specific for each plant and developmental stage (Huang et al., 2014). Previously, roots exudation was believed to be a passive process however, recent research has suggested that the ATP-binding cassette transporters in the roots which are involved in the translocation of phytochemicals into the rhizosphere, indicates that this process is actively secreting metabolites into the environment (Badri et al., 2008).  A wide variety of plants have specialized root cells that contain mitochondria, Golgi stacks and Golgi derived vesicles, all of which indicates active secretion of metabolites by theses specialized root cells (Vicré et al., 2005).  These cells were designated as border cells, detaching from the root and becoming entangled into the mucilage layer surrounding the surface of the roots (Hawes et al., 1998).  The proposed functions of the border cells are attracting beneficial microorganisms, reduction of sensitivity to heavy metals, and entrapment of pathogenic bacteria and nematodes within the mucilage layer surrounding the roots (Hawe, 1990)
The plant-microbe interactions differ between plant species and results in different rhizospheric communities.  Smalla et al. (2001) observed bacterial rhizospheric communities within strawberries, oilseed rape, and potatoes for two years using culturable-independent fingerprinting methods (Doorbos et al., 2012).  DNA analysis was completed using denaturing gradient gel electrophoresis (DGGE) showed that the plant-dependent shift was relative to the abundance of bacterial population within the rhizosphere which in the second year of growth became more pronounced (Smalla et al., 2001).  The DGGE analysis of the oilseed rape and potato rhizosphere results showed similarities within the rhizospheric communities in comparison to that of the strawberry plants (Smalla et al., 2001).  In both years of growth, the seasonal shifts were seen in abundance and composition of the bacterial rhizospheric community (Smalla et al., 2001).  They also noted that gram-positive bacteria may be more dominant in rhizospheric regions (Smalla et al., 2001).  A study conducted by Fierer and Jackson (2006) analyzed close to 100 soil samples collected from across North and South America using DNA fingerprinting methods to compare composition and diversity of the bacterial communities across each site (Doornbos et al., 2012).  This research team found that the diversity of the soil bacterial communities differed by edaphic variability, particularly pH, while site temperature and latitude were of little influence (Doornbos et al., 2012).  They noted that in general, soils that were pH-neutral had a higher diversity when compared to acidic soils (Doornbos et al., 2012).  Such extensive molecular studies into soil microbial population diversity has been made possible because of machines such as PCR-fingerprinting techniques which is based on differences in nucleotide sequences of the phylogenetic markers, where small subunit 16S rDNA is primarily used, and widely employed (Doornbos et al., 2012)
There is increased evidence that shows that the quantity and quality of the exudate released into the soil is determined by the species of plant, developmental stage of the plant, stress factors, and various environmental factors (pH, temperature, and presence of microbes) (Huang et al., 2014; Latour et al., 1996).  A study conducted by Kamilova et al. (2012) on tomatoes, cucumbers and sweet peppers grown under gnotobiotic conditions on rock wool results showed that exudates released contained a higher amount of organic acids than sugars.  Of the organic acids citric, succinic, and malic acids were the major acids present, while fructose and glucose were the major sugars (Kamilova et al., 2006b).  The composition of the root exudates is also influenced by the rhizospheric microflora (Doornbos et al., 2012).  The application of bacterial biocontrols strain Pseudomona fluorescens WCS365 on the tomato plants resulted in an increase in organic acid levels, whereas there was a decrease in succinic acid (Kamilova et al., 2006a).  When the plants were inoculated with pathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici the fungus caused severe foot and root rot which then lead to decreased citric acid levels and increased succinic acid in comparison to control plants (Kamilova et al., 2006a).  When plants were induced with both WCS365 and the pathogenic fungus, the effects of the disease was decreased and the succinic acid content within the roots exudates was lowered compared to the pathogen infected plants (Kamila et al., 2006a).  The results of this study show that the availability and composition of the nutritional diet for the microbes within the rhizosphere is highly dynamic (Doornbos et al., 2012).  This study also shows that the root exudations is dependent on the erratic interactions between microbes, and the analysis of root exudates within a gnotobiotic system is the beginning to understanding the interactions and conditions of the rhizosphere (Doornbos et al., 2012).

2.3. Effects soil microbes have on soil health and plant productivity  effects microbes have on plant health

o        Discuss microbes being studied (intro of the importance of a diverse microbiome) role of the microbes in relations to the plants (ISR, SAR responses)

Nitrogen fixing

Phosphate solubilizing

Flavin secreting

Heat resistant fungi

Soil bacteria play an important role in the biogeochemical cycles and have been used in crop production for decades (Hayat et al., 2010).  Soil bacterial populations efficiently multiply rapidly and can utilize the wide range of nutrients within the soil and/or obtained from the surrounding plants with the concentrations of rhizospheric microbes being 100-1000 times greater than that of bulk soil microbes (Goswami et al., 2016).  Bacterial floras can be found in different regions of the soil; they are often attached to soil particles and roots systems.  The dispersal of these microflora will range depending on nutrient concentrations and locations, motility ability, and chemical signaling (from plant and other microbes). The interactions between plants and bacteria (within the rhizosphere) influence the plant health and soil fertility (Hayat et al., 2010).  Bacteria have the ability and versatility (metabolically) to adapt and utilize the root exudates.  The main functions of these soil bacteria are to (i) supply nutrients to the plant, (ii) stimulate plant growth, (iii) to control or inhibit the activity of pathogens, (iv) improve soil structure, and (v) the bioaccumulation or microbial leaching of inorganics (Brierley 1985).
Vessey (2003) indicated that there are many species of soil bacteria which thrive in the rhizosphere surrounding plants, they may grow within, on, or in close proximity to the plant root and shoot tissues; these bacteria have the ability to stimulate plant growth by numerous mechanisms these microbes are collectively known as plant growth promoting rhizobacteria (PGPR) (Jha and Saraf, 2015). PGPR have the ability to create a beneficial effect on the plant upon inoculation through contributions to sustaining plant growth and development.  PGPR aid through synthesizing specialized compounds for the plant, facilitating the uptake of specific nutrients from the soil, and aiding the plant in lessening or preventing the effects from pathogens (Hayat et al., 2010).  Roughly 2-5% of the rhizospheric bacterial population are considered PGPR (Antoun and Prévost, 2005).  Gray and Smith (2005) have suggested that the associated PGPR is dependent on the degree of bacterial proximity to the root and shoot systems.  The majority of the PGPR to date belong the genera Acinetobacter, Agrobacterium, Arthobacter, Azotobacter, Azospirillum, Burkholderia, Bradyrhizobium, Rhizobium, Frankia, Serratia, Thiobacillus, Psedomonads, and Bacillus (Vessey, 2003).  Within the last ten years, defining the role of the rhizosphere as an ecological niche, has allowed PGPR to gain more significance aiding research efforts in understanding the mechanisms of PGPR within the rhizosphere (Goswami et al., 2016).
2.3.1. Symbiotic N2-fixing Bacteria
Nitrogen is essential for all plants, it is a requirement for the synthesis of enzymes, proteins, amino acids, chlorophyll, DNA, and RNA.  Although nitrogen may be found in abundance within the environment, it is a limiting nutrient for plants as the abundance of nitrogen found within the environment is most commonly found as atmospheric di-nitrogen gas (an inert gas), containing a triple bond which consumes a great deal of energy to break.  This limited nutrient has been overcome by farmers through the application of nitrogen based fertilizers, however, the cost of application of these fertilizers has an excessive cost, both environmentally and economically.  Economically nitrogen fertilizers cost is greatly affected by the cost of breaking the triple bond of the inert gas. Although nitrogen fertilizers have the ability to providing plants with this difficult to obtain nutrient.  The downfall of these nitrogen fertilizers is the negative impact they have on the environment through run-off of excess nitrogen in the form of nitrates.
Bacterial strains which have developed the ability to fix nitrogen can be classified into two categories root/legume associated bacteria and free living bacteria (Goswami et al., 2016).  Root/legume associated bacteria infect the root system and produce nodules such as Rhizobium, while free living nitrogen fixing bacterial do not penetrate the plant.  For nodulating legumes, nitrogen can be obtained through the creation of symbiotic relations between the plant and nitrogen fixing microbes.  Microbes such as Rhizobia (species of Rhizobium, sinorhizobium, Mesorhizobium, Brandyrhizobium, Azorhizobium, and Allorhizobium) create symbiotic relations with legumes through the secretion of the signaling molecules (flavonoids), the microbes respond to these chemicals through the secretion of lippo-chitooligosaccharides (LCOs) this signals cell division (mitotic) in roots, thus creating nodules (Dakora 1995).  Free-living nitrogen-fixing bacteria have the ability to live in close proximity of the plants and the nitrogen fixed from the atmosphere which is not utilized by the microbes can be used by the plants, this relationship is describes as a non-specific and loose symbiosis (Goswami et al., 2016).  Some examples of free-living nitrogen fixing bacteria include Azospirillum, Azotobacter, Burkholderia, Herbaspirllum, Bacillus, and Paenibacillus (Goswami et al., 2015). Stacey et al., (1992) reported that the nitrogen fixing microbes in fertile soils have the ability to fix 20-30kg per hectare per year.  Species which belong to the genus Azotobacter and Azospirillum are the most commonly used nitrogen fixing bacteria in agricultural trials, with the first reported use in 1902 and has been widely used around the world to date (Goswami et al., 2016; Bhattacharyya and Jha, 2012).
2.3.2. Phosphate Solubilizing Bacteria
Phosphorus is one of the essential macronutrients for plants, for both growth and development (Jha and Saraf, 2015).  Much like nitrogen, phosphorus in its natural state is inaccessible to plants, as plants have the ability to absorb mono-, and dibasic phosphates (soluble forms of phosphates) (Jha and Saraf, 2015).  Thus, plant growth promoting microbes convert the insoluble forms of phosphorus (both inorganic and organic forms) to accessible forms for the plants to increase plant yield.  Maintaining plant-available phosphorus is essential for plants however, it is also crucial to avoid over exploitation of the soil natural phosphorus reservoirs, as this will lead to deficiency and low plant yield (Jha and Saraf, 2015).  Holford (1997) reported that there are 3 important soil components in controlling the supply of phosphorus from the labile pools to replenish the crop extractions (Jha and Saraf, 2015).  These include the concentration of phosphorus within the soil solution, the concentration of phosphorus within the replenishment source that enters the equilibrium of the soil solution phase, and the phosphorus buffering capacity of the soil (Jha and Saraf, 2015).
Phosphate solubilizing microbes mineralize phosphorus within the soil by solubilizing complex structured phosphates (tricalcium phosphate, rock phosphate, and aluminum phosphate) into accessible forms for the plants to uptake (Goswami et al., 2016). These bacteria use different mechanism(s) to solubilize the insoluble forms of phosphates, the primary mechanism used for solubilizing phosphates is based on the use of organic acid secretions to break down the phosphates by sugar metabolism by the microbes (Goswami et al., 2016).  Rhizospheric microbes obtain sugars from the root exudates; and through this metabolism organic acids are produced.  It has been estimated that the 20-40% of the culturable population of microbes has been found to be phosphate solubilizing microorganisms, with a significant proportion of these microbes can be isolated from the rhizosphere (Chabot et al., 1993).  The majority of the phosphate solubilizing bacteria isolated from the rhizosphere of various plants tend to be metabolically more dynamic than those located in other soil regions (Jha and Saraf, 2015).  Phosphates are reactive with calcium, iron, and aluminum this reaction tends to lead to a precipitation making phosphates unavailable to plants for uptake (Goswami et al., 2016).  Inorganic phosphates found in acidic soils associate with aluminum and iron molecules while calcium phosphates are associated with calcareous soils (Goswami et al., 2016).  Organic phosphates make up a large percentage of soluble phosphates which can be as high as 50% in the soil that contains high levels of organic matter (Barber, 1995).
In previous research bacteria belong to genera such as Achromobacter, Agrobacterium, Bacillus, Enterobacter, Erwinia, Escherichia, Flavobacterium, Mycobacterium, Pseudomonas, and Serratia are highly efficient at solubilizing complex phosphates into accessible forms (inorganic phosphate ions) (Goldstein 2000).  While strains from Pseudomonas, Bacillus, and Rhizobium genera are among the most powerful phosphate solubilizing bacteria (Arora and Gaur 1979).  While in recent research ectorhizospheric strains of PseudomonasBacilli, and endosymbiotic Rhizobium has been found as effective phosphate solubilizers, while Bacillus megateriumB. circulansB. coagulansB. subtilisP. polymyxaB. sircalmous, and Pseudomonas striata are referred to as the most important and effective strains (Govindasamy et al., 2011).
2.3.3. Other mechanism of plant growth promotion
Soil bacteria have the ability to convert atmospheric nitrogen gas into ammonia which is essential to a plants health and development these microbes play an essential role in nutrient cycling within the soil. The soil microbiome contains dynamic genera of bacteria, many which take part in nutrient cycling while some have the ability to help protect plants against diseases.  PGPR have the ability to aid the plants growth and development through direct and indirect mechanisms.  Root morphology can be manipulated through the production if secondary metabolites resulting in increased root surface area, siderophores production, antagonism to soil-borne pathogens, nitrogen-fixation, and phosphate solubilization (Hayat et al., 2010). While bacteria play an important role in plant growth, they are not the only microbial group which is beneficial to a plants growth and development.
Fungi are important within the soil as they are heavily involved in organic matter decomposition and nutrient cycling providing a wide range of nutrients to both the surrounding soil and plants (Hannula et al., 2014).  Mycorrhizal fungi form symbiotic relations with the roots of plants, the preferred niche for mycorrhizal fungi is within hypogeous plant organs (roots mostly) (Parniske, 2008; Bonfante and Anca, 2009).  Mycorrhizal fungi increase roots surface area as the hyphae act as an extension of the root system allowing for the increase in nutrient and water uptake (Powell et al., 2009; Nadeem et al., 2014).  Mycorrhizal fungi also increase plant growth, by providing phosphate and decrease negative effects from pathogens as they become entangled within the hyphae of the mycorrhizal fungi (Powell et al., 2009; Sykes, 2010).  Arbuscular mycorrhizae are the most abundant and widely distributed fungi to be found within agricultural soils (up to 70-90% of land species) and are important for the cycling of nutrients within the soil (Nadeem et al., 2014; Parniske, 2008).  For mycorrhizal fungi to provide and execute its beneficial effect on the plants it must first obtain carbon from the host plant, relations between plants and microbes are a give take relationship where a microbe infects the plant and obtains required nutrients from the plant but in the process releases nutrients the plant cannot obtain on its own (Makarian et al., 2016).
The two most common mycorrhizal fungi are arbuscular mycorrhizae (AM) and ectomycorrhizae (ECM) (Nadeem et al., 2014). AM are endomycorrhizae (part of their hyphae reside intracellular within the root) these fungi form symbiotic relations mostly with leafy green plants and commercially produced plants (Parniske, 2008).  While ECM reside intercellular within the roots and these fungi form symbiotic relations mostly with woody plants and trees.  AM fungi reside within the soil as spores until a host plant has been detected (Tkacz and Poole, 2015).  The extension of the AM fungal hyphae aids in the structural stability of the soil and the ability of the plant to acquire nutrients and water from its surrounding (Nadeem et al., 2014).  AM hyphae uptake approximately 80% of the phosphorus required by the plant, the hyphae can also provide the plant with the required macro- and micro- nutrients (K, N, Mg, Cu and Zn) in soils where the nutrients are present in a less soluble form (Nadeem et al., 2014). Ericoid mycorrhizae (ERM) are known to be associated with the V. augustifolium Ait, these endomycorrhizae are important for nitrogen uptake and may also supply the host plant with phosphorus (Goulard et al., 1993; Jeliazkova and Percival, 2003b). ERM are unique in their ability to adapt to stressful environments, these fungi have the ability to enhance the host plants fitness through the reduction of the stressors on the plants (Cairney and Meharg 2003). A study conducted by Jeliazkova and Percival (2003a) considering the effects of drought on the EMR associated with the wild blueberry suggested that the distribution of EMR stayed the same throughout the drought conditions.  The ERM were not affected by the drought conditions but an indication of ERM associated adaptive mechanisms that helped the wild blueberries withstand the drought conditions and other such unfavorable conditions.
While mycorrhizal fungi may form symbiotic relations with plants they also form loose or tightly associations with bacteria, which are most likely to play a role in the functions of the mycorrhizal fungi (Bonfante and Anca, 2009).  Garbaye research in 1994 was a game changer for this field as he introduced the word “helper bacteria”, in which the “helper bacteria” assist and support mycorrhizal establishment (Bonfante and Anca, 2009).  Since Garbaye’s research more research has been completed noting that the interactions between bacteria and fungi are much more dynamic and complex than expected and their interactions may be crucial in the ecosystem (Artursson et al., 2006).  Bacteria which associate with mycorrhizal fungi tend to colonize the extraradial hyphae, and in some fungi taxa reside as cytoplasmic endobacteria (Bonfante and Anca, 2009).  Mycorrhizal-bacterial community studies have revealed that there is a widespread repertoire of bacterial taxa predominantly of species from genera Pseudomonas, Burkholderia, and Bacillus,
while Streptomycetes have been associated with ECM fungi and been discussed as modulators of plant symbiosis and Archaebacteria thrive within the rhizosphere when mycorrhizal fungi are present (de Boer et al., 2005; Schrey and Tarkka, 2008; Bomberg and Timonen, 2007)
2.4.0. Profiling microbial communities
Historically the discovery of a new microbial species was achieved through cultivation and subsequent characterization of strains (Cardenas and Teidje, 2008). Recently, the methods developed for the detection of non-cultivable or difficult to culture microbes has become a revolutionary technique within the field of biology (Fadrosh et al., 2014). This has been enabled by recent advances in next generation DNA sequencing methods that include pyrosequencing and shotgun metagenomics, which have boosted scientific interest in understanding the complexity of microbial community in a wide range of environments (Shokralla et al., 2012). Next generation sequencing technology allows for sequencing of the whole genome, this provides aid in understanding the molecular genetic of many microbial species (Gupta et al., 2014).
Profiling of the culture-independent bacterial and fungal communities relies on the amplification and sequencing of the 16S and 18S ribosomal RNA (rRNA) gene (Fadrosh et al., 2014).  The 16S rRNA gene is present in all bacteria and archaea while the 18S rRNA gene within eukaryotic cells is a homolog to the 16S rRNA gene, 16S are components of the 30S ribosomal small prokaryotic subunits and 18S are components of the 40S ribosomal small eukaryotic subunit (Ouvrard et al., 2000).  Bacterial 16S and fungal 18S rRNA markers are frequently used to characterize the phylogenetic diversity and taxonomic composition of environmental samples (Chakravorty et al., 2007). The use of 16S rRNA gene profiling allows for large amounts of samples to be sequenced and examined in greater depth at a lower cost (Fadrosh et al., 2014). Sequencing cannot directly detect the metabolic or the functional capability of the organisms being studied (Langille et al., 2013).  However, bioinformatic methods such as PICRUSt (Langille et al., 2014) can allow 16S gene profiling to provide a prediction of the functional composition of the microbial community being studied similar to shotgun metagenomics, creating a more comprehensive picture.
Shotgun metagenomics provides a comprehensive list of the genes present within the given sample.  Unlike 16S rRNA gene profiling, shotgun metagenomics provides information about the functions of all the organisms being studied.  However, since the whole genome from each member in the microbial community is being sequenced the cost per sample is much greater in comparison to a single gene approach such as 16S gene profiling.  The development of metagenomic approaches provides an unprecedented level of access to the microbial genomes from many different environments, making it possible for the characterization of phylogenetic and functional diversity of difficult to cultured microbes from various biomes of interest (Kakirde et al., 2010).
Metagenomics provides information about the microbes present and their genomic potential while metatranscriptomics provides information into the activity though the measuring of transcript levels the microbes in the environmental sample (Mutz et al., 2013). The use of 16S profiling, metagenomics, and metatranscriptomics can be generated through several commercially available techniques such as: Roche 454, Illumina and ABI SOLiD (Steward et al., 2010; Van Vliet et al., 2010). Each one of these techniques have positive and negatives, however, access to equipment, facilities, and cost tend to influence the final selection.
2.4.1. Alpha and Beta Diversity
Alpha diversity can be defined as the diversity within a particular area, habitat, or ecosystem, which is expressed by the number of taxon or species (i.e. species richness or evenness) (Whittaker, 1972).  While beta diversity can be measured as the variation in microbial communities’ composition that occurs between samples (Whittaker 1972).  Beta diversity examines the changes in species diversity between the ecosystems being examined. Beta diversity counts the total number of species which are unique to each ecosystem being compared. Alpha diversity can be calculated using several different metrics such as Shannon and Simpson index. Simpson’s index is used to measure the number of species present and the abundance of each species, the index proportion value obtained ranges between 0 to 1 and assumes the importance of diversity within the given sample (Sea Grant Maryland, 2016).  Simpson’s index will measure the probability of the two individual (species) in the community belong to the same category (Sea Grant Maryland, 2016) Shannon index calculates richness (number of species) and the evenness (proportion) of each species within the environmental sample.  The index shows that as the richness increases, or the distribution becomes more even causes the increase in biological diversity (Sea Grant Maryland, 2016). Beta diversity can be calculated with phylogenetic diversity and non-phylogenetic diversity. Phylogenetic beta diversity uses a phylogenetic tree to measures the phylogenetic distances among the members within the microbial communities, while a non-phylogenetic beta does not require a tree and considers all organisms as independent entities (Graham and Fine, 2008).  Unique fraction metric (UniFrac) is a phylogenic method that is used to measure the phylogenetic distances between sets of taxa in a given phylogenetic tree (Lozupone and Knight, 2005).  Using rRNA as a marker (phylogenetic) which is capable of indicating the amount of sequence evolution that has occurred in the environment being studied (Lozupone and Knight, 2005).  UniFrac can be either weighted and unweighted, weighted provides appropriate weight to each sample, while unweighted considers each sample to be evenly weighted.  UniFrac can also produce a similarity matrix which describes the pairwise phylogenetic distances between sets of sequences from the given sample, which then can be place onto principal coordinate analysis (PCoA) (Lozupone and Knight, 2005). While Bray-Curtis is a non-phylogenic non-parametric multivariable method that is used to describe the dissimilarities between two samples by measuring the ecological distances (Anderson, 2001).  Bray-Curtis aims to calculate a distance matrix, selection of two reference points for to be used for each axis, and to project all samples onto the previously determined axis (Clarke et al., 2006).  Bray-Curtis assumes that the measurements which the samples are taken from are the same physical size and is measured on a scale of 0 to 1 where 0 the who samples have the same composition while 1 means they do not share the same composition.

2.5. Management practices and the implications of agriculture

o        What they are doing now and how it has improved from previous management practices

The costs of the management practices (fertilizers, pesticides, herbicides)

What fertilizers are being used and do they actually assist the growth of the plant or more benefit the weed production

The use of pollinators

Soil scientists are beginning to have a better comprehension of the dynamic interactions within the soil microbiome (East, 2010).  A recent study conducted by Zolla et al. (2013) showed that when Arabidopsis thaliana (seedling)planted in soils which were previously exposed to A. thaliana, corn, and pine; allowed the seedlings to grow more vigorously in soils exposed to drought conditions (Zolla et al., 2013).  The majority of microbes that were identified through pyrosequencing were members of the phyla Actinobacteria and Proteobacteria which have been previously reported to aid in abiotic stress conditions (Zolla et al., 2013; Mayak et al., 2004). The use of microbiota transplantation (that colonize other species) has the potential to increase the stress tolerance of food crops (East, 2010; Zolla et al., 2013). The goal of the U.S National Regional Council is the development of a farming system that can be productive profitable, energy conservative, environmentally sound, conservative of natural resource and ensures the food safety and quality (Lakshmanan et al., 2014).  The Council has suggested that the use of pre-selected beneficial microbes as a replacement for hazardous agrochemicals shows promising effects through the nutritional benefits to the soil, plant, and livestock as well as providing protection from biotic and abiotic stresses (Lakshmanan et al., 2014). Mendes et al. (2011) analyzed and compared the microbiomes of disease suppressive and conductive against Rhizoctonia solani within the soil of sugar beets (Lakshmanan et al., 2014). The soils were subsequently treated for the removal of suppressiveness or mixed to obtain six different types of soil (based on PhyloChip-based metagenomic approach) (Lakshmanan et al., 2014).  They noted that there was no significant difference within the number of operational taxonomic units (OTUs) however, there was significance differences in the particular soil types.  The major microbial abundances found in the soil suppressants were - and -proteobacteria and Firmicutes (Lakshmanan et al., 2014).  These microbes were found in the host plant in higher concentrations during Rhizoctonia solani infection, inferring the possibility of a host-induced microbiome to combat pathogenic attacks (Lakshmanan et al., 2014).
While the application of chemicals (fertilizers, herbicides and pesticides) may assist plant growth and reduce pathogens, the harm comes from the potential side effects of decreasing beneficial soil microbial diversity and creating resistance pathogens (Krauss et al., 2011).  An alternative method using naturally occurring microbiota available in the soil, using derivatives, secondary metabolite, and microbes for the protection of crops including pest management, disease protection, plant productivity enhancement, and increase fertility (Schäfar and Adams, 2015). Krauss et al. (2011) showed that organic farming contributes to the maintenance of biodiversity and enhances the selection of microbial communities that can provide beneficial contributions to the microbiome and to the farmer through better top-down control of pest species. They also noted that the use of preventative insecticides on conventional fields had negative effects on the plant’s natural antagonists (Krauss et al., 2011).  Studies have been conducted on the use of microbes as a replacement for agrochemical, which has increased the microbial diversity within the soils and plant health and productivity (Krauss et al., 2011).
While the application of fertilizers (N, P, and K) is a routine procedure for any farmer these fertilizers are being over used within the developed agricultural community (Tkacz and Poole, 2015). Nitrogen fertilizers are used in agricultural practices to provide the plant with nitrogen, however, approximately 60% of the applied fertilizer is not absorbed by the plant (Tkacz and Poole, 2015).  The unabsorbed nitrogen fertilizers leach into groundwater and cause drastic changes the marine microbial population, thus affecting the whole marine food chain in the process (Tkacz and Poole, 2015).  Additionally, the application of nitrogen fertilizers halts the biological nitrogen fixation process (Omrane et al., 2009).  It has become crucial to understand how to improve plant growth and development without becoming solely dependent on expensive and environmentally damaging fertilizers (Tkacz and Poole, 2015).  During the vegetative state of blueberry development fertilizers are applied to the fields to assist in plant growth, blueberry have a lower fertilizer requirement in comparison to other berry plants (Starast et al., 2008).  Jeliazkova and Percival (2003a) looked at the effects N and P fertilizers had on the mycorrhizae within wild blueberries, they suggested that neither of the fertilizers used in the experiments significantly affected the ERM during both vegetative and cropping cycles.  They also noted that nitrogen had marginal increased effects of total stem length and numbers, total dry weight.  The use of fertilizers alone on blueberry crops is not ideal as the addition of fertilizers will help promote the growth of both the plants and weeds within the fields, thus the use of herbicides is required (Penney and McRae, 2000).
While many researchers tend to focus on the aboveground portion of the plant, insufficient understanding of the belowground region of the plant has become a limiting factor for the prediction of crop growth and productivity (Eissenstat et al., 2006). Previous studies have attempted to simplify the interactions that occur between the plant and the surrounding microbes, however these interactions are much more complex with a vast variety of microbes and symbiotic effects (Chaparro et al., 2012).  The roots, soil, microbes, surrounding plants, soil type, and pH are just a few important facts that must be placed into consideration when studying a plant, as they all play an important role directly or indirectly in the growth and development of a plant.  Susanne C. Brink in her editorial “Unlocking The secrets of the Rhizosphere” describe the relationship between the plant and microorganisms within the soil as a single entity a “super-organism”.  This is a very important approach in observing plant growth and development as it will allow researcher to look at a plant health, growth, and development from a different angle.  Allowing for the manipulations that will benefit both the plant and microbes rather than just the plant.  Treating these components as a single entity will allow for a better understanding of each separate component, as the environment surrounding the plant is complex entity, thus having more pieces to the puzzle will provide a clearer picture of “super-organism” (Rellan-Alvarez et al., 2016).

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