Impact of Nitrogen Fertilization on Arbuscular Mycorrhizal Fungi Abundance in Association with Panicum Virgatum

Growing concerns about environmental degradation and the limited supply of fossil fuels have led scientists to investigate the possible use of biofuels for energy. Switchgrass (Panicum virgatum), a C4 perennial prairie grass, is an ideal plant to be used as a biofuel due to its widespread geographic distribution (Elberson et al., 2001; Jefferson et al., 2002), ability to withstand acidic environments (Bona & Belesky, 1992; Hopkins & Taliaferro, 1997; Stucky et al., 1980), high nitrogen utilization efficiency (Brown, 1999), and ability to recycle nitrogen and other nutrients (Chapin, 1980; Clark, 1977; Heckathorn & Delucia, 1996; Killingbeck, 1997).

However, use of switchgrass as a biofuel will require the cultivation of large amounts of biomass. Unfortunately, the common agricultural method of increasing biomass through the use of inorganic nitrogen (N) fertilizers has not proven continuously effective in promoting the growth of switchgrass. Although some studies have shown increased shoot and root dry weight with the addition of N fertilizer (Shroeder-Moreno et al., 2012), more studies have shown varying results when N is applied (Ma et al., 2001; Muir et al., 2001; Suplick et al., 2002; Vogel et al., 2002), which may be a result of differences in soil or site-specific agroecology (Parrish & Fike, 2005). A study by Jach-Smith and Jackson (2015) concluded that N fertilizer cannot be considered a sustainable method of increasing switchgrass yield due to its variable responses.

An alternative method of increasing plant yield is the use of symbioses between switchgrass and arbuscular mycorrhizal fungi (AMF). An estimated 90% of all plants have symbioses with AMF (Smith & Read, 1997), which supply carbon to the fungi in exchange for nutrients such as phosphorus and nitrogen (Finlay, 2004; Rillig & Steinberg, 2002). The presence of AMF is also thought to be important in maintaining carbon within the soil through the creation of water stable aggregates (Rillig & Steinberg, 2002; Wilson et al., 2009; Wright & Upadhyaya, 1998). AMF may also have the ability to recycle N within the soil (Shroeder-Moreno et al., 2012). The presence of AMF has been shown to increase primary production (van der Heijden et al., 1998) and increase shoot dry weight (Shroeder-Moreno et al., 2012). Specifically in switchgrass, the presence of AMF has been shown to increase production and stress resistance (Al-Karaki et al., 2004).

It is possible that these two methods could be combined for optimal biomass yield of switchgrass. Some research has been conducted to examine the effect of N fertilization on AMF presence in soil; however, results have been variable. Some researchers have witnessed a decrease in the presence of AMF in response to an increase in N (Egerton-Warburton & Allen, 2000; Treseder & Turner, 2007) or the increase of AMF with a suppression of N (Oehl et al., 2004), possibly as a result of fertility directing photosynthate away from AMF (Mosse & Phillips, 1971) or by increasing competition among AMF (Liu et al., 2014). Still, other research has shown variation in AMF abundance in response to N application (Chen et al., 2014; Qin et al., 2015; Treseder et al., 2007; Wuest et al., 2005). One researcher even found an increase in AMF abundance in response to N presence (Wilson et al., 2009).

There has been little research specifically into the growth of switchgrass using either inorganic fertilizer or AMF symbiosis. The purpose of this study is to examine the effects of nitrogen application on the abundance of AMF in association with switchgrass. I predicted that increasing N in the soil would increase presence of AMF within the soil. To test this hypothesis, samples from eight treatments of nitrogen from two years were examined for presence of glomalin related soil protein and extra-radical hyphae within the soil to determine AMF abundance. Results from this study will provide key information to understanding methods through which I can promote growth of switchgrass to be used in the production of biofuels.

Methods

2.1 Sample Treatment

This study is part of an ongoing experiment at the Intensive Site of the Long-Term Ecological Research plots at the Kellogg Biological Station operated by Michigan State University. Switchgrass is grown in four replicate blocks each consisting of eight 15.2 m by 4.6 m plots laid in a randomized split-plot design differing by nitrogen input treatment (Table 1). Samples for this study were collected in May 2014 and May 2015.

2.2 Glomalin Extraction and Analysis

Glomalin is a glycoprotein secreted by the AMF living within the soil. Therefore, a measurement of the concentration of glomalin within the soil reflects a measurement of the abundance of the AMF within the soil. Glomalin was extracted from 1.00 g of soil using 8 mL of 50 mM sodium citrate (pH = 7.0) for a measurement of glomalin related soil protein (GRSP). The extractions were autoclaved for 60 min at 121°C and then centrifuged at 5,000 x g for 10 minutes. The extraction was performed 3 or 4 times until the solution was straw-colored. The supernatant was collected and stored at 4°C. GRSP analysis was completed using a Bradford Assay with a kit from Thermo Scientific. A BioTek ELx800 plate reader was used to read the GRSP at 595 nm using a standard of bovine serum albumin.

Each extraction was read using four pseudo-replicates. Readings were revised to eliminate any pseudo-replicate readings that fell above or below one standard deviation from the mean. A series of ANOVAs were performed using GRSP as the dependent variable and treatment group as the independent variable. Significance was determined at P<0.05.

2.3 Extra-Radical Hyphae Extraction and Analysis

Extra-radical hyphae (ERH) are filamentous structures of fungus. Therefore, a measurement of the total length of ERH found within soil reflects a measurement of the abundance of AMF within the soil. ERH were isolated using 20 g soil in 500 mL of water decanted through a 3’’ sieve set of 710 μm and 212 μm. Isolated ERH were stained in 10 mL of 0.05% trypan blue for 60 min. ERH were stored in distilled water at 4°C. Slides were created from 20 mL aliquots of stained ERH suspended in 200 mL of water.

Slides were analyzed using two pseudo-replicates at a magnification of 100x using 25 fields of view, as per the VET method (Boddington et al., 1999). Hyphal length was determined using the equation by Tennant (1975). A series of ANOVAs were performed using ERH as the dependent variable and treatment group as the independent variable. Significance was determined at P<0.05.

Results

3.1 Effects of treatments on glomalin related soil protein

Nitrogen input did not have any significant effect on the concentration of GRSP, as seen in Table 2. Figure 1 shows a downward trend between treatments F3 and F6 followed by an upward trend between treatments F6 and F8 for the year 2014 as well as a downward trend between treatments F3 and F5 followed by an upward trend from treatment F5 to F6 in the year 2015; however, the large error values in the data suggest there is no difference between the treatments and the control. Table 2 shows no significant difference in GRSP between years. Figure 1 suggests a trend for a slight decrease in total concentrations from 2014 to 2015.

3.2 Effects of treatment on extra-radical hyphae length

Nitrogen input did not have any significant effect on the length of ERH, as seen in Table 2. Figure 2 shows that ERH length remained relatively stable between treatments for both 2014 and 2015. The large error values in the data suggest there is no difference between the treatments and the control. Table 2 shows a significant decrease in ERH length from 2014 to 2015 (p<0.000).

Discussion

According to the results of this study, N application has no effect on the abundance of AMF within the soil. These findings support the work of several previous studies, such as the findings of Wuest et al. (2005) that found EE-IRSP and IRSP, alternative glomalin abundance metrics to GRSP, were not affected by N fertilization within an agricultural system.

However, these findings are in opposition to the findings of Wilson et al. (2009) that suggested an increased production of fungal structures, such as ERH, in response to N input, the findings of Oehl et al. (2004) that found an increase in AMF abundance in the absence of N, and the findings of Egerton-Warburton and Allen (2000) that found a decrease in abundance of AMF in the presence of N. Still, these studies do not necessarily need to be viewed as contradicting.

Within one study by Treseder et al. (2007), IRSP was seen to increase, decrease, and remain unaltered in response to the presence of N within different types of forests. Given that different species of AMF colonize with different species of plants, it is reasonable to conclude that the variation in responses of AMF abundance to N input are due to the fact that each experiment examined a different species of plant, and thus different species of AMF. Thus, it could be possible that the AMF species that colonize switchgrass roots are resistant to change in N while other AMF species are not. The results from two other studies, Avio et al. (2013) and Chen et al. (2014), indirectly support this hypothesis.

Respectively, these studies found that N input had no effect on GRSP or AMF abundance however both did find a change in AMF community composition. This idea could further be influenced by competition for nutrients within the soil, leaving only the fittest AMF species that are able to withstand changes in N to reproduce and expand. It has already been suggested that introducing nutrients to soil increases competition between AMF and the host plant (Liu et al. 2014).

Perhaps within each treatment group, by the time the soil samples had been selected only those fittest AMF that were able to withstand competition with the switchgrass remained, reaching some sort of carrying capacity within the soil which is equal to that found within the control treatment. To determine if this hypothesis is correct, future studies should explore AMF diversity within the soil, measuring the AMF prior to treatment, directly after treatment, and several months after treatment to see if there is any response in community composition. Future studies should also examine the possibility of a carrying capacity within the soil for AMF.

If these suggestions are correct, this means good news for the sustainable production of switchgrass to be used as a biofuel. A major concern among researchers today is the reduction of soil pollution caused by the excessive use of N fertilization in conventional agriculture. The use of fertilizers in agriculture, particularly in the large-scale agriculture that will be necessary to grow switchgrass as a biofuel, often leads to N pollution (Drinkwater & Snapp, 2007), which can lead to detrimental effects on plants, soil, water, air, and human health (Galloway et al., 2003; Galloway et al., 2008; Schlesinger, 2009; Johnson et al., 2010).

Although we did not witness an increase in AMF abundance, as predicted, the ability of the AMF colonizing the switchgrass to remain constant throughout different treatments of N suggests that the AMF may be used in agriculture to counteract the effects of N pollution in the soil. Overuse of N fertilizers can lead to soil acidification (Galloway et al., 2003), which results in a loss of nutrients available in the soil.

However, the presence of AMF is known to increase in the availability of nutrients within the soil (Wright & Upadhyaya, 1998; Rillig & Steinberg, 2002; Wilson et al., 2009) and recycle N within the soil (Shroeder-Moreno et al., 2012). In fact, AMF have already been used to restore nutrients to depleted landscapes, such as the experiment that was completed by Báez-Perez et al. (2010) to increase carbon availability to plants within nutrient deficient tepetates. The relatively stable abundance of AMF within the soil in response to N treatment suggests that the AMF within the soil were able to consistently carryout their life functions, those functions which result in the production of water stable aggregates within the soil which cause carbon to become sequestered.

If that is the case, then perhaps in addition to using AMF as a restoration tool to promote increases in nutrients within depleted soils we can use the AMF to maintain the structure of the soil while fertilization is taking place. If possible, agriculturalists could use as much nitrogen fertilizer as necessary to cause optimal growth of the switchgrass plants while the AMF colonizing those plants continue to recycle that nitrogen and replenish nutrients, eliminating fear of destroying soil fertility in the process.

To determine the validity of this assumption, future researchers should collect data about nutrient availability within the soil prior to fertilization and several months after fertilization. Researchers should also attempt to measure factors such as nitrogen runoff that could have detrimental effects on the surrounding environment to determine if the presence of AMF can truly decrease environmental degradation caused by nitrogen use.

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Acknowledgements

I would like to thank Dr. Sarah Emery of the University of Louisville and the members of her lab for guiding me in this project. I would also like to thank the researchers at Kellogg Biological Station for maintaining the study sites necessary to carry out this experiment. This research was funded by an NSF REU grant.

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