The beneficial effects of silicon application on plant growth and crop yield are well documented (for a recent review see Guntzer et al., 2012), but it is in the mitigation of both abiotic and biotic plant stresses, where the application of silicon demonstrates its real potential (Keeping and Reynolds, 2009). Notably, biochemical or molecular responses (and frequently growth/yield responses) due to silicon fertilization, are usually not apparent unless in the presence of a biotic (or abiotic) stressor. Studies have shown resistance to a range of abiotic stress factors including drought and salinity stress, heavy metal toxicity, excess nitrogen and phosphorous, and lodging (for a recent review see Liang et al., 2015). Biotic stressors may come in the form of plant pathogens, including fungi, bacteria, viruses, and animals (vertebrate and arthropod herbivores). Defense against biotic stress, has centered around two main mechanisms, mechanical (physical), and biochemical or molecular.

There is a dominance of work on fungal pathogens, compared with other disease-causing agents. Those fungal pathogens defined as biotrophic or hemibiotrophic, including the powdery mildews and blast fungus (Magnaporthe grisea (T.T. Hebert) M.E. Barr), appear to be better controlled by silicon than are necrotrophs (Liang et al., 2015). The reasons for this are increasingly becoming apparent, with a recent study showing that while silicon contributes to Arabidopsis defense priming following pathogen infection, that silicon will confer protection even when priming is altered, indicating other mechanisms may be involved (Vivancos et al., 2015). Evidence suggests that silicon may interfere with effector proteins released by these pathogens,

permitting the plant to mount better defense reactions (Vivancos et al., 2015). Other work has confirmed the role of silicon in priming plants in plant–pathogen interactions (Fauteux et al., 2005; Chain et al., 2009; Van Bockhaven et al., 2013). It is thought that the work on silicon and effector proteins may assist in developing a unifying theory around the mode of action of silicon in alleviating biotic stresses (Vivancos et al., 2015). A recent, comprehensive review of silicon and plant–pathogen interactions in agriculture is provided by Liang et al. (2015).

Vertebrate herbivores are probably the least studied biotic stressors, against which silicon provides some protection, and research in this area has largely focused on natural ecological systems. We briefly review this field because it has some relevance to arthropod pests given that plant defenses are at the heart of the phenomenon. The majority of studies have been on field voles, Microtus agrestis L (Rodentia: Cricetidae), showing reductions in the body weight and growth rate of juveniles and adults when fed on silicon-treated grasses (Massey and Hartley, 2006; Massey et al., 2008). Recent laboratory work demonstrated that grasses employ several defense strategies against M. agrestis including silicon, endophytes, and secondary metabolites (Huitu et al., 2014). It is hypothesized that induction of silicon-based plant defense in response to herbivore damage may influence rodent population cycles (Massey et al., 2008). In sites where M. agrestis population density was high, silica levels in the leaves of their food plant, collected several months later were also high and vole populations afterward declined, while population density increased where vole population density was initially low and silicon levels were also low (Massey et al., 2008). A key food species, Deschampsia cespitosa L., of M. agrestis exhibits a delayed defensive response to grazing by increasing silica concentrations (Reynolds et al., 2012). Further, the authors presented theoretical modeling that predicts that this response alone could lead to population cycles observed in M. agrestis and in other graminivorous rodent populations, where populations that reach sufficiently high densities can induce silica defenses in their food source.

Studies on the root vole, Microtus oeconomus (Pallas, 1776), have shown that changes in the silicon content of tussock sedges may be induced by variations in vole population densities (Wieczorek et al., 2015). However, no correlation was shown between the silicon content in the faeces of M. oeconomus and survival rate (Wieczorek et al., 2015). A very recent study in Poland demonstrated that the amount of silica in plants, fed upon by voles, leaves a traceable record in their dental microwear textures, and that these differ through different phases of vole population cycles (Calandra et al., 2016). The authors hypothesize that the high quantity of phytoliths, produced due to intense grazing in peak years, can result in malocclusion and other dental abnormalities, and may explain how these silicon-based plant defenses contribute to population crashes. Silicon-treated wheat plants showed enhanced resistance to feeding by the wild rabbit (Oryctolagus cuniculus L.), a major vertebrate pest of cereals in the United Kingdom (Cotterill et al., 2007). Further, severe, potentially lethal feeding damage due to rabbit browsing, was reduced in silicon-treated wheat by over 50%. Feeding preference in sheep (Ovis aries L.), in response to silicon availability, did not differ within a grass species; however, there were differences in the bite rate and feeding preference between grass species, with these differences more obvious in silicon-treated plants (Massey et al., 2009). Further, silicon influenced grass preference less in palatable species, compared to less desirable species, an effect that appeared to be due to the most palatable species containing relatively little silicon even after supplementation, and being less tough (Massey et al., 2009).

 However, there is no consistent evidence for silicon having a greater effect in any particular feeding guild or taxon (Keeping and Kvedaras, 2008). The vast majority of studies are at two trophic levels, with few studies at the third trophic level (Reynolds et al., 2009; Gurr and Kvedaras, 2010; Kvedaras et al., 2010). A comprehensive review of earlier work on the role of silicon against herbivorous insects was provided by Reynolds et al. (2009).

This paper explores the more recent advances in the role of silicon in ameliorating the effects of biotic stress, particularly that caused by arthropods from agricultural systems, and the response of their natural enemies, together with the mechanisms involved in bi- and tri-trophic interactions. We also review literature relating to the effects of silicon on plant pathogens where this helps illustrate underlying mechanisms of plant defense that may have relevance to arthropods. Understanding the role and function of silicon against arthropod pests, will ultimately enable us to optimize the use of this element in the context of sustainable agriculture.

#### BI-TROPHIC INTERACTIONS

Silicon fertilization of plants has proven to be effective in controlling insect herbivores and other arthropods. Indeed, silicon application has become a routine practice in rice production in some countries, including Japan, where a silicon fertilizer was first applied to any crop worldwide (Ma and Takahashi, 2002). In agricultural systems, silicon is typically applied to the soil, or as a foliar spray to the vegetation. It is feasible that foliar application of silicon can have an effect on arthropods, e.g., via surface pH or osmotic effects. However, there is now considerable evidence, notably in fungal systems, that soil applied silicon leads to significantly more silicon accumulation in plant tissues, than foliar applications and produces much better results against biotic stressors (Liang et al., 2005, 2015; Guével et al., 2007; Dallagnol et al., 2015). Details of the mechanisms underlying silicon-mediated plant resistance against biotic stress are increasingly becoming clear, with an increase in the number of publications in this area in recent years.

#### Physical Mechanisms

fpls-07-00744 June 9, 2016 Time: 14:36 # 4

An increased physical barrier produced by silicon deposition beneath leaf cuticles has long been considered to represent a major component underlying silicon-mediated plant resistance to insect pests. Silicon deposition contributes to increased rigidity and abrasiveness of plant tissues, thereby forming a mechanical barrier and reducing their palatability and digestibility to both vertebrate (Massey and Hartley, 2006, 2009) and invertebrate herbivores (Goussain et al., 2005; Kvedaras et al., 2007a; Massey and Hartley, 2009). Increased abrasiveness of leaves due to silicon deposition reduces food quality for herbivores and may cause wear of herbivore mouthparts, which further reduces feeding efficiency and growth rates (Massey and Hartley, 2009). Conversely, using a simple method to determine mandibular wear (Smith et al., 2007), it was shown that although there was a trend for increased wear in Eldana saccharina larvae that developed on silicon-treated sugarcane, the ability of larvae to renew their mandibles at each moult probably allows them to compensate for increased wear (Kvedaras et al., 2009). Finely ground wollastonite (CaSiO3) in artificial diets at rates of up to 3.3% silicon had no significant effect on larval growth of Helicoverpa armigera (Hübner; Lepidoptera: Noctuidae) and Helicoverpa punctigera Wallengren, suggesting that silicon may not be directly deleterious to insects via ingestion and other mechanisms may be involved in silicon-mediated plant resistance (Stanley et al., 2014). It should be noted, however, that by grinding the silicon, this has likely removed potential abrasive attributes, in addition to the potential effects of soluble-silicon-induced plant defenses.

Using energy-dispersive X-ray (EDX) and X-ray mapping, it was shown that the pattern of silicon deposition in sugarcane, especially at the internode and root band, is likely the reason (at least, in part) for enhanced resistance of silicon-treated sugarcane to penetration and feeding by E. saccharina at these sites (Keeping et al., 2009). Further, epidermal silicon was higher in the control (i.e., no silicon treatment), E. saccharina resistant cultivar, than the susceptible control cultivar, suggesting that such differences in silicon-mediated resistance exist to a large extent due to the varying ability of cultivars to deposit silicon within the stalk epidermis (Keeping et al., 2009), thus preventing E. saccharina penetration (Kvedaras and Keeping, 2007). A more recent study using scanning electron microscopy and EDX compared four grass species, and showed that spine and phytolith morphology both within and between species may be more important than leaf silicon concentration in determining the abrasiveness and/or digestibility of leaves and thus the effectiveness of anti-herbivore defense (Hartley et al., 2015). The authors showed that all the grasses tested were able to deposit new types of silicon-based structures when silicon supply was increased. These changes were particularly evident when the leaves were mechanically damaged; however, damage in the absence of additional silicon did not produce such structures (Hartley et al., 2015).

#### Biochemical/Molecular Mechanisms

McNaughton and Tarrants (1983) were the first to show induction of silica. They showed that plants growing in a more heavily mammal-grazed grassland in the Serengeti, Tanzania, accumulated more silica in their leaf blades relative to plants from a less heavily grazed site, and blade silica content was higher when plants were defoliated, suggesting that silicification is an inducible defense against mammalian herbivores. Massey et al. (2007) demonstrated in a laboratory study, that feeding by both a mammal, M. agrestis and an insect, Schistocerca gregaria Forskal (Orthoptera: Acrididae) led to increased levels of silica in grass leaves. Other recent studies on arthropods have demonstrated that silicon-mediated antiherbivore defense is both inducible and allelochemical-mediated (Gomes et al., 2005; Kvedaras et al., 2010; Costa et al., 2011) and these effects can complement the physical effects described above, leading to impaired feeding, growth, and development (**Figure 1**).

Increasing evidence shows that silicon treatment increases transcript levels of defense-related genes, thereby enhancing the activities of plant defensive enzymes (Liang et al., 2003; Cai et al., 2008; Rahman et al., 2015) leading to increased accumulation of defensive compounds, such as phenolics, phytoalexins, and momilactones (Fawe et al., 1998; Rodrigues et al., 2004; Rémus-Borel et al., 2005). Gomes et al. (2005) showed that the addition of silicon strongly enhanced wheat resistance to greenbug Schizaphis graminum (Rondani; Hemiptera: Aphididae). Further, silicon pre-treatment increased the activities of the defensive enzymes peroxidase, polyphenoloxidase, and phenylalanine ammonia lyase. In particular, silicon facilitated the strongest resistance if wheat plants had previously been infested with aphids. Chérif et al. (1994) found that silicon-treated cucumber plants show increased activity of the enzymes peroxidase, polyphenoloxidase, β-1,3 glucanase, and chitinase in response to infection by pathogens. Perennial ryegrass (Lolium perenne L.) grown in silicon-amended soil exhibited greater activity of peroxidase and polyphenoloxidase, higher levels of several phenolic acids, including chlorogenic acid and flavonoids, and enhanced expression levels of genes encoding phenylalanine ammonia lyase (PALa and PALb) and lipoxygenase (LOXa) in response to infection by Magnaporthe oryzae (T.T. Hebert) M.E. Barr (Rahman et al., 2015). Histological and ultrastructural analyses revealed that silicon mediates active localized cell defenses, and epidermal cells of silicon-treated plants displayed specific defense reactions including papilla formation, production of callose, and accumulation of glycosilated phenolics in response to pathogen infection by the fungus Blumeria graminis f. sp. tritici (DC.) Speer (Bélanger et al., 2003). Silicon-mediated brown spot resistance in rice plants is independent of the classic immune hormones, salicylic acid and jasmonic acid (JA; Van Bockhaven et al., 2015). Conversely, silicon mounted rice resistance to the brown spot fungus Cochliobolus miyabeanus (Ito and Kuribayashi) Dastur, by interfering with the production and/or action of fungal ethylene, prevents the fungus from suppressing the rice innate immune system (Van Bockhaven et al., 2015).

Pre-treatment with certain chemicals or previous biotic stressor may provoke a specific physiological state in plants called "priming" (Fauteux et al., 2006; Hao et al., 2012;

Worrall et al., 2012; Aimé et al., 2013). Primed plants are thus physiologically prepared to induce quicker and/or stronger defense responses upon subsequent attack, providing plants with a more effective means to respond to challenges (Ton et al., 2006; Jung et al., 2009; Slaughter et al., 2012; Ye et al., 2013). A recent study demonstrates that silicon is able to prime jasmonate-mediated defense responses and rice defense against a chewing herbivore, the rice leaffolder, Cnaphalocrocis medinalis (Lepidoptera: Pyralidae; (Ye et al., 2013). More interestingly, activation of jasmonate signaling in turn promotes silicon accumulation in rice leaves, indicating a strong interaction between silicon and jasmonate in rice defense against insect herbivores. Some recent studies have shown that silicon can also prime plants for alleviating biotic stress imposed by pathogens (Ghareeb et al., 2011; Rahman et al., 2015). Vivancos et al. (2015) showed that priming is also an important mechanism of siliconmediated resistance of Arabidopsis thaliana (L.) Heynh. against powdery mildew caused by Golovinomyces cichoracearum (DC.). Further, this work has also revealed that silicon may interfere with effector proteins released by such biotrophic pathogens, suggesting that mechanisms other than salicylic acid-dependent plant defense priming are involved (Vivancos et al., 2015). It has been suggested that priming of plant defense responses, alterations in phytohormone homeostasis, and interaction with defense signaling components are all potential mechanisms involved in regulating silicon-triggered resistance responses (Van Bockhaven et al., 2013). Silicon has also been demonstrated to prime plants for resistance against abiotic stresses (Ahmed et al., 2013). Research on silicon-mediated herbivore resistance lags

far behind that on silicon-mediated disease resistance. Further studies are needed to determine the exact nature of siliconprimed anti-herbivore defense and indeed other mechanisms that may play a role in plant resistance to biotic stressors. For example, effectors that modulate plant defenses have also been identified in the saliva of insects (for a review see Hogenhout and Bos, 2011) and it is feasible that a similar mechanism proposed for plant pathogens, also operates for insects, although this remains to be elucidated.

Recent developments regarding the understanding of molecular mechanisms controlling silicon accumulation and the discovery of silicon transporters have enabled a ready ability to classify a plant as Si-competent, or not. This will enable a better understanding of the role of silicon in several fundamental aspects of ecology concerning plant fitness under stress (Deshmukh and Bélanger, 2015).

## TRI-TROPHIC INTERACTIONS

Natural enemies of herbivores can be important in the management of agricultural pest species. Evidence for this includes the wide literature on biological control using predators, parasitoids and entomopathogens. In this section we consider what is currently the least thoroughly investigated aspect of plant–silicon–herbivore interactions: the mechanisms by which the application of silicon compounds may affect the impact of natural enemies on herbivores.

#### Entomopathogenic Microorganisms

Entomopathogens are increasingly used in arthropod pest management. However, as this approach uses applications of live organisms rather than chemicals, as in conventional insecticide use, particular attention needs to be given to maximizing the viability and impact of the treatment on the target pest. In work with the fungus Beauveria bassiana (Bals.-Criv.) Vuill., 1912, potassium silicate was added to nutrient solutions applied to plant roots seven days after inoculation with spider mite, Tetranychus urticae Koch (Gatarayiha et al., 2010). Potassium silicate alone did not kill the pest mites, but when used at the higher rates, equivalent to 80 and 160 mg of pure silicon per liter, pest mortality caused by B. bassiana was up to 92%. The authors of that study hypothesized that silicon application primed biochemical defenses in the plants (see above) which interfered with the feeding of mites making them more susceptible to the entomopathogen (**Figure 1**).

#### Predators

Of particular relevance to the possible effects of silicon on nonentomopathogenic natural enemies is a study of induced defense in rice (Ye et al., 2013). This study, employing rice mutant lines in which genes for jasmonate synthesis or jasmonate perception were silenced, showed a strong interaction between soil-applied silicon and JA in defense against insect herbivores. This involved priming of JA-mediated defense responses by silicon and the promotion of silicon accumulation by JA (Ye et al., 2013). While that work did not extend to considering natural enemies it is significant for third trophic level effects because it identified a relationship between silicon and JA. Silicon is translocated within plants in the form of monosilicic acid, Si(OH)4 which is reported as an elicitor for systemic stress signals including JA (Fauteux et al., 2005). JA, in turn, is the primary signaling pathway that is activated by chewing herbivores leading to herbivore-induced plant volatiles (HIPV) production (Dicke et al., 1999, 2009).

The first published study of the effects of silicon on plant defense in which HIPV-mediated effects has been the focus was in cucumber (Kvedaras et al., 2010). That work demonstrated that soil-applied silicon enhanced the attraction of the predator Dicranolaius bellulus (Guerin-Meneville; Coleoptera: Melyridae) to Helicoverpa armigera (Hubner; Lepidoptera: Noctuidae) infested cucumber plants in a Y-tube olfactometer bioassay. Further, a small-scale field trial, using H. armigera eggs affixed to potted cucumber plants, before they were placed in a field plot of lucerne, showed that increased biological control by "wild" predators was significantly higher for soil-applied, silicon-treated plants than for control plants (Kvedaras et al., 2010; **Figure 2**). The authors hypothesized that this was due to a change in the plant volatile profile (HIPVs) produced by cucumber plants when attacked by an herbivore. Additional studies to measure and identify the compounds produced by pest-infested silicontreated and untreated cucumber plants are worthwhile. Similar work on grapevines has yielded preliminary evidence for volatilemediated defenses to promote predator attraction to pest-infested plants (Connick, 2011). A study of the volatiles produced by grapevines infested by the Lepidoptera pest, grapevine moth

Phalaenoides glycinae (Lewin; Lepidoptera: Noctuidae) found that soil applied potassium silicate had profound effects. Seven volatile compounds emitted from P. glycinae-infested grapevines were identified and n-heptadecane found to be produced in significant amounts only by silicon-treated plants. Cis-thio rose oxide production, in contrast, was significantly lower in silicontreated grapevines. A second study in that thesis found that the attractiveness of grapevines infested with the lightbrown apple moth (Epiphyas postvittana (Walker; Lepidoptera: Tortricidae) was positively correlated with plant foliar tissue concentration of silicon (Connick, 2011).

The impact of natural enemies on herbivores may be enhanced by mechanisms other than induced, indirect defenses based on HIPVs. By extending development time, and particularly the period over which neonate larvae feed on the exterior of plants before being able to penetrate the plant cuticle and commence mining or boring, herbivores are exposed to a higher risk of attack by predators. Delayed penetration was evident in a study of sugarcane borer, E. saccharina (Kvedaras and Keeping, 2007). Massey and Hartley (2006) reported similar findings for Spodoptera exempta Walker feeding on grass with high silicon levels. Many natural enemies forage for prey by locomotion over the foliar surface, so the practice of applying silicon treatments to the above ground plant parts could have physical or chemical effects on natural enemy foraging (**Figure 1**). Examples of recent studies that included treatments with foliar applied silicon are Dalastra et al. (2011) and de Assis et al. (2012, 2013), and in the latter of those studies, there was no effect of foliar treatments to potato plants on predatory beetles, although the plants treated with silicic acid were less preferred by defoliators. Further work needs to test for the strength of such effects on a wider range of natural enemy taxa.

Foraging of predators may also be affected by foliar pubescence, especially glandular trichomes. The latter produce

irritant, toxic and adhesive liquid secretions from the tips that can provide high levels of protection from foliar-associated herbivores (Gurr and McGrath, 2002) but can also affect natural enemies (Simmons and Gurr, 2004, 2005). When subject to herbivores, plants have the capacity to regenerate new leaves that exhibit enhanced densities of trichomes, an induced defense that is under the control of JA (Yoshida et al., 2009). This form of induced defense is remarkable in taking place over days rather than the timespan of hours as in the case of induced production of semiochemical volatiles. This phenomenon has relevance to the interplay between silicon and plant defense because plant-available silicon influences the JA signaling pathway (Ye et al., 2013). Accordingly, the phenomenon of herbivore-attacked plants producing more hirsute foliage is another form of plant defense that we hypothesize may by amplified by silicon pretreatment (**Figure 1**).

Not only might plant-available silicon promote the density of trichomes on young foliage, work on deposition patterns of silica in the leaf epidermis suggests that the bases of trichomes is a major site in cucumber (Samuels et al., 1991a,b), while in the grasses D. cespitosa and Festuca ovina L., silica was particularly evident in the tips of spines under control conditions, but was distributed throughout the spine and the leaf surface when silicon fertilized (Hartley et al., 2015). The epicarp hairs present on the mature caryopses of the four cereals, barley, oats, rye, and wheat (Bennett and Parry, 1981) are also important silicon deposition sites, particularly in the tips of hairs where it is most likely to promote adverse effects on herbivores including – potentially – human consumers of grain products (Parry et al., 1984). It remains to be tested whether the potentially adverse effects of trichomes on predators are exacerbated by silicon supplementation and the extent to which any such effects are offset by stronger effects on herbivores.

Among studies of the effects of silicon on pests that do consider third trophic level effects, these tend to use designs that are not well suited to detecting the full range of possible mechanisms that may operate. An example is work by Moraes et al. (2004), with the lacewing Chrysoperla externa Steinmann in which wheat aphid (Schizaphis graminum (Rondani; Hemiptera: Aphididae) prey were removed from the test plants before being exposed to the predators. Since predators were not exposed to plants or their volatiles, they would have been unable to detect HIPV-mediated effects, though effects related to prey quality could be assessed.

A major limit on our current understanding of the effects of silicon on natural enemies is the apparent absence of studies on below-ground effects. Many arthropod pests cause important damage to plant roots so studies of how silicon might promote natural enemies such as predacious beetle larvae and entomopathogenic nematodes would be valuable.

#### Parasitoids

Of the three types of natural enemies, parasitoids are the least well studied in relation to plant available silicon, though many of the comments made above, for established and possible effects on predators (**Figure 1**), will apply to parasitoids. Of particular significance is the wealth of evidence for HIPVs attracting parasitoids to pest-infested plants (Dicke et al., 2009). The only study with silicon-treated and un-treated plants in which a parasitoid was considered is that by Moraes et al. (2004) with Aphidius colemani Viereck (Hymenoptera: Aphididae). Unfortunately, this confined wasps to narrowly spaced wheat plants and, because it used non-choice conditions, would not have allowed HIPV-mediated effects to be apparent.

### HOW "OMICS" SUPPORT PLANT DEFENSE STUDIES?

To understand how the addition of silicon to a plant's environment can improve plant defense, the plant as a whole must be considered through global analysis of the major responsive components of the DNA, RNA, proteins, and metabolites which are then holistically viewed using bioinformatics (**Figure 3**).

While system-wide analysis has long been applied to plants, their application to analyzing plant defense has been limited (Chen et al., 2005; Giri et al., 2006; Thivierge et al., 2010; Lewandowska-Gnatowska et al., 2011; Duceppe et al., 2012; Timbo et al., 2014) and analyzing silicon's role even more so. Numerous reductionist experiments targeting specific proteins or enzymes have shown that silicon treatment induces plant defensive enzymes (Liang et al., 2003; Cai et al., 2008), leading to the accumulation of defensive compounds and metabolites (Fawe et al., 1998; Rodrigues et al., 2004).

But the power of -omics approaches lies in its nontargeted nature, allowing the unearthing of unexpected changes. Transcriptome analysis represents the only -omic analysis of silicon's effects, with a study on challenged A. thaliana showing silicon treatment causes a decrease in primary metabolism that allows a more efficient defense response (Fauteux et al., 2006). A similar analysis was also conducted on rice (Ye et al., 2013), as indicated above. Recent work has sought to establish the "Prime-ome", or the mechanism behind how a plant defends itself or is in a "primed state" to rapidly respond to attack by insects and microbial pathogens (Balmer et al., 2015). Not surprisingly, the available -omics scale data shows that the plant's response depends on the priming inducer and the pathogen, which is also observed in defense against arthropods (Balmer et al., 2015). Silicon's role in defense against herbivores remains vastly understudied by -omics methodologies which would reveal the role of, as yet, untargeted molecules, including proteins and metabolites, through global analysis.

Transcriptomics alone is insufficient to understand an organism's phenotype (Barah and Bones, 2015) as it is the proteome and metabolome that provide the molecular mechanisms that allow a plant to defend itself (Oliveira et al., 2014). While proteomics and metabolomics are rapidly maturing fields, they are still limited by the issues of throughput and the depth of proteome and metabolome coverage due to the dynamic range of concentration of the molecules present (Jorge et al., 2015). The abundance of proteins can vary by 7–10 orders of magnitude (Ly and Wasinger, 2008; Zubarev, 2013) and the existence of a proteoform is often reported by the detection of

only a single peptide (Mallick et al., 2007). Without an equivalent of PCR utilized in genomics and transcriptomics, the only way to reliably detect and quantify the abundance of low copy number proteins is to start with more material (Zubarev, 2013) and fractionate the proteins to isolate those of high abundance from the rest (Stasyk and Huber, 2004; Righetti et al., 2005; Ly and Wasinger, 2011). The same logic applies to metabolites but in both cases the number of fractions requiring analysis increases.

In the case of proteomics, fractionation of intact proteins reduces this increase compared to "shotgun" peptide-centric methods while retaining the option of utilizing 2D-PAGE as a further fractionation and quantification method (Coorssen and Yergey, 2015). To determine plant defense responses as a result of silicon treatment, 2D-PAGE has the distinct advantage of quantifying protein abundance changes prior to identification. This is contrary to LC/MS/MS methodologies where identification of peptides and their assignment to a protein isoform needs to be performed prior to quantitation. Thus, 2D-PAGE can decrease the number of samples requiring analyses by mass spectrometry (MS), freeing valuable instrument time. In proteomics, the issue of throughput is being addressed somewhat by faster instrument scan speeds (Richards et al., 2015), the adoption of ultra high-pressure chromatography (Kocher et al., 2011; Thakur et al., 2011) and data-independent acquisition (DIA) techniques in LC/MS/MS (Huang et al., 2015). DIA methodologies have also been applied to measure nitrogen flux and metabolism (Ullmann-Zeunert et al., 2012) indicating that DIA could have application in quantitative metabolomics, in order to assess how changes in the levels of specific metabolites can be related to observed plant defensive phenotypes.

ungrazed pastures.

#### CONCLUSIONS AND FUTURE DIRECTIONS

There is now considerable literature supporting the role of silicon as a physical defense mechanism, and a growing number of published works on the role of silicon-mediated biochemical defense. However, there are few references on the role of silicon in tri-trophic interactions.

Research should focus on understanding the relative importance of both physical and biochemical defence and how (if) this differs between herbivores. A meta-analysis of the literature would be valuable to discern if silicon has a greater effect in certain feeding guilds or taxons. Understanding the interaction between silicon and the plant defense pathways, and if there is a similar mechanism acting against insects, and pathogens, will also be paramount, as there is a wealth of literature on silicon/pathogen interactions that can inform arthropod work.

Future researchers need to address the lack of knowledge on below-ground effects of silicon application to plants on predators. There is a more general dearth of knowledge on how silicon might alter root toughness and chemical defenses. There is also a need to test for the effects of foliar deposits from foliar applied silicon on natural enemy foraging and impact. Work also needs to consider the possibility that changing the plant surface, by denser or more robust trichomes, may have negative effects on natural enemy foraging (**Figure 1**). More generally, workers need to consider the effects of silicon under field conditions (something done quite extensively for mammals in natural ecological systems) and be less reliant on greenhouse and laboratory studies, especially those that make it impossible for natural enemy mediated effects on herbivores to be apparent. Finally, there are currently no published studies of the effects of silicon on HIPV production but such work is known to be underway. If strong evidence is forthcoming for effects on the blend of HIPVs, this will add impetus to the need for greater attention to be given to the third trophic level in studies of silicon on plant defenses.

#### REFERENCES

Using system-wide analysis or -omics technologies would permit us to not only understand silicon's role in the production of defense-related compounds, but in the production of HIPVs, in addition to the associated energy costs to the plant. This could potentially inform the manipulation of plants to minimize herbivory and maximize the impact of natural enemies.

Modern approaches of transcriptomics, proteomics, metabolomics, and transgenic mutants will serve as powerful tools for dissecting the underlying mechanism/s involved in silicon and plant defense. In an era when sustainable pest management is receiving more attention than ever before, due largely to restrictions or the withdrawal of toxic pesticides, because of their negative impacts on human and environmental health, silicon treatment should be more widely considered and tested as a pest management option.

#### AUTHOR CONTRIBUTIONS

OR and GG developed the concept, drafted, and critically revised the manuscript. MP and RZ drafted and critically revised the manuscript.

#### FUNDING

GG is supported by the National Thousand Talents Program in China and the Advanced Talents of SAEFA, and OR is supported by the Jinshan Scholar Program at Fujian Agriculture and Forestry University, Fujian Province, China.

#### ACKNOWLEDGMENTS

The authors thank Mrs. Anne Johnson for assistance with manuscript production and two reviewers, who made useful comments on the manuscript.

rice against the rice brown planthopper (Delphacidae: Homoptera). Entomol. Exp. Appl. 26, 314–322. doi: 10.1111/j.1570-7458.1979.tb0 2932.x

Zubarev, R. A. (2013). The challenge of the proteome dynamic range and its implications for in-depth proteomics. Proteomics 13, 723–726. doi: 10.1002/pmic.201200451

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The reviewer JD and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.

Copyright © 2016 Reynolds, Padula, Zeng and Gurr. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# The Use of Arbuscular Mycorrhizal Fungi to Improve Strawberry Production in Coir Substrate

Louisa Robinson Boyer 1 \*, Wei Feng1 , Natallia Gulbis 2 , Klara Hajdu2 , Richard J. Harrison1 , Peter Jeffries 3 and Xiangming Xu1

*1 NIAB-EMR, East Malling, UK, 2 Plantworks Ltd., Sittingbourne, UK, 3 School of Biosciences, University of Kent, Canterbury, UK*

Strawberry is an important fruit crop within the UK. To reduce the impact of soil-borne diseases and extend the production season, more than half of the UK strawberry production is now in substrate (predominantly coir) under protection. Substrates such as coir are usually depleted of microbes including arbuscular mycorrhizal fungi (AMF) and consequently the introduction of beneficial microbes is likely to benefit commercial cropping systems. Inoculating strawberry plants in substrate other than coir has been shown to increase plants tolerance to soil-borne pathogens and water stress. We carried out studies to investigate whether AMF could improve strawberry production in coir under low nitrogen input and regulated deficit irrigation. Application of AMF led to an appreciable increase in the size and number of class I fruit, especially under either deficient irrigation or low nitrogen input condition. However, root length colonization by AMF was reduced in strawberry grown in coir compared to soil and Terragreen. Furthermore, the appearance of AMF colonizing strawberry and maize roots grown in coir showed some physical differences from the structure in colonized roots in soil and Terragreen: the colonization structure appeared to be more compact and smaller in coir.

Keywords: strawberry, yield, growing substrate, AMF, coir, Class I yield quality

## INTRODUCTION

Strawberry is an important horticultural crop in the UK and is a highly nutritious and important food source. Strawberry accounted for 67% of all soft fruit production worth an estimated £247 million in 2013 (DEFRA, 2015), and this is set to rise significantly over the coming years. Recently, a significant trend in commercial strawberry cropping has been to move away from traditional field cultivation toward production into substrate. Industry estimates that more than 50% of the UK strawberry production is produced in substrates, usually coir (coconut fiber) and mostly under protection (polythene tunnel or glasshouse). This change was intended to mitigate the threat of soilborne fungal pathogens, principally wilt (Verticillium dahliae Kleb). Chemical treatments have been an indispensable tool for controlling soil-borne pathogens; however, several of these treatments are already banned or face an uncertain future due to legislation (Martin, 2003). There are many significant benefits to the adoption of substrates in commercial strawberry cropping, such as to extension of the growing season, increased ease of picking and better control of the crop from

#### Edited by:

*Rex Brennan, James Hutton Institute, Scotland*

#### Reviewed by:

*Inger Martinussen, Norwegian Institute of Bioeconomy Research, Norway Tania Wyss, University of Lausanne, Switzerland*

#### \*Correspondence:

*Louisa Robinson Boyer<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Crop Science and Horticulture, a section of the journal Frontiers in Plant Science*

Received: *15 December 2015* Accepted: *04 August 2016* Published: *19 August 2016*

#### Citation:

*Robinson Boyer L, Feng W, Gulbis N, Hajdu K, Harrison RJ, Jeffries P and Xu X (2016) The Use of Arbuscular Mycorrhizal Fungi to Improve Strawberry Production in Coir Substrate. Front. Plant Sci. 7:1237. doi: 10.3389/fpls.2016.01237*

fertigation and pollination regimes. However, this practice relies heavily on high inputs of water and fertigation; these inputs are estimated to be more than doubled compared to a field grown crop, with an increased cost of up to £1800 per Hectare.

Arbuscular mycorrhizal fungi (AMF) penetrate the roots of plants to form a mutualistic symbiotic relationship. Mineral nutrients, mainly phosphorus, nitrogen and water are extracted from the soil via the extensive hyphal network and transferred to the plant. Organic carbon compounds are transferred to the AMF in return. They are known to improve plant nutrient uptake, protect plants from pathogens (Borowicz, 2001; Ismail and Hijri, 2012; Ren et al., 2013) and buffer against adverse environmental conditions, especially drought (Smith et al., 2010; Robinson-Boyer et al., 2015). A number of studies have reported the beneficial effects of mycorrhiza on strawberry plants (Castellanos-Morales et al., 2010) and commercial AMF inoculum has been shown to increase both growth (crowns, roots and leaf area) and tolerance to water stress in micro-propagated strawberry (Borkowska, 2002).

The maintenance of a developed and diverse population of AMF and other soil micro-organisms is important in achieving sustainable agriculture (Jeffries et al., 2003) thus reducing the requirement of such high levels of fertigation. However, products containing AMF are rarely used in commercial agriculture because of (a) difficulties in producing AMF inoculum in large quantities, (b) their variable beneficial effects, and (c) uncertainties in the benefits with added AMF in the presence of resident AMF populations. Substrates such as coir are usually devoid of beneficial microbes such as AMF; thus introducing them into substrate production is more likely to generate benefits.

This paper reports results from three studies on the use of AMF in strawberry production in coir substrate. First, we assessed whether use of AMF in substrate could improve strawberry fruit yield in respect to water stress and nutrient input. This work showed positive effects of the addition of AMF in fruit production despite observing low levels of AMF colonization and compact, immature mycorrhizal structures inside colonized roots of strawberry. Thus, we conducted further experiments to better understand the extent and structure of root colonization in different types of substrate (including soil). Furthermore, to establish if the effects observed on root colonization were limited to strawberry only, we included maize in the experiment as maize is a common, highly mycorrhizal host plant of AMF.

## MATERIALS AND METHODS

### Effect of AMF Inoculation of Strawberry in Coir

The experimental design was a full factorial design with three factors: AMF inoculation, irrigation and nutrient. For AMF inoculation, there were three treatment levels: negative control with no inoculum added, and application of either granular or liquid formulation of AMF (G\_AMF or L\_AMF). There were two irrigation regimes: well-watered, to capacity (WW), and regulated deficit irrigation (RDI, 60% of the WW). There were two nutrient input regimes: standard or reduced nitrogen input (60% of the standard). Thus, there were 12 treatments in total (see **Figure 1**). This experiment was conducted on three separate occasions, with two replicates of each treatment each time.

Agronomical management of strawberry (including dosatron setting, nutrient composition and irrigation) followed previously established protocols (Xu et al., 2013), which were based on current commercial practices. From a combination of visual assessment of water leakage from coir bags and moisture content measurements, estimated using a Delta-T "WET" sensor (Delta-T Devices, Cambridge, UK), the amount of irrigation water was adjusted as necessary via a Galcon irrigation timer. Overall, the volume of irrigation water applied increased gradually over time, reached the maximum at the first week of blossom and thereafter remained at this level, equivalent to 2 L per day per bag.

Irrigation and fertigation were delivered to plants via eight irrigation lines using drippers, two of which were controlled by a separate irrigation controller (dosatron). Each dosatron was randomly allocated to one of the four nutrient and water combinational treatments. Within each irrigation line, there were three replicate bags, each allocated to one of the three AMF treatments. Thus, the experiment was a split plot design—the main plot was the dosatron (two irrigation lines) and the subplot was the individual coir bag. All coir bags (BotaniCoir, England) prior to planting were saturated with water over a period of 2 weeks in order to re-hydrate the coir. Inoculum of AMF was supplied by PlantWorks Ltd, Kent, UK, the granular formulation applied as commercially available "Rootgrow" (Funneliformis mosseae, F. geosporus, Claroideoglomus claroideum, Glomus microagregatum, Rhizophagus irregularis), containing propagules of spores, hyphal and root fragments. The liquid application was an in-vitro produced preparation of R. irregularis DAOM197198 (consisting of sterile water and spores).

Cold-stored (−2 ◦C) runners of cv. Elsanta (Hargreaves Plants, UK) were planted in coir bags. At the time of planting, for the G\_AMF treatment, 20 g of granular AMF was placed to a single planting hole before the plant was planted; for L\_AMF, a liquid AMF suspension [4 ml estimated to be taken up per plant] was applied to the roots of individual runners and for the control nothing was added. For both G\_AMF and L\_AMF each plant received ca. 6650 propagules of AMF estimated using MPN analysis (Cochran, 1950). After the onset of flowering a mini hive of bees, Bombus terrestris, (Agralan, UK) was introduced to the compartment to pollinate (with the exception of the first replicate experiment). Plants were grown in a GroDome compartment (Unigro, UK) set at 22◦C day/20◦C night with a 16 h day/8 h night cycle with supplementary lighting.

A sample of roots from a number of plants was assessed prior to planting to check for colonization by AMF. Roots were cleared with KOH before being stained using Trypan Blue and assessed microscopically for root length colonization (RLC) using the grid-line intersect method (McGonigle et al., 1990). Colonization was expressed as a percentage of the root colonized by AMF. Ripe fruit were picked regularly (2–3 times weekly). Except for the first experiment, fruit were divided by size into Class I (above 18 mm diameter) & II and weighed separately for individual bags and the number of fruit was recorded. For the first experiment, because of smaller fruit (lack of pollinators), fruit were not divided into Class I or II. After harvesting, fresh weight of individual plants (both above- and below-ground parts) was determined. A composite sample of roots was taken for each coir bag at harvest to check colonization by AMF. Only fresh, recently formed roots were sampled, and the original runner roots were avoided. Roots were stained and assessed as above.

#### The Effect of Substrate on Colonization by AMF

#### Substrate Effect and Time of Inoculation

Maize (cv Thalys, Cotswold Seeds, UK) was used in this experiment since it is known to be highly responsive to AMF and a common host for production of commercial AMF and is used here to study the effect of substrate on root colonization. There were three treatment factors: pre-emergence inoculation with AMF (PreAMF: Yes or No), post-emergence inoculation with AMF (PostAMF: Yes or NO). Substrates compared were Top soil: S, Terragreen: T, coir: C, and peat-free compost: PF. These substrates are commonly used for commercial cropping with the exception of Terragreen (attapulgite clay; OilDry, UK) which is routinely used in the study of AMF, giving a clear indication of "expected" colonization. A randomized block design was used with five blocks. There were two pots per treatment in each block: one for destructive sampling 4 weeks after transplanting, and the other after 10 weeks.

Maize seeds were first soaked in sterile water for 24 h. Multicell trays (cell volume 250 ml) were filled with Terragreen. Half of the cells were amended with 10 g of granular AMF inoculum to allow inoculation of seedlings (pre-emergence). One seed was manually sown 2 cm deep per cell. Seedlings for the Pre-AMF inoculation treatment were sampled and checked microscopically for colonization by AMF (as above) and only those seedlings with colonization were retained. Prior to transplanting, a sufficient number of coir bags were thoroughly wetted; coir from these bags was then used to fill pots. Similarly, top soil (ca top 10–15 cm) from a plot at East Malling Research was obtained to fill pots; peat-free compost was purchased commercially (Dobbies, UK). Seedlings were transplanted approximately 2 weeks after sowing. On the day before transplanting, all pots were thoroughly watered to reach the fully-wet state. A planting hole was made in each pot and 10 ml AMF sprinkled into the hole for those pots allocated to the Post-AMF inoculation treatment. Then a single seedling was transplanted to each single pot (4 L). All plants were fed with Vitafeed 102 (Vitax, UK) 1 gL−1 every 2 weeks. The height and stem diameter, just above the substrate surface and colonization by AMF (RLC) were assessed destructively 4 and 10 weeks after transplanting. The root samples analyzed at 10 weeks were sampled from two positions on the plants, firstly very close to the inoculation site and secondly from the peripheral roots, and stained and assessed as above.

#### Effect of Coir Substrate on AMF Colonization

For this study, both maize and strawberry were used to compare colonization by AMF in coir and in Terragreen. Maize seedlings (cv. Jubilee F1, B&Q, UK) were obtained as in the previous experiment, except Terragreen was not amended with inoculum of AMF. Strawberry module plants (cv. Elsanta), produced from tipping in compost, were obtained from a commercial nursery (Hargreaves plants, UK); plants derived in this way have shown Robinson Boyer et al. AMF to Improve Strawberry Production

in previous work to be free from colonization by AMF (Xu, unpublished data), although a few plants were tested prior to the experiment to confirm this. Individual plants were transplanted to 1 L pots [one plant per pot]; all plants were inoculated with 20 g of granular inoculum of AMF at the time of transplanting. There were 10 replicate pots per treatment (substrate [Terragreen or coir] and host [strawberry or maize]). A complete randomisation design was used. A standard commercial fertigation scheme of N-P-K = 120-45-176 for strawberry was used to manage the plants (J. Atwood, ADAS, England, per. comm.). Only eight strawberry and four maize plants per substrate (randomly selected from 10 plants in each treatment) were sampled to assess root colonization 10 weeks after transplanting; the amount of vesicles, arbuscules and hyphae were also recorded.

#### Data Analysis

ANOVA of a split-plot design was applied to strawberry data from all three experiments treating individual experiments as a blocking factor, using GenStat 13 (VSN International, England). In addition to total Class I yield and number of Class I fruit, average individual fruit weight was also analyzed. Fresh plant weight was also used as a co-variate in ANOVA but it did not alter the main results; therefore, only results from ANOVA without the co-variate are presented. Interactions between three factors: AMF, irrigation and nitrogen input were statistically tested. For the data on AMF colonization in different substrates, standard ANOVA were used to compare treatment effect. In all analyses, once ANOVA indicated significant effects of a specific treatment factor or interaction, pairwise comparison was then carried out based on the LSD test. Common diagnostic plots (e.g., q-q plot, residuals-fitted value plot) did not reveal apparent violation of the normality and homoscedasticity assumptions. Hence no transformation was necessary in order to satisfy ANOVA assumptions for fruit yield and RLC data.

## RESULTS

### AMF on Strawberry Production in Coir

Strawberry plants in all treatments grew normally and there were no visual differences in plant growth between treatments. Fewer and lighter fruit were produced in the first replicate experiment than in the other two experiments, due to less developed fruit from the lack of insect pollination. Plants in each coir bag on average produced 57, 127, and 108 fruit for the 1st, 2nd , and 3rd replicate experiment, respectively; the corresponding average fruit weight was 4.7, 16.8, and 11.3 g. There was a large variation in fruit yield among individual picks in all three replicate experiments but the three AMF treatments followed a similar trend over time (**Figure 2**). AMF-treated strawberries (particularly G\_AMF) had increased fruit production in the mid to late harvest period (**Figure 2**).

Because of the lack of bee pollinators, the 1st replicate experiment was excluded from statistical comparisons, hence all the subsequent presentations were from statistical analysis of replicate experiments 2 and 3 (**Table 1**). There were significant differences in the yield [total class I fruit weight; F(2, 24) = 3.43, P < 0.05], and number of fruit per plant [F(2, 24) = 3.30, P < 0.05] among AMF treatments. The G\_AMF treatment led to higher (P < 0.05) yields than the control but not different from the L\_AMF treatment (**Figure 3**). There were also no significant differences in yields between the L\_AMF and the control treatments. On average the G\_AMF and L\_AMF treatments led to a greater (P < 0.05) number of fruit than the control (**Figure 3**). In both experiments, G\_AMF had higher yield than L\_AMF, although this difference is not statistically significant. Higher yields and more fruit were obtained in both high nitrogen and well-watered treatments than the low nitrogen and RDI treatments but these differences were not statistically significant (**Table 1**). For both the average fruit weight and plant fresh weight, none of treatments resulted in significant differences (**Table 1**).

All interactions involving any two factors were not statistically significant (**Table 1**). However, the three-way interaction was significant for both the total yield [F(2, 24) = 4.59, P < 0.05] and average number of fruit per plant [F(2, 24) = 7.45, P < 0.01]. The interactions mainly resulted from the fact that the increase in the yield and number of fruit associated with AMF application was for the high nitrogen input under the deficit irrigation but the low nitrogen input under the wet treatment (**Figure 3**).

Prior to planting, runners were colonized by AMF and the RLC ranged from 20 to 40% for all three experiments. However, after the final harvest, there was almost no colonization (average <1%) found in roots of the control, non-inoculated, plants and the level of AMF colonization found in the roots of the treated plants was low (average <15%) and highly variable among samples; many samples failed to show any colonization. There were no differences between treatments in RLC.

## The Effect of Substrate on Colonization by AMF

#### The Effect of Substrate on RLC of Maize

There was no AMF colonization in non-inoculated plants when assessed 4 or 10 weeks after transplanting. For inoculated plants, RLC at 4 weeks ranged from 0 to 75.0% (with an average of 25.3%); only for five plants were AMF not observed. At 10 weeks, average AMF colonization over all roots, regardless of the position of sampled roots in relation to the inoculation site, was 49.2%; only for a single plant were AMF not observed.

At 4 weeks after transplanting, RLC did not differ significantly between plants inoculated twice at both pre- and post-emergence and those plants only inoculated once (24.4% [both] vs. 25.5% [single]; **Table 2**). In contrast, inoculation during sowing resulted in greater [F(1, 43) = 5.5, P < 0.05] RLC than inoculation during transplanting: 30.0 vs. 20.9%. There were large [F(3, 43) = 19.6, P < 0.001] differences in RLC between four substrates, accounting for ca. 44% of the total variation. Average RLC for Terragreen was 40.9%, significantly (P < 0.01) greater than coir (25.5%), and peat (27.1%), which in turn was greater (P < 0.001) than the peat-free substrate (6.8%). In addition, RLC differences between two inoculation timings varied with

TABLE 1 | Summary of ANOVA (F-values) of strawberry class I yield in two replicate experiments where granular and liquid AMF products were applied to strawberry plants grown in coir bags.

*Plants were also subjected to two irrigation (high, low) and two nitrogen (high, low) treatments, delivered through automated fertigation pipe lines. A split-plot design was used in which the fertigation line and individual coir bags were the main and sub-plots, respectively.*

\**,* \*\**Significant at the level of 0.05 and 0.01, respectively.*

substrates [F(3, 43) = 6.6, P < 0.001]: for both coir and Terragreen, inoculation in sowing led to greater RLC than during transplanting, which was opposite to the situation for peat (**Figure 4**), and the difference for peat-free was very small.

When assessed 10 weeks after transplanting, the only significant difference in RLC was related to the four substrates [**Table 2**; F(3, 42) = 27.9 and F(3, 43) = 91.7 (P < 0.001)] for RLC near and further away from the inoculation site, respectively. For roots near the inoculation site, RLC was greatest for Terragreen (81.2%) and least for peat-free (16.2%; **Figure 4B**) and RLC did not differ significantly between Terragreen (81.2%) and peat (70.8%). The relative differences in RLC in roots further away from the inoculation site between the four substrates were similar as for the near-inoculation-site, except that the difference between Terragreen and peat was significant (P < 0.05). RLC differences between the two root positions also varied [F(1, 42)

= 4.5, P < 0.01; **Table 2**] with substrates: for both coir and peat-free, RLC was less on the roots far from the inoculation site whereas no such differences were observed for Terragreen and peat (**Figure 4B**).

#### Effect of Coir Substrate on AMF Colonization

Overall AMF colonization was lower [F(1, 23) = 5.6, P < 0.05] in coir (13.1%) than in Terragreen (29.3%); average colonization was greater [F(1, 23) = 10.5, P < 0.01] in maize (36.8%) than in strawberry (13.4%). There were no significant interactions between hosts and substrates in affecting AMF colonization. Although, there were significant differences between treatments, the level of AMF colonization varied considerably within individual treatments.

The morphology of the mycorrhizal structures in coir was different from those in Terragreen (**Figure 5**), in which

normal colonization by AMF with fully-formed clear arbuscule structures was observed. Because of the changed structure in coir, a much larger root sample was assessed for colonization using a grid line technique. In strawberries growing in coir, the arbuscules and vesicles were small, underdeveloped, and their presence was inconsistent in the colonized roots—in many cases only hyphae were observed.

## DISCUSSION

Inoculation with a commercial AMF product in coir increased yield and number of class I fruit of strawberry, particularly under stress conditions of deficient irrigation or low nitrogen input. The granular product of mixed AMF species resulted in a consistent limited benefit (though not statistically significant) to strawberry plants than the liquid inoculum, although the same number of infective propagules was added at planting in either formulation. This difference could be because the liquid formation has only a single species (R. irregularis), whereas the granular has four species of AMF. Thus, there could be synergistic interactions among AMF species in promoting plant growth (Wagg et al., 2011). However, recent work suggested that addition of two AMF species/strains did not result in improved performance of strawberry plants in compost or Terragreen relative to the use of individual species/strains (Robinson-Boyer et al., 2015). Another possible cause could be that with the liquid formulation there may be considerable losses of inoculum with irrigation water to the bottom of the bag that was not reachable by roots. The increase in fruit production was mainly associated with either of the two stress conditions singly, but not when combined. This suggests that AMF can alleviate the negative effects of either drought or low nitrogen. However, the positive effects of AMF on plant may be limited if plants are subjected extreme or combined stresses, which needs further research. Currently, we

TABLE 2 | Summary of ANOVA (F-values) of AMF root length colonization (RLC) where granular AMF products were applied to maize plants grown in four different types of substrates at sowing time only (SO), or transplanting time only (TO), or both (ST).

*AMF colonization was assessed at 4 and 10 weeks post-transplanting; for the 10 week assessment, two types of roots were sampled—those near or further away from the AMF inoculation site.*

\**,* \*\**,* \*\*\**Significant at the level of 0.05, 0.01, and 0.001, respectively.*

+*For RLC near the inoculation sites, there was only 42*◦ *of freedom for the residuals.*

are conducting transcriptomics research trying to shed light on this AMF-strawberry interaction in coir.

Surprisingly, with such an appreciable increase in plant growth/yield, there were very low levels of root colonization by AMF under any of the conditions in coir. This is remarkable given that the original native colonization found in the planting material (runners lifted from field-grown mother plants) had ca. 20% RLC by AMF, which did not appear to spread and establish into the roots produced post-planting in the control plants. The detection of AMF was by root staining and thus does not give an indication of the viability of the AMF colonization present. This may be important considering these runner plants would have overwintered at −2 ◦C prior to planting. Root colonization by AMF in the treated plants was also mostly found in roots that had developed in close proximity to the inoculation site. Reduced root colonization by AMF in coir was also shown for maize, normally recognized for high RLC levels of AMF. Here we clearly show that the level of root colonization in maize was affected by the substrate in which the plant was grown and was significantly reduced when grown in coir. Again the colonization detected in maize grown in coir was largely close to the site of inoculation.

Such low colonization of roots in coir could indicate that (1) coir is a harsh environment for mycorrhiza to colonize roots, (2) movement of inoculum in coir is limited (as irrigation is well controlled to prevent run-off), (3) spore production from colonized roots is limited and (4) changes to plant root physiology in substrate. In addition to the low level of colonization by AMF in coir, the AMF structures appear to be more compact and immature in coir than in soil and Terragreen on both maize and strawberry. This compact AMF structure in coir was also observed in clover (data not shown).

Studies (Isayenkov et al., 2004; Hart et al., 2013) have shown that the level of colonization of a root is not necessarily an indicator of mycorrhizal benefit; however it is notable to record such low levels providing large and consistent plant growth promoting effects. It is possible that in a substrate environment, which is highly artificial for plant growth containing no background level of beneficial micro-organisms, colonization by AMF, even at a low level, may be highly beneficial for plant production.

Another consideration in applying AMF in commercial agriculture is to what extent there is a specific interaction of plant growth environment with AMF species or strain genotype. Multiple variants of sequences have been shown to occur within individual spores and isolates, as well as within and between species of the Glomeromycota (Rodriguez et al., 2001). It is important to determine how different growing environments and host plants could influence AMF genomic changes (Krüger et al., 2015) and consequently their beneficial effects on plant growth, enabling specific AMF products under specific conditions to maximize their beneficial effects. Further research is needed to investigate the inter-relationship of AMF effect, colonization structure and colonization levels in different types of substrates.

It is known that in other crop plants, e.g., wheat, genotypes and cultivars can differ in the extent to which they form an association with AMF (Al-Karaki and Al-Raddad, 1997). Further work is needed to assess to what extent the benefit associated with AMF inoculation in coir is dependent on strawberry genotypes (cultivars). To fully exploit the positive effect of AMF on strawberry in coir substrate, further work is needed to clarify to what extent the ability of specific strawberry genotypes being colonized by AMF in coir is heritable. If this trait is controlled genetically, this could be exploited to breed strawberry plants that can be easily colonized by AMF in substrate to increase their cropping potential and tolerance to pathogens, e.g., powdery mildew and Phytophthora diseases.

In conclusion this work demonstrates that there is a role for AMF in the commercial production of strawberry when grown in substrate and they could be a valuable tool for sustainable cropping of this important fruit crop especially under low-input productions systems. Current levels of high intensity agriculture are no longer sustainable primarily due to energy costs of N fertilizers and the decreasing supplies of P (Cordell et al., 2009), along with a decreasing armory of pesticides (due to legislation) and water limitation. Further studies such as this are needed to improve our knowledge of how best to apply and use these beneficial organisms to successfully incorporate them into sustainable commercial cropping systems for soft fruit and other commercial crops. With a greater understanding of the application and benefits of these beneficial microbes there is a real possibility for their use in aiding sustainable crop production.

#### AUTHOR CONTRIBUTIONS

LR, is the lead author, and undertook the bulk of project management, practical work and writing. WF contributed largely to the metagenomics studies undertaking practical work and writing. NG, KH worked on inoculum preparation and the strawberry experiments with practical work, data collection, analysis, and writing. RH contributed to the metagenomics analysis and writing. PJ was a PI on the metagenomics work, contributed to analysis, interpretation and writing. XX was PI on all the work, contributing to analysis, metagenomics analysis, interpretation, and writing.

#### REFERENCES

#### ACKNOWLEDGMENTS

The authors thanks East Malling Trust for providing funding for LR for her PhD studies to complete this work, the China Scholarship Council for the financial assistance of WF, and Interreg (project: VégéDurable 2) funding for the AMF work in the substrate strawberry.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2016 Robinson Boyer, Feng, Gulbis, Hajdu, Harrison, Jeffries and Xu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.


**1. Introduction**

96 Gel Electrophoresis – Advanced Techniques

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Microsatellite Loci for a Threatened Reef Fish *Plectropomus leopardus*. Conservation

Enzymes are vital biological catalysts with essential action in the metabolism of all living beings. Moreover, enzymes have a very significant role in various industrial sectors, including baking, brewery, detergents, textile, pharmaceutical, animal feed, cellulose pulp biobleaching, biofuels and others (Polizeli et al., 2005; 2009; 2011). Industrial enzymes are especially produced from microorganisms, such as bacteria, yeasts and filamentous fungi.

Here, we describe some basic knowledge about microbial enzymes with potential application in industry, their properties and some biochemical methods for the detection of amylase, pectinase, proteases, and xylanase activities on polyacrylamide gel electrophoresis. These methods are more qualitative procedures than quantitative, once they may be used to confirm the electrophoretic homogeneity of purified enzymes through chromatographic process. In addition, we described the principle of each method approached in this chapter, which grants a better understanding of each procedure.

### **1.1 Overall considerations about industrial enzymes**

Enzymes are optimum biological catalysts present in all living beings, and they, under adequate conditions, catalyze in their active sites the natural substrates from the metabolic route reactions. Quite often, the metabolic enzymes act in a sequence, and the product generated in a reaction becomes the substrate for the following phase, as diagramed:

$$\text{Substrate} \, 1 \xrightarrow{\text{enxyzme1}} \text{Product1} + \text{Enzyme} \, 1 \tag{1}$$

$$\text{Substrate} \newline 2 (= \text{Product1}) \xrightarrow{\text{enzyme 1}} \text{Product2} + \text{Enzyme 2} \tag{2}$$

Some properties of the enzymes make them excellent competitors against traditional chemical catalysts, due to their great catalytic efficacy (kcat), considering that the main

Gel Electrophoresis for Investigating Enzymes with Biotechnological Application 99

Proteases and lipases also have a role in the dairy industry, acting in the production of cheese (Gupta et al., 2002). Chemokine, extracted from the stomach of calves, acts in the Milk coagulation, leading to the formation of cheese. Lipases give the aroma and hot flavor in cheese (Hasan et al., 2006). Still in the food industry, pectinases and cellulases increase the extraction of oils through pressing (coconut, sunflower, soybean,

One sector that has been widely economically explored is the application of amylases in processes of starch saccharification (Silva et al., 2009a, 2009b). For so, there is the need of several enzymes such as α-amylase, which forms maltooligosaccharides; glucoamylases and β-amylases, which hydrolyze starch to glucose and maltose and the glucosyltransferases with the production of cyclodextrins. With synergistic action of the amylolytic system, there is the production of maltose syrup used in breweries, as well as the glucose syrup which is preferably converted by glucose isomerase to fructose syrup, due to the high sweetening power. Such compounds may be used in the manufacturing of sauce, child feeding, gums, candies, ice-cream, pharmaceutical products, canned products etc. Amylases also act in the textile industry to remove starch added to cotton to increase resistance (Gupta et

In the pharmaceutical industry, the use of enzymes is increasingly growing, reflecting the *in vivo* catalysis potential. We can highlight the use of pancreatin, obtained from the swine pancreas, which is used as adjuvant in the digestive process of people who have genetic disorders leading to digestive problems or who, due to surgical removal of the pancreas or precocious aging, present digestive problems. Many enzymes are used as therapeutical agents, such as asparaginase and glutaminase, collagenase, hyaluronidase, ribonuclease,

Semi synthetic penicillins (ampicillin and amoxicillin) were launched in the market to replace penicillin, given the acquired resistance of some microorganisms. All kinds of penicillin have the same basic structure: 6-aminopenicillanic acid (6-APA), a thiazolidine ring bound to a beta lactam that takes a free amino group. In the synthesis of semi synthetic penicillin there is the enzymatic hydrolysis of G penicillin with the penicillin G acylase. After purified and concentrated, the 6-APA released in the hydrolysis is used as an

The enzymes can be used in the cosmetics industry in creams against skin aging and acne,

Enzymes may be used in analytical applications, due to their high specificity, identifying substances in complex mixtures such as blood, urine and other biological fluids. They participate in tests for glucose (glucose oxidase), urea, amino acids, proteins, ethanol, etc

In fine chemistry, the list of compounds produced by enzymatic biocatalysis is huge, and we highlight, as an example, vitamin C and several L-amino acids. Acrylamide is used as a monomer in the production of polyacrylamide, widely used as flocculating polymer. Acrylamide was initially produced chemically, but the technological disadvantages such as the formation of toxic residuals and the costs in the purification process made the enzymatic

intermediary in the synthesis of amoxicillin or ampicillin (Cabral et al., 2003).

streptokinase, uricase and uroquinase (Prakashan, 2008).

buffing cream, oral hygiene and hair dying.

(Godfrey & West, 1996).

way a viable process.

olive, etc.).

al., 2003).

objective of any biological transformation is to obtain a short-term high conversion of substrate into product. Besides this characteristic, enzymes present a high specificity and selectivity, according to their metabolic role, acting in optimum conditions of pH and temperature. Also, they do not pose damage to the environment because they are biodegradable.

Applied biological catalysis had its origins in ancient China and Japan, in the manufacturing of foods and alcoholic beverages in which amylases and proteases of vegetal and microbial origins were employed. It dates back to the end of the XIX century with the introduction of standardized preparations of rennilases in the production of cheese. After this period, the implementation of new industrial applications of enzymes was slow, arising intensively in the last 40 years.

The main reasons for the current importance of enzymes in the industrial scenario are due to the development of application processes of proteases in detergents, pectinases in juices and glucoamylase in the production of glucose from starch. Additionally, the employment of recombinant DNA techniques allowed the obtainment of high productivity and the most suitable design of enzymes.

Following, we present the application of enzymes in several technical sectors justifying the development of methods for the visualization of enzymatic activities in electrophoresis gels.