A Functional View of Plant Microbiomes: Endosymbiotic Systems That Enhance Plant Growth and Survival

(Chapter for Book: Advances in Endophytic Research (Springer-Verlag)

Eds: VC Verma and AC Gange)

A Functional View of Plant Microbiomes: Endosymbiotic Systems that Enhance Plant Growth and Survival

Authors: James F. White, Jr.1, Mónica S. Torres1, Holly Johnson2, Ivelisse Irizarry1

1Department of Plant Biology and Pathology, School of Environmental and Biological Sciences, Rutgers University, New Brunswick, New Jersey, 08901, USA; 2Department of Engineering Technologies, Safety, and Construction, Central Washington University, Ellensburg, Washington, USA

Correspondence to: J. F. White; email [email protected]

Abstract

Over the past several decades it has become clear that numerous non-pathogenic or weakly-pathogenic microbes inhabit plants both internally and externally. The challenge for plant biologists who study endophytism lies not only in the discovery of endophytes in plants, but also in articulating the precise mechanisms whereby these endophytes function to support the growth and survival of their plant hosts. In this chapter, we discuss the phenomenon of microbial endophytism from a functional perspective. We propose that endophytic microbes in plants comprise a critical and symbiotic part of the plant’s functional systems. We propose three broad categories of endosymbiotic systems, including: 1.) Defensive Endosymbiotic Systems; 2.) Stress Tolerance Endosymbiotic Systems; and 3.) Nutritional Endosymbiotic Systems. We will also consider the potential interactions between endosymbiotic systems of plants. Further, a particular endophyte may serve multiple functions in the ecology of its host plant and predominant functions of an endophyte may change depending on the ecological circumstances affecting its host. We are only beginning to realize how important endophytic microbes are to plants, and we start to approach the study of endophytes not only as individual species associations but as microbial endophyte consortia. Much research remains to be performed to elucidate the roles that endophytes play in modulating host plant ecology.

Key Words: associative nitrogen fixation, reactive oxygen, stress tolerance, defensive symbiosis.

Introduction

Since the development of the germ theory, most scientists, working with plant and animal infecting microbes, have focused their research efforts on microbes that cause disease (Ainsworth, 1981). Endophytes (fungi and/or bacteria that live within tissues of plants) largely appear to be the inverse of plant pathogens since generally they represent cryptic benign infections of healthy plants. Although we cannot cite any statistics, experience with endophytic microbial populations in plants suggests that they outnumber plant pathogens many times to one (Bills, 1996; Suryanarayanan et al., 1998; Arnold et al., 2001). Recent diversity studies of microbial endophytes would seem to support this view (Arnold et al., 2001; West et al., 2010; Lucero et al., 2011; Porras-Alfaro and Bayman, 2011; Zimmerman and Vitousek, 2012). In fact, endophytism is so common among microbes (as compared to pathogenicity), that it may well be that endophytism is the nominal state for most plant-infecting microbes and pathogenicity is the ‘out-of-balance’ condition (Schulz and Boyle, 2005). In this chapter we discuss the current state of knowledge in the domain of endophyte biology and highlight those areas that we believe represent future fertile ground for expanding our understanding of the functioning of ‘endosymbiotic systems’ and their roles biology or ecology of host plants. We further posit that analyzing the phenomenon of endophytism from a functional systems perspective will permit us to develop a better understanding of the ecological context in which these endosymbioses function.

Endophytic microbes have for much of history been perceived to be ‘nigoda’, to use a term borrowed from the religion Jainism. The Jain ‘nigoda’ are microscopic forms of life that inhabit plants and animals, have limited function, and are generally evolutionary dead ends (Jaini, 1998). This describes fairly well how many biologists have viewed microbes found in tissues of plants. Scientists have viewed fungal endophytes in particular as parasites, weak pathogens, or saprophytes that enter plants but cannot function until the host is weakened or senescent (Saikkonen et al., 2004). Other endophytes are speculated to be degenerate pathogens whose life cycles have been curtailed by genetic phenomena or partial host incompatibility resulting in microbes that are trapped in hosts and unable to reproduce or evolve (White, 1988; Schardl and Phyllips, 1997; Schardl and Craven, 2003; Moon et al., 2004). If these models of endophyte functionality were, in fact, correct, endophytes would be largely non-functional and perhaps have only negative impacts on host plants. However, there exists a body of empirically-anchored research that indicates that the reverse appears to be correct. Endophytic microbes are increasingly being found to have positive impacts on host plant fitness, with infections frequently resulting in greater growth, fecundity, herbivore deterrence, disease resistance, and abiotic stress tolerance, etc. (Cheplick and Clay, 1988; Clay, 1988; Bashan et al., 1989; Wilkinson et al., 2000; Malinowski et al., 2005; Malinowski and Belesky, 2006; Waller et al., 2005; Clarke et al., 2006; Feng et al., 2006; Ortíz-Castro et al., 2008; Puente and Bashan, 2009; Álvarez-Loayza et al., 2011; Bacon and Hinton, 2011). Although we previously proposed that fungal endophytes were trapped in host plants, we have since discovered that they actually possess cryptic conidial states on surfaces of plants where they disseminate horizontally (White et al., 1996; Moy et al., 2000; Dugan et al., 2002; Tadych et al., 2012). An increasing body of research further suggests that endophytes are not functionless nigoda at all, but instead

have definable functions in plants and ecosystems (Clay, 1988; Puente and Bashan, 1994; Saikkonenen et al., 1996; White et al., 2001; Rudgers et al., 2004, 2005; Kuldau and Bacon, 2008). It is becoming evident that endophytes are adapted to hosts, express different life cycle stages at distinct stages of host development, and transmit with seeds to succeeding generations of the hosts (Latch et al., 1987; White, 1987; White et al., 1991; Afkhami and Rudgers, 2008; Rodriguez et al., 2009b; Álvarez-Loayza et al., 2011; White et al., 2012a, b). The adaptation to hosts and seed transmission aspects of many endophytes both emphasize the importance of endophytes to their plant hosts. Endophytic microbes, whether bacteria or fungi, inhabit niches within plants that result in enhancements of host fitness and the subsequent ability to enable hosts to colonize and reproduce in a particular ecological niche within a larger ecosystem (Matthews and Clay, 2001; Rudgers et al., 2005; Rodriguez et al., 2009b). Rodriguez et al. (2009a, b) demonstrated that endophytic associations with plants were habitat-adaptive symbioses, serving to enable host plants to survive and reproduce in habitats where hosts could not otherwise grow (see also Redman et al., 2011). Thus, it has become clear that a more accurate and appropriate view of endophytic microbes has emerged: They constitute endosymbiotic systems of plants that enable plants to thrive in particular ecological niches. We believe that one important course for future lines of investigation will be to better define the endosymbiotic systems and elucidate how they function to enable plants to compete with other species and adapt to their environments. In this chapter we will provide evidence to support the existence and functioning of three broad categories of endosymbiotic systems, including: 1.) Defensive Endosymbiotic Systems; 2.) Stress Tolerance Endosymbiotic Systems; and 3.) Nutritional Endosymbiotic Systems. Lastly, we look to move the study of endophytes beyond individual species associations in order to recognize the complexity of interactions between endophytes and effects of microbial endophyte consortia on plants.

1. Defensive Endosymbiotic Systems

There exists a large body of work that supports a defensive function for certain fungal endophytes (Cheplick and Clay, 1988; Lewis et al., 1993; White et al., 1993; Azevedo et al., 2000; Clay and Schardl, 2002; Arnold et al., 2003; Schardl et al., 2004; Spiering et al., 2005; Álvarez-Loayza et al., 2011). Much of this work focuses on various clavicipitaceous fungal endophytes found within grasses. In cool-season and some warm-season grasses, species of the clavicipitaceous fungal genera Epichloë (including Neotyphodium) and Balansia systemically colonize plants and produce alkaloids (and possibly other metabolites) that reduce or alter herbivory (Panaccione, 2005; Panaccione et al., 2006; Clay and Cheplick, 1989).

Certain species of morning glories (Ipomoea spp.; Convolvulaceae) have long been regarded as toxic and avoided by herbivores due to possession of high levels of ergot alkaloids (Austin, 1973). In recent years, it has been shown that ergot alkaloids that toxify certain species of morning glories are produced by clavicipitaceous endophytes or epiphytes (Steiner et al., 2003; Markert et al., 2008; Leistner and Steiner, 2009). Even though much of the work on endophytes in Convolvulaceae has yet to be done, it is difficult to ascribe any function other than defense to these symbionts.

While some controversy arises with regard to defining fungal endophytes universally as anti-herbivorous (Faeth et al., 1999; Saikkonen et al., 1999), it seems

apparent that some do appear to function in this capacity and thus may constitute an herbivory defensive system (Clay, 1988). Herbivory defensive systems may function through production of toxins by the endophytes themselves or through endophyte-induced up-regulation of host defensive compounds. Clavicipitaceous fungal endophytes in grasses have been shown to have a ‘reprogramming effect’ on host plants. Grasses bearing these particular endophytes have increased levels of phenolics and other potential anti-feeding compounds (Waller et al., 2005; Sullivan et al., 2007; Kumar et al., 2009; White and Torres, 2010; Torres et al., 2012). This ‘reprogramming effect’ is a topic of current interest since it may be key to understanding the mechanism of endophyte-host interactions. For any given endophyte-plant association, observed anti-herbivory could be the result of 1.) endophyte-induced anti-herbivore compounds; 2.) endophyte-produced anti-herbivore compounds, or 3.) a combination of both.

Another specific example of a defensive endosymbiotic system is the Diplodia-palm association. Diplodia mutila (Botryosphaeriaceae; Ascomycota) is an endophyte of the neotropical palm Iriartea deltoidea (Álvarez-Loayza et al., 2011). Diplodia is an asymptomatic endophyte in the leaves and stems of mature palm populations. The fruits of the palm transmit the fungus, which often forms a black carbonaceous mycelium on the surface of fruits. Mature plants containing Diplodia were also found to be resistant to stem borer insects. While mature palm plants show no symptoms of infection by Diplodia, the fungus may be mortally pathogenic to seedlings of the palm under certain environmental conditions. In high sunlight conditions (e.g. under the gaps in the rainforest canopy), Diplodia expresses a pathogenic phase where the fungus causes extensive necrosis of seedling tissues to such an extent that many seedlings in high light areas do not survive. If seedlings bearing the endophyte grow in the shaded areas of the forest understory, the fungus does not cause disease. Instead, it remains as an asymptomatic endophytic defensive mutualist of the plant (Álvarez-Loayza et al., 2011). Studies conducted on cultures of Diplodia provide an possible explanation for how light affects the expression of the pathogenic phase of the endophyte. Cultures of Diplodia, that were exposed to high light, were shown to have enhanced secretion of hydrogen peroxide (H2O2). Hydrogen peroxide acts as an important defense signal molecule that triggers a hypersensitive response in some plants that results in cell/tissue death (Heath, 1968). The extent to which endophytes produce hydrogen peroxide in plant tissues appears to determine whether the fungus remains a defensive mutualist or becomes a pathogen capable of killing plant tissues, colonizing them, and later producing conidia and ascospores on the necrotic tissues.

Defensive protection that results from endophyte infection, may include pathogen protection. Bacterial endophytes are frequently found to protect hosts from fungal pathogens (Cho et al., 2007). For example, Clavicipitaceous endophytes in grasses have been shown to protect grass hosts from certain fungal diseases including dollar spot, caused by Sclerotinia homoeocarpa, and red thread disease caused by Laetisaria fuciformis (Clarke et al., 2006, Bonos et al., 2005). Defensive mechanisms against pathogens are generally not clear, but could involve antibiosis-like antagonisms (White and Cole, 1985), physical exclusion phenomena (White et al., 1996), or physical colonization of the pathogens by endosymbiotic microbes. We have observed numerous instances in which fungal hyphae within plant tissues becomes colonized by endophytic

bacteria (Unpublished data). What interactions occur between fungi and bacteria in such circumstances is completely unknown.

2. Stress Tolerance Endosymbiotic Systems

An increasing body of work suggests that many endophytes enhance host plant tolerance to abiotic stresses; however the mechanism that facilitates this enhancement is not clear (Zhang and Nan, 2007; Kuldau and Bacon, 2008; Hamilton et al., 2012). This endosymbiotic system has a parallel in animal biology where it has been shown that intestinal microbes may enhance the ability of animals to cope with stress but the faciliating mechanism also remains unclear (Bravo et al., 2011). It is likely that these endosymbiotic systems in plants increase oxidative stress resistance and enhance host tolerance to soils having high salinity, heavy metals, extreme heat, extremely arid conditions, and various biotic and abiotic assaults to plants that manifest as increased oxidative stress. In animals or plants, enhanced oxidative stress resistance could stem from nutrients that the host obtains directly from the endophyte (Bravo et al., 2011). This is a logical hypothesis to explain increased stress tolerance in plants and cannot be discarded; Unfortunately, we have little evidence for this mechanism at the present time.

Some research suggests that fungal endophytes of plants may produce antioxidants that could modulate oxidative stress through the scavenging of reactive oxygen generated during biotic or abiotic stress events. Endophytic fungi have been shown to be the producers of numerous antioxidant compounds that may play a role in enhancing stress tolerance in host plants (Schulz et al., 2002, Rasmussen et al., 2008). Huang et al. (2007) examined the total antioxidant capacity and total phenolic content of 292 endophytic fungal isolates and demonstrated a high correlation between phenolic content and antioxidant capacity, suggesting that the endophytes themselves may be producing phenolic antioxidants. These investigators identified phenolic acids, flavonoids, tannins, hydroxyanthraquinones, and phenolic terpenoids as potential antioxidants. From the endophyte Pestalotiopsis microspora, the potent antioxidants pestacin and isopestacin have been identified. These compounds scavenge superoxide and hydroxyl free radicals (Strobel and Daisy, 2003).

Fungal endophytes may also produce carbohydrate compounds that have antioxidant capacity. The fungal sugar alcohol mannitol has been shown to have antioxidant activity (Jennings et al., 1998). Richardson et al. (1992) reported higher concentrations of mannitol and other potential fungal carbohydrates with antioxidant activity in the apoplasts of tall fescue grass tissues infected by the endophytic fungus Neotyphodium coenophialum. Mannitol is used by fungi as a common storage sugar and it has been hypothesized that it functions as an osmoprotectant in plants that also produce it. Mannitol is produced by the endophytic pathogen Alternaria alternata. Some scientists have suggested that mannitol suppresses reactive oxygen species (ROS)-mediated plant defense responses in Alternaria’s tobacco host (Jennings et al., 1998). Fungal antioxidants of all forms may contribute to enhance overall oxidative stress tolerance in plants. The ‘habitat-adapted symbiosis’ phenomenon proposed by Redman et al. (2002) could be explained by this mechanism. Here different endophytes may produce antioxidants that differ in their capacities to quench various types of reactive oxygen or may differ in their capacities to reach specific tissues undergoing stress.

Other research proposes a more general mechanism for stress tolerance in endophyte-infected plants. Torres et al. (2012) and Hamilton et al. (2012) proposed that enhanced oxidative stress tolerance in grasses infected by clavicipitaceous endophytes was the result of induced up-regulation of plant produced antioxidants and other stress defensive compounds due to secretion of ROS and auxins by the endophyte into plant tissues. In particular, the secretion of hydrogen peroxide into plant tissues, a known plant defense signal molecule, may be responsible for increasing the readiness of many endophyte-infected grasses to endure biotic and abiotic stresses.

Antioxidants may increase the tolerance of plants to many oxidative stresses and also increase the resistance to pathogens that use ROS to incite disease (Clarke et al., 2006). Therefore, ROS-producing endophytes may increase the hardiness of plant hosts in multiple ways. In some food crop plants, ROS-producing endophytes may increase the nutritional value of the crop by enhancing production of antioxidant nutrients. An example of such an application could be in a crop like cranberries where endophytic fungi are common in fruits and leaves (Jeffers, 1991). Some of these fungi may be latent pathogens and responsible for fruit rot or other diseases, but others appear to be non-pathogenic (Oudemans et al., 1998). In a preliminary study of seven of the most common endophytes in cranberry we identified several that secreted observable amounts of ROS in cultures. The leaf, stem, and fruit endophyte Pestalotia vaccinnii produced notable quantities of superoxides in potato dextrose agar cultures, while the endophytic field rot pathogen Phyllosticta vaccinii produced significant amounts of hydrogen peroxide in potato dextrose agar cultures as well. Several other endophytic pathogens (Colletotrichum gloeosporioides, Physalospora vaccinii and Strasseria geniculata) produced weak reactions to stains for peroxides and superoxides.

An understanding of the mechanism by which endophytic microbes enhance stress tolerance of host plants remains elusive. New approaches to answer the ‘mechanism’ question will most likely involve carefully controlled experiments combined with genome expression analyses to determine precisely what genes in the host and endophyte up-regulate under particular stress conditions.

3. Nutritional Endosymbiotic Systems

Healthy plants are colonized by many different endophytes and epiphytes, both fungal and bacterial (Döbereiner, 1992; James, 2000; Alvarez-Loayza et al., 2011; Bacon and Hinton, 2011; Stone et al., 2000; Fürnkranz et al., 2012; Taulé et al., 2012). The ability of many bacterial endophytes to fix atmospheric nitrogen implicates them as key components of nutritional endosymbiotic systems (Rosenblueth and Martínez-Romero, 2006; Reinhold-Hurek and Hurek, 2011). There is a large body of research on ‘associative nitrogen fixation’ that seeks to determine if plants are obtaining fixed nitrogen from endophytic diazotrophic bacteria (Döbereiner, 1992; Döbereiner et al., 1994; James et al., 1994; Kloepper, 1994; Hurek et al., 1988, 1994; James, 2000; Mantelin and Touraine, 2004; Zhang et al., 2008; Dakora et al., 2008; Magnani et al., 2010). Generally, investigators either use an assay such as acetylene reduction or isotopic nitrogen tracking to ascertain whether gaseous nitrogen is being assimilated into plants (Stewart et al., 1967; Radajewski et al., 2000). However, the question of whether nitrogen moves into plant tissues or remains associated with microbes in any substantial

manner is generally not answered clearly. Other studies focus on plant growth enhancements due to the presence of specific microbes on plants (Kim et al., 2012). This work is complicated by the fact that endophytes frequently produce growth regulators. Furthermore, any growth enhancements in plants may be attributed to the growth regulatory compounds rather than to the nitrogen derived from the microbes (Barazani and Friedman, 1999). Recently, we have found evidence for a mechanism whereby grass seedlings obtain nutrients from seed-transmitted diazotrophic bacteria through oxidation using plant secreted reactive oxygen (White et al., 2012a). We denominated this mechanism ‘oxidative nitrogen scavenging’ (ONS) since the plant employs ROS (specifically H2O2), to degrade microbes and oxidize their constituent protein components prior to proteolysis and absorption. Our studies have centered on documenting ONS in pooid grasses, including Poa annua, Poa pratensis, Festuca arundinacea, Festuca rubra and Lolium perenne. In tall fescue, we have identified two endophytic diazotrophic bacteria that may be part of a nutritional endosymbiotic system, including Pantoea agglomerans and an unidentified species of Pseudomonas both of which are seed-transmitted and colonize germinating seedlings. Bacteria may be transmitted on the caryopsis surface on glumes and paleas that closely adhere to caryopses. All seed collections of these grass species obtained from natural populations or from commercially available samples bear similar diazotrophic bacteria. Our experiments suggest that the bacteria proliferate in meristems of seedlings, but are degraded predominantly on seedling roots to provide organic forms of nitrogen and perhaps other nutrients that are needed for the rapid seedling growth. Roots secrete H2O2 onto bacterial populations on and within roots. Microscopic examination of bacteria on root hairs and other root epidermal cells has shown that the rod-shaped bacterial cells first swell to become spherical, lose their nucleic acid and protein contents, and eventually disappear from plant surfaces. In experiments using grass seedlings, we have found that proper seedling root development depends on the presence of the bacteria on roots of seedlings (unpublished). Using seeds that were rigorously surface disinfected to remove all bacteria, those that were germinated on water agarose medium produced seedlings whose roots did not develop properly when compared to roots germinated from non-surface sterilized seeds. With bacteria present, seedling roots showed proper gravitropic response with roots growing downward into the agarose medium and developed root hairs that extended into the agarose medium. Without bacteria, seedling roots frequently did not grow downward and the few roots that found their way into the medium did not produce root hairs.

In other experiments using similarly sterilized seedlings, we were able to restore proper root development by incorporating 0.1% proteins (egg albumin, lipase or cellulase) into the agarose medium. These simple experiments suggest that the grasses, at least in the seedling stage, require bacteria largely as a nutrient source to fuel early seedling development. The fact that proteins are sufficient enough to restore root development indicates that the effect is nutritional one rather than the result of a microbial-produced hormone that affects development.

There is evidence that ONS, or a more developed phagocytic digestive system to extract nutrients from bacteria, may be widespread in plants. Paungfoo-Lonhienne et al. (2010) demonstrated that tomato plants internalized exogenously applied bacteria into

root cortical cells that were then degraded and their nutrients transported into shoot tissues. We conducted a preliminary survey of seedlings of 23 species of plants in 16 families of vascular plants for evidence of ONS. All seeds used in that study were rigorously disinfected to remove all exogenous bacteria and were subsequently germinated on sterile water agarose medium to reduce any contamination or exogenous nutrients. Under these low nutrient conditions we frequently observed bacteria within vesicles in the cytoplasm of root hairs and root epidermal and cortical cells. Using ROS and protein probes (White et al., 2012a), we confirmed oxidative degradation of the intracellular bacteria, where degrading bacterial cells swelled and lost capacity to stain for protein contents. Although our survey is preliminary, we were able to determine that oxidative degradation of intracellular bacteria is a phenomenon that occurs in many plant species in diverse habitats. Desert plants that show this phenomenon include Agavaceae and Cactaceae. Vines in many temperate and tropical plant families (e.g., Anacardiaceae, Araceae, Araliaceae, Caprifoliaceae, Orchidaceae, Polypodiaceae, Ranunculaceae and Vitaceae) also may rely on nutrient scavenging from bacteria to provide sufficient nutrients to fuel plant development. In vines, bacteria can be visualized in meristematic cells and are distributed intracellularly throughout tissues of plants (Unpublished data). Oxidative degradation occurs generally in tendrils and stem epidermal tissues or aerial roots.

Nutritional endosymbiotic systems may occur where the nutrients obtained from microbes are not strictly nitrogen-based, but, instead, provide other forms of nutrients that plants require. Specific examples here could include biotin, folic acid, niacin and thiamine. When plants are grown axenically in tissue culture these nutrients must frequently be provided exogenously. This proposed function has a parallel in animal systems where bacteria in the gut supply the host with vitamin K, vitamin B12, biotin, folic acid and pantothenate (Hooper, Midtvedt and Gordon, 2002). In a study of whiteflies (Bemisia spp.) and their bacterial endosymbionts (Portiera spp.), the whitefly host was hypothesized to obtain carotenoids from its endosymbionts (Sloan and Moran, 2012). The transfer mechanisms of nutrients from microbes in animals to host tissues are still unknown but they could be comparable to mechanisms in plant systems where endosymbionts are degraded to extract nutrients. Similarities between plant and animal endosymbiotic systems could provide the basis for using plant nutritional endosymbiotic systems as models to understand nutrient acquisition from microbes in animal systems. It is entirely possible that some of the nutrients that we believe are produced by plants may in fact be acquired from endosymbiotic microbes within plants. We hypothesize that most plants obtain at least some of their nitrogenous nutrients from nutritional endosymbiotic systems (White et al., 2012b). However, our preliminary evidence suggests that plants differ with respect to the extent that they rely on endosymbiotic microbes and the ONS process to provide nitrogen. Epiphytes, vines and some desert species appear to heavily utilize nutritional endosymbiotic systems to obtain nitrogen (Unpublished data).

The study of nutritional endosymbiotic systems has the potential to impact fields of agriculture, ecology, evolutionary biology and human health. Nutritional endosymbiotic systems could form the foundation of the nitrogen cycle in ecosystems, such as deserts, where limited available nitrogen is present in soils (Whitford, 2002). A

thorough understanding of these systems could lead to applications in agriculture. For example, new strategies may emerge for cultivation of more efficient food, fuel and fiber crops with reduced inorganic nitrogen applications. From the perspective of evolutionary biology there are several potential impacts. The earliest land plants (e.g., Cooksonia, Sawdonia, Rhynia, Horneophyton, etc…) in the Ordovician, Silurian, and Devonian lacked root systems to absorb nutrients from soils efficiently (Taylor and Taylor, 1993). It is possible that they employed nutritional endosymbiotic systems involving internal oxidation and digestion of diazotrophic bacteria as a source of nitrogen and other nutrients. This possibility could explain why we see oxidation of intracellular bacteria in diverse plant families ranging from ferns to dicots. These early plants are also known to have associated with glomalean fungi that may have functioned like mycorrhizae in assisting with absorption of some nutrients (Taylor et al., 1995). However, at present we know almost nothing about how nutritional endosymbiotic systems work, and further studies must be undertaken to develop our understanding of impacts they have on plant nutrition, development and ecology.  

4. Interactions Between Endosymbiotic Systems of Plants

Plants generally have multiple endophytes and these may constitute multiple endosymbiotic systems. For example, pooid grasses may have clavicipitaceous fungal endophytes in the shoot meristems, leaves and culms that function in defense and perhaps provide stress tolerance. Plants may also contain diazotrophic bacterial endophytes that are oxidized on the surface and interiors of roots to provide nutrients, such as nitrogen, for growth. Thus a particular grass plant individual may possess at least two different endosymbiotic systems, one that is defensive and the other that is nutritional. One of the interesting phenomena with regard to clavicipitaceous endophytes in grasses is that infection will frequently result in enhanced growth of plants compared to plants that are not infected. This effect has been attributed to increased photosynthetic efficiency or to growth regulator production by the fungal endophyte (Spiering et al., 2006). Because some clavicipitaceous endophytes have been documented to produce auxins, increased growth of some hosts has also been attributed to auxin-induced growth stimulation (Yue et al., 2000; Vadassery et al., 2008).

Investigators have only recently begun to examine interactions between symbiotic systems in plants. Novas et al. (2011) examined the effects of a clavicipitaceous endophyte, Neotyphodium, on growth of VA-mycorrhizae, a nutritional symbiotic system, on roots of the host Bromus. These investigators reported an increase in the colonization of mycorrhizae as a result of clavicipitaceous endophyte infection. Many pooid grasses also oxidize diazotrophic bacteria in order to utilize them as a nutrient source (White et al., 2012a). Any enhancement of oxidation of bacteria in roots by the clavicipitaceous endophytes would be expected to increase nitrogenous nutrients available to plants and increase growth. There is some indirect evidence that Neotyphodium coenophialum endophyte infection in tall fescue grass may alter oxidative reactions on roots of the host. Malinowski and Beleski (2005) demonstrated that tall fescue plants bearing the endophyte secreted higher levels of antioxidant phenolics from roots. The phenolics may be secreted to protect plant roots from reactive oxygen secreted by roots onto bacteria. Additionally, Lyons et al. (1990) found that organic and inorganic

forms of nitrogen increased in tall fescue grasses as a result of endophyte infection. Stimulation of nutritional endosymbiotic systems in grasses directly or indirectly by endophytic fungi could account for enhanced nitrogen content and increases in the efficiency of photosynthesis as a result. This is an interesting possibility that will require further experimentation in order to evaluate.

5. Microbial Consortia and Other Factors Affecting Endosymbiotic Systems Nutritional endosymbiotic systems are often composed of microbial consortia. These microbes provide their plant hosts with key nutrients [such a nitrogenenous compounds] that may cause an increase in plant biomass, fecundity, and crop yield. Consortia of nitrogen fixing bacteria have been documented in a number of plant hosts such as wild rice, sugarcane, and grasses (Minamisawa et al., 2004; Miyamoto et al., 2004; Zhang et al., 2008; Taulé et al., 2012). Nitrogen fixing endophytic bacteria typically include common diazotrophic soil species such as Azotobacter diazotrophicus, Azoarcus spp., Pantoea agglomerans, and Klebsiella oxytoca, among many others. However, recent evidence has uncovered the presence of novel endophytic anaerobic clostridia along with the presence of common diazotrophs (Miyamoto et al., 2004; Minamisawa et al., 2004). This finding stresses the importance of utilizing nontraditional microbiological isolation techniques in endophyte research. Future research challenges include the development of isolation and cultivation techniques for these novel bacteria and assessing the diversity of endophytes utilizing culture independent methods that may detect the presence of unculturable or novel microorganisms that have been overlooked previously. Consortia of nitrogen fixing bacteria have the potential of improving plant growth in marginal soils and serve as biofertilizers for agricultural crops, thus reducing the environmental impact of fertilizing practices. How microbial consortia function is unknown, however, it is conceivable that a consortium of endophytic microbes may interact with hosts in order to complete an endosymbiotic system. Any given endophytic microbial consortium may potentially confer more than one benefit to its plant host. Therefore, these systems could be categorized within more than one type of endosymbiotic system. Endosymbiotic consortia may be composed of fungi, bacteria, and even viruses. It seems feasible that favorable combinations of microbial endophytes may have a synergistic effect that could be more efficient at enhancing plant growth than the individual symbionts. Microbial consortia may interact with phytopathogenic fungi or bacteria to complete a defensive endosymbiotic system. This could alter the physiology of the pathogen and induce a nonpathogenic endophytic state, thereby avoiding disease in the plant host. Fusarium oxysporum is commonly documented as a wilt-causing phytopathogen in some plants or a nonpathogenic plant growth promoting endophyte of other plants. Minerdi et al. (2008) determined that the virulence of a pathogenic strain of F. oxysporum can be reduced by a bacterial consortium. Fusarium oxysporum MSA 35 was isolated from wilt-suppressive soils and was observed microscopically to contain numerous bacterial cells attached to the hyphae. The species of bacteria in this consortium were Serratia marcescens, Stenotrophomonas maltophilia, Achromobacter xylosoxidans, and Bacillus halodurans. This consortium had numerous effects on the growth and pathogenicity of F. oxysporum. Among the effects observed were changes in pigmentation, sporulation, aerial hyphae production, and hyphal thickness. Further studies showed that members of this microbial consortium communicate with each other

and their plant hosts through emissions of volatile organic compounds. Volatiles emitted by co-cultures of the fungus with the microbial consortium appeared to promote growth in lettuce, more so than volatiles emitted by F. oxysporum lacking the consortium of bacterial symbionts (Minerdi et al., 2011). The idea that microbial consortia are able to alter the virulence of pathogens that devastate or destroy economically important crops is one that must be further explored. Understanding the mechanisms by which microbial consortia are able to decrease the virulence of specific phytopathogens may be crucial when seeking solutions for disease control. Because of potentially complex population interactions applying this knowledge in order to implement successful biocontrol strategies for agricultural crops vulnerable to disease will be a challenge. Recent research has suggested viruses as possible modulators of stress tolerance endosymbiotic systems. A unique endophytic system was documented by Márquez, et. al. (2007) where a mycovirus infecting the fungus Curvularia protuberata resulted in enhanced thermal tolerance in the host plant, panic grass (Dicanthelium lanuginosum). The Curvularia thermal tolerance virus (CThTV) appeared to alter the physiology of the fungal endophyte. A further study of this three-way symbiotic system indicated that the virus induced the expression of fungal genes that are thought to be involved in thermotolerance. CThTV altered the expression of fungal genes involved in the biosynthetic pathways of various osmoprotectants such as trehalose, glycine betaine, taurine, and melanin (Morsy et al., 2010). The production of these osmoprotectants may be a strategy used by this and many other endophytes to protect themselves from environmental stress, such as increased temperature. Mycoviruses and bacteriophages are often overlooked and may be indispensable parts of successful endosymbiotic systems in plants. Viruses that alter the gene expression of microbial endophytes are likely to differ in a case-by-case basis, and thus the genes they express may differ. It is possible that many endophytic fungi may be infected and altered by the presence of viruses. Understanding specifically how these viruses are able to alter fungal endophyte physiology could lead to the design of microbial consortia that allow their plant hosts to inhabit otherwise inhospitable environments.

Studies have uncovered a number of bacteria that are symbiotic with mycorrhiza and are commonly referred to as 'mycorrhiza helper bacteria' (Bonfante and Iulia-Andra, 2009). Arbuscular mycorrhizal fungi, such as Gigaspora margarita, have been described as a niche for many rhizobacteria, some of them being vertically transmitted endohyphal symbionts (Bianciotto and Bonfante, 2002; Bianciotto et al., 2004). Some species of bacteria described as mycorrhizal symbionts are common soil bacteria such as Pseudomonas aeruginosa and Burkholderia cepacia (Sundram et al., 2011). Other symbionts may be novel species such as the endohyphal symbiont 'Candidatus Glomeribacter gigasporum' (Bianciotto et al., 2003). Mycorrhizas are common endophytes of plant roots that may also be part of a nutritional endosymbiotic system by partnering with certain bacteria.

6. Conclusions

The plant microbiome consists of bacterial and fungal endophytes, many of which have not been identified. In this chapter we advocate a functional view of the plant microbiome where the microbes function to enhance survival and growth of the host

plants. We propose that plant endophytic microbes are critical to plant growth and development, providing nutrients, enhancing stress tolerance and defending plants from herbivores. There is good support for this moderate functional view in the large body of research on endophytes and beneficial microbes of plants. However, we also propose that endophytes were critical to the evolution of plants. Perhaps, without endophytes land plants may never have evolved or would be very different than the plants we see today. Developing a full understanding of plant microbiomes and the endosymbiotic systems of plants may permit us to produce hardier and more resistant food, fiber and fuel crops using reduced agrichemical inputs. 7. References

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Acknowledgements

We are grateful to the New Jersey Agricultural Experiment Station, the Rutgers University Turf Science Center and Central Washington University for resources and financial support.