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9/22/2015 11:10 AM
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Functions, Mechanisms and Regulation of Endophytic and Epiphytic Microbial
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Communities of Plants
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Charles W. Bacon1 and James F. White, Jr.2
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Unit, Athens, Georgia 30605; Email:
[email protected]
USDA, ARS, US National Poultry Research Center, Toxicology & Mycotoxin Research
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Jersey 08901; Email:
[email protected]
Department of Plant Biology, Rutgers University, 59 Dudley Road, New Brunswick, New
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Abstract: Over the past several decades, we have come to appreciate that healthy plants
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host, within and on the surfaces of their tissues, endophytic and epiphytic fungi and
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bacteria that do not cause disease. Individual species (typically endophytes) of plants
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have been found to fall largely into one or more of three major functional groups: 1)
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Microbes that alleviate abiotic stress of the host; 2) Microbes that defend hosts from
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biotic stress (pathogens and herbivores); and 3) Microbes that support the host
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nutritionally through increased nitrogen, phosphorus, iron, etc. This functional aspect of
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plant microbiomes raises the potential to design and construct microbiomes for crop
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plants in order to enhance their cultivation with reduced agrochemical inputs and at lower
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cost. In order to design and construct functional microbiomes, we must first develop an
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understanding of the mechanisms by which plant microbiomes function. Examples of
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hypotheses for the abiotic stress tolerance mechanism include: 1) Oxidative stress
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protection by increased production of antioxidants produced either by the microbes or by
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hosts in response to microbes; 2) Ethylene reduction by production of ACC deaminase;
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and 3) Ammonia or ammonium detoxification and consequent oxidative stress avoidance.
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Mechanisms to explain biotic stress resistance generally include production of anti-
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herbivore or anti-pathogen defensive compounds by the microbe or by the host in
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response to the microbe (i.e., induced systemic resistance). Examples of hypothesized
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mechanisms to explain microbe-mediated enhanced plant growth include: 1) Stimulation
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of plant growth due to growth regulator production by microbes; 2) Increased absorption
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of nutrients by plants from the rhizosphere due to activities of microbes on roots; and 3)
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Increased supply of nitrogen obtained directly from diazotrophic microbes in plants.
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Factors by which plant endophyte communities are regulated are hypothesized to involve
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host-produced compounds that modify behavior of endophytic microbes, often reducing
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growth rates and suppressing pathogenic behaviors. These behavior-modifying
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compounds are proposed to include phenolic acids, quorum quenching compounds, and
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perhaps other secondary metabolites.
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Key Words: Endophyte Community, Plant Stress Tolerance, Pathogenicity Suppression
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1 Introduction Since confirmation of the germ theory of disease by Louis Pasteur in the late 19th
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century, microbes on plants have been primarily studied as causative agents of disease.
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Non-pathogenic microbes in or on plants were largely ignored as contaminants—or
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considered to be latent pathogens or early colonizing saprophytes that would become
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active as plant defenses waned or the plant senesced (Petrini, 1991). However, there
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were anomalous microbes colonizing plants that did not appear to be pathogenic under
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any circumstances. Freeman (1902) described an endophytic fungus inhabiting the
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tissues of the grass Lolium temulentum. Neill (1941) reported presence of a similar
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fungal endophyte in healthy tissue of the grass Festuca arundinacea. At that time no
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harmful or beneficial effects on the host plants could be documented for the endophytes
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and they were largely considered commensals of the plant host (Neill, 1941). Even root
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endophytic arbuscular mycorrhizae were considered commensals or weak pathogens of
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plants (Dangeard, 1900; Koide and Mosse, 2004). To most biologists microbes isolated
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from healthy plants were inconsequential and without any significant ecological function.
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Endophytic and epiphytic microbes comprise the non-pathogenic components of
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the plant microbiome. A close examination of the interactions of these microbes and
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plant metabolome may help us to gain a better understanding of the functions,
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mechanisms and regulation of plant microbiomes. Among the questions that we seek to
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answer in this review are the following: 1) Are plant microbiomes functional or are they
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rather non-functional? 2) What are the functions of plant microbiomes? 3) What are the
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biochemical interactions or signals that regulate behaviors in the community of microbes
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within plant tissues? How are plant microbiomes regulated?
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Endophytes are typically non-pathogenic fungi or bacteria that at some point in their life cycles colonize the interior spaces of plant tissues, including roots, stems,
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leaves, flowers or seeds (Stone et al., 2000; Schulz and Boyle, 2006). Endophytes may
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be restricted as to their distributions and metabolic activities within plant tissues,
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localized in tissues in a nearly dormant phase; or endophytes may be systemic through
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multiple tissues of plants (Rodriguez et al., 2009). In terms of host cell and tissue
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locations, endophytes are largely intercellular; bacterial endophytes in particular may
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become intracellular and enter into host cells in cytoplasm or become situated in
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periplasmic spaces, between the cell wall and the cell plasma membrane (Paungfoo-
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Lonhienne et al., 2010; Thomas and Sekhar, 2014; White et al., 2014a). Epiphytes are
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non-pathogenic fungi, bacteria, or algae that remain restricted to the plant surface without
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interior penetration throughout their life cycles (Zambell and White, 2014).
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A recent concern is the unknown levels of microbial colonizers and precise intra-
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tissue titer of endophytic organisms that inhabit plants. The microbe load is likely to be a
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co-regulated function of both the host and microbes. It is our contention that plants
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produce compounds by which they actively regulate the communities of microbes that
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enter into their tissues. We suggest that phenolic compounds that alter microbe growth
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rates and suppress pathogenicity traits are among the host-produced endophyte regulatory
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compounds. We also suggest that ‘quorum quenching’ compounds may be important in
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modifying endophyte behaviors and maintenance of endophytic communities in plants
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(Fuqua et al., 1994; Miller and Bassler, 2001; Bassler, 2002; Hogan, 2006; Hank and
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Bessler, 2004; Visick and Fuqua, 2005).
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2 Endophytic and epiphytic microbes as functional components of plant
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microbiomes
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With the advent of metagenomic analysis it has become clear that plants are host
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to many microbes that become endophytes or epiphytes of roots or aerial parts of plants
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(Bulgarelli et al., 2013). Further, it has been proposed that many of the microbes are
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recruited from soil populations of microbes in a process that involves plant enrichment of
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the rhizosphere with root secretions followed by colonization by compatible microbes
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into internal tissues of the plant (Zarraonaindia et al., 2015). Other plant microbiome
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inhabitants (e.g., Epichloë endophytes) do not originate in the soil but instead grow
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exclusively on plants (Spatafora et al., 2007; Torres and White, 2009).
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Several decades of investigation of microbial endophytes (Petrini, 1991; Clay,
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1988) and rhizobacteria (Kloepper et al., 2004) have left us with a fragmentary picture of
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the important role that the plant microbiome plays in supporting plant growth and
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survival. Although incomplete, it is now clear that endophytic and epiphytic microbes
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are functional components of the plant microbiome (White et al., 2014b). Endophytic
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microbes have been reported to fall into one or more of three major functional groupings:
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1) Microbes that alleviate abiotic stress of the host plant; 2) Microbes that defend host
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plants from biotic stressors; and 3) Microbes that support the host nutritionally, either
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through increased nitrogen, phosphorus, iron or vitamins. This functional aspect of non-
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pathogenic endophytes and epiphytes of plants raises the potential to design and construct
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microbiomes for crop plants in order to enhance their cultivation with reduced
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agrochemical inputs and at lower costs. In order to synthesize functional microbiomes
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we must first understand how they function.
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3 Abiotic stress alleviation functional group
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Enhanced abiotic stress tolerance due to inoculation with non-pathogenic
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microbes has been noted for mycorrhizae (Hameed et al., 2014), and fungal and bacterial
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endophytes (Malinowski et al., 2005; Waller et al., 2005; Rodriguez et al., 2008; Saraf et
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al., 2014; Gond et al., 2015). Waller et al. (2005) demonstrated that the root endophyte
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Pyriformospora indica enhances salt tolerance in its host plant. Malinowski et al. (2005)
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found that the fungal endophyte Epichloë coenophiala confers increased drought
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tolerance to its grass host. Saraf et al. (2014) and Gond et al. (2015) demonstrated
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enhanced salt tolerance in plants due to infection with bacterial endophytes.
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Mechanistically, the phenomenon of microbe enhanced stress tolerance is not well
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understood. In terms of functions, Rodriguez et al. (2008) proposed that different
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endophytes might adapt plants to different stresses. Evidence in support of this
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hypothesis was that endophytes in coastal plants provided salt tolerance to plants, while
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those near hot springs provided heat tolerance to plants; microbes conferring tolerance to
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one type of stress did not confer tolerance to the other type of stress. We will not
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understand the phenomenon of endophyte enhanced stress tolerance until we understand
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the precise mechanisms of stress tolerance enhancement.
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What exactly are the microbes doing to increase abiotic stress tolerance in plants?
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Several mechanisms for microbe-enhanced plant stress tolerance have been proposed.
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Some examples of proposed mechanisms are as follows: 1) Oxidatave stress protection
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by increased production of antioxidants produced either by the microbes or hosts in
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response to microbes; 2) Regulation of ethylene levels by microbial production of ACC
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deaminase, the enzyme that degrades the precursor of ethylene; 3) Ammonia or
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ammonium detoxification and consequent oxidative stress avoidance; and 4) Osmotic
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adjustment through production of osmolytes by microbes.
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4 Oxidative stress protection via antioxidants This hypothesis came about because it was recognized that many endophytes
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cause an increased expression of antioxidant enzymes and antioxidant phenolic
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compounds in plants. Increased production of antioxidants is important because the
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result of many abiotic stress events is an increase in reactive oxygen in plant tissues
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(White and Torres, 2009). Endophyte-induced increases in antioxidants would counteract
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stress-induced reactive oxygen. Waller et al. (2005) demonstrated that in the infection of
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barley roots with the fungal endophyte Pyriformospora indica the host shows an increase
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in anti-oxidative capacity due to activation of the host’s glutathione-ascorbate cycle.
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White and Torres (2009) suggested that oxidative stress avoidance through production of
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anti-oxidative compounds could account for Epichloë-endophyte enhanced stress
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tolerance in host grasses. The production of antioxidant substances by many endophytic
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microbes (Schulz et al., 2002; Huang et al., 2007) seems to provide further support for
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this hypothesized mechanism. Hamilton et al. (2012) reviewed literature on diverse types
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of fungal endophytes in diverse host plants and found support for the hypothesis that
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endophyte mediation of reactive oxygen may play a key role in endophyte induced
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changes in host plant oxidative stress susceptibility. Hamilton and Bauerle (2012) further
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showed that antioxidant activity in endophyte containing host plants was elevated under
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abiotic stress compared to endophyte-free hosts. These authors hypothesized that abiotic
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stress protection was largely a function of oxidative stress protection.
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5 Enhanced stress tolerance through ethylene regulation From research on the effects of plant growth promoting bacteria (PGPB) on plants
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an elegant mechanism for enhanced stress tolerance was proposed to explain increases in
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abiotic stress tolerance (Saraf, Jha and Patel, 2014). This mechanism is based on the
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production of excess ethylene when plants are under stress. Normal levels of ethylene
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are important for proper growth regulation in plants; stress-generated ethylene in high
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concentrations results in plant growth inhibition, although the precise mechanism is not
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clear (Saravanakumar and Samiyappan, 2007). Some bacterial endophytes that enhance
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stress tolerance produce an enzyme ACC deaminase that degrades the precursor of
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ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC). Degradation of ACC releases
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ammonia and α-ketobutyrate and prevents formation of ethylene. ACC deaminase
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appears to be among the metabolites produced by some non-pathogenic bacterial and
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fungal symbionts of plants. Microbe-produced ACC deaminase cleaves ACC leaving the
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microbe with important nutrients to support growth. It has been proposed that growth
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stimulation of plants bearing some bacteria may be the result of ACC deaminase activity
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where the bacteria remove ethylene that represses plant growth, permitting growth
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stimulation that results from auxin secretion by the microbes. However, experiments that
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involved comparing performance of plants transformed with genes to express ACC
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deaminase to non-transformed plants bearing symbiotic bacteria under stress conditions
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showed that non-transformed plants with bacteria outperformed transformed plants (Saraf
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et al., 2014). This seems to indicate that the mechanism of microbe-enhanced stress
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tolerance and plant growth stimulation may involve more than ACC deaminase.
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6 Ammonia or ammonium detoxification hypothesis This mechanism involves ammonia/ammonium scavenging by endophytic or
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epiphytic microbes. Most fungi and bacteria that associate with plants have ammonia
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transporters that they may use to absorb free ammonia from host tissues and cells.
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Ammonia is one product that builds up in tissues during photorespiration when levels of
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oxygen in plants increase and carbon dioxide levels fall. High levels of ammonia that are
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generated during photorespiration in tissues results in generation of high levels of
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reactive oxygen and oxidative stress (Kiraly et al., 2013). The high affinity of some
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microbial endophytes for ammonia (see White et al., 2015) makes them ideal
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scavengers/detoxifiers of excess ammonia. Reduction in levels of ammonia in plant
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tissues and cells would have the consequence of reducing oxidative stress in plants. One
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interesting aspect of this hypothesized mechanism along with the previous ethylene
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regulation mechanism is that both mechanisms actually relate to mechanisms whereby
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the microbe extracts organic nitrogen from host cells. It is reasonable to expect to see a
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flow of nutrients from plant to microbe and that excesses of any nutrient might be drained
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off by the microbial symbionts, resulting in host cell detoxification.
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7 Stress tolerance through endophyte-mediated osmotic adjustment
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One of the early ideas regarding endophyte-enhanced tolerance to drought or salt
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stress was that it involved osmotic adjustment (Arachevaleta et al., 1989; Elmi and West,
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1995). Osmotic adjustment is the capacity of plant cells to adjust their cytoplasmic
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osmotically active solutes in order to maintain turgor pressure in response to dehydration
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stress (DaCosta and Huang, 2006). In tall fescue infected by the endophytic fungus
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Epichloë coenophiala osmotic adjustment was measured to occur in tiller meristems of
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the grass during periods of drought, enabling the grass to resume growth more quickly
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after the cessation of the period of drought (Elmi and West, 1995). The significance of
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endophyte-mediated enhanced drought tolerance in tall fescue is that it enabled
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cultivation of the grass in drought prone areas where the grass could not be cultivated
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without the fungal endophyte. It has been proposed that secreted fungal endophyte
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alkaloids such as lolines or secreted fungal sugar alcohols (e.g., mannitol or arabitol) may
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be partially responsible for increased osmotic adjustment capacity in tall fescue grass
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(Richardson et al., 1992).
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Endophyte-enhanced salt stress tolerance may also relate to the osmotic
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adjustment mechanism. Recently, Gond et al. (2015) showed that a rhizobacterium
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Pantoea agglomerans conferred tolerance to salt stress in tropical corn and demonstrated
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up-regulation of several plant aquaporin genes. The aquaporins are proteins that function
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in water flow through cell membranes (Peng et al., 2007). It is logical that aquaporins
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and water movement between cells and the apoplast would be critical in adjusting
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osmotic potentials in plant tissues (Peng et al., 2007). In this respect it is interesting to
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note that P. agglomerans has been demonstrated to produce and secrete 1, 3-propanediol,
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a compound that is osmotically active (Barbirato et al., 1996). Whether 1,3-propanediol
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or other secreted metabolites could be osmoprotectants, inducing osmotic adjustment in
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tropical corn has not been ascertained.
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8 Poly-mechanistic microbe-enhanced abiotic Stress tolerance in plants The mechanisms for microbe-enhanced abiotic stress tolerance previously
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described could be considered competing hypotheses. On the other hand, they may not
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be mutually exclusive and multiple mechanisms could be in effect to induce abiotic stress
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tolerance. In this model for microbe-induced abiotic stress tolerance, multiple
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mechanisms could be in operation to differing degrees during stress events to ameliorate
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negative effects on plant hosts. Different mechanisms could be in operation depending
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on the microbes involved. For example, nitrifying bacteria that associate with roots may
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be particularly adept at capturing ammonia and converting it to nitrate. Nitrifiers could
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be working via the ammonia detoxification mechanism in plants that grow in aquatic
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habitats where ammonia may accumulate in high concentrations (Kiraly et al., 2013).
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Most microbes that associate with plants produce auxin. In many of these plant-
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associated microbes, ACC deaminase could be produced to degrade ACC as one avenue
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whereby microbes can extract nutrients from plants. Abiotic stress incited by soil heavy
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metals could be counteracted by antioxidants produced directly by microbes or by hosts
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in response to microbes. In situations where drought severely restricts plant growth
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osmotic adjustment facilitated by microbes might protect fragile meristems in which
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microbes grow so that recovery may occur rapidly once the drought ends. It seems likely
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that microbe-induced abiotic stress tolerance could be the result of more than one
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mechanism. Multiple mechanisms could explain the ‘habitat-adapted stress tolerance
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symbiosis phenomenon’ described by Rodriguez et al. (2008) where microbes adapt host
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plants to particular stresses in the environment of the host. Using the example provided
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by Rodriguez et al. (2008), the mechanisms that adapt a plant to tolerate heat stress may
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be different from the combination of mechanisms that adapt a plant to tolerate salt stress.
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9 Protection from pathogens
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Bacterial endophytes in genus Bacillus are known to produce a suite of antifungal
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and antibacterial lipopeptides, including iturins, bacillomycins, fengycins, and surfactins
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(Gond et al., 2014; White et al., 2014c). Lipopeptides inhibit fungal and bacterial
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pathogens by imbedding in cell membranes and producing pores that make cells leaky
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and prevent cellular growth or metabolic activity. The lipopeptide surfactin is also
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known to inhibit viruses through dissolution of the lipid envelope and the protein shell of
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the virus particles (Ongena and Jacques, 2008). The frequent occurrence of Bacillus
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endophytes in natural populations of plants suggests that many wild populations may
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have disease protection employing lipopeptides, compliments of their Bacillus
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endophytes (White et al., 2014c). Endophyte infection in general may result in
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expression of disease resistance genes in the host plant. In Bacillus endophytes in corn it
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was shown that Bacillus endophytes resulted in induced expression of defense related
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genes in the plant (Gond et al., 2014). In this study Gond et al. (2014) found that corn
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seedlings inoculated with the endophytic bacterium Bacillus subtilis showed an increased
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expression of defense-related genes PR1 and PR4 compared to seedlings that were not
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inoculated with the bacterium. The details of the interactions between endophytes and
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host plants that result in up-regulation of resistance genes are presently unknown.
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Enhanced fungal disease protection has been shown to occur in several grasses
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infected by fungal endophytes of genus Epichloë, including grasses Festuca rubra, F.
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arundinaceae, F. pratensis, Elymus cylindricus, and Lolium perenne (Clarke et al., 2006;
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Li et al., 2006; Gwinn and Gavin, 1992; Wiewiora et al., 2015). Ambrose and Belanger
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(2012) identified antifungal protein genes in fungal endophytes of fine fescue grasses.
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They hypothesized that the antifungal protein genes were a case of horizontal transfer of
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antifungal genes from genera Penicillium or Aspergillus (order Eurotiales), where the
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genes are widespread, into a narrow group in genus Epichloë (order Hypocreales).
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Experiments suggested that the antifungal genes in Epichloë were functional and
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expressed within the grass host.
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Moy et al. (2000) proposed a pathogen exclusion mechanism for Epichloë
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endophyte enhanced resistance to fungal pathogens. This mechanism was based on
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observations of a superficial network of mycelium belonging to the endophytes that
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developed on leaf blade surfaces of grasses. The superficial mycelium was hypothesized
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to be defensive in function, ‘defensive nets’ that physically excluded entry of fungal
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pathogens into leaves (Tadych and White, 2007).
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10 Anti-herbivory effects Anti-herbivory is one of the earliest documented effects in plants infected with
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endophytic microbes. These reports describe in considerable detail toxicity from
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endophyte-infected grasses to cattle, sheep, and other livestock (Bacon et al., 1977;
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Belesky and Bacon, 2009; White et al., 2003). Following these reports, it was shown that
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numerous herbivorous organisms were deterred from consuming endophyte-infected
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grasses, including insects, arachnids, nematodes and mammals (Belesky and Bacon,
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2009; Bacetty et al., 2009a; Bacetty et al., 2009b; Siegel et al., 1991; Rowan et al., 1986;
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Bush et al., 1997). These reports relate to the agricultural importance of endophytes both
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at the ecological and agricultural levels of concern. In this regard, the nature of
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antiherbivory induced by endophyte-infected pasture grasses has received the most
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attention, with most work focused on endophyte-infected-tall fescue and perennial
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ryegrass. The ecological importance of antiherbivory suggests a role in determining host
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species abundance, and perhaps could play a role in host speciation. It has been
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established that numerous ergot alkaloids in the foliage of endophyte-infected grasses are
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defensive in nature, serving to repel herbivores of all types (Lyons et al., 1986; Porter et
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al., 1977). Endophytic species of bacteria and fungi often produce in planta one or more
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compounds that serve to deter grazing, although under strict agricultural management
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natural grazing deterrence is not possible. Cattle and other livestock under pasture
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situations are forced to consume endophyte-infected forage.
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A diversity of biologically active compounds that deter feeding by animals, plant
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pathogens, and other pests are produced by microbial populations within plants. The
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chemistry, pharmacology, and functions as antibiotics, growth enhancers, nematicides,
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and host activity are diverse, complex, and have been reviewed (Belesky and Bacon,
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2009; Porter et al., 1977; Snook et al., 2009; Tanaka et al., 2014; Ongena and Jacques,
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2008), and will not be repeated here.
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Bacterial endophytes are also important in producing defensive compounds that in
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most instances have not been demonstrated to occur in planta. Such strains are however
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used as biocontrol agents to prevent or reduce infections from pathogens particularly on
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crops. In the case of the genus Bacillus, most of the defensive compounds identified to
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date include a combination of isomers related to the lipopeptide and related biosurfactants
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(Grangemard et al., 1999). Thus, we find that the surfactins are found in association with
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the fengycins, and or the iturins, bacillomycin D, and multiple isomers of each (Snook et
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al., 2009; Bacon et al., 2012; Tanaka et al., 2014; Ongena and Jacques, 2008). The
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abundance of each within a plant will exert an overall effect on a pathogen. The biological uses of biosurfactants are primarily those related to the ability of
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surfactins to cause membrane dissolution of pathogenic microbes and this ability is
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effective at very low concentrations. Biosurfactants are effective controls of some Gram-
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positive and most Gram-negative bacteria, and they are anti-viral, anti-fungal, antitumor,
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and anti-mycoplasma (Desai and Bonat, 1997; Heerklotz and Seelig, 2001). The
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revelations that the surfactins at very low concentrations can serve as elicitors of induced
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systemic resistance in plants (Jourdan et al., 2009; Ongena et al., 2007) adds complexity
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to mechanisms of action for the surfactins as biocontrol substances.
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11 Nutritional functions of the plant microbiome It is already clear that mycorrhizal, actinorhizal and rhizobial components of plant
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microbiomes function nutritionally. The ubiquitous mycorrhizal associations have been
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demonstrated to enhance access of hosts to soil reserves of organic and inorganic
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nutrients. Actinorrhizae (also called Frankia) have evolved in just three orders of dicots,
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including Fagales, Cucurbitales, and Rosales; Rhizobia evolved in the dicot order
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Fabales. Actinorhizal and rhizobial symbioses provide nitrogen to host plants via
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nitrogen fixation from the atmosphere (Wall, 2000).
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The mechanism that evolved in actinorhizal and rhizobial symbioses for nitrogen
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fixation involved nodules and restriction of oxygen in the nitrogenase active areas. In
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contrast, many plants bear endophytic and epiphytic bacteria that may be found in all
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parts of plants, but nodules are not formed. Some of these endophytic microbes are
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diazotrophic. It has been proposed that endophytic or epiphytic diazotrophs could
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provide nutrients to plants even though they lack nodules (Baldanicteria and Baldani,
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2005). This process has been called associative nitrogen fixation. However, the
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importance of associative nitrogen fixation to the nitrogen budgets of most plants has
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been disputed (James, 2000). The biggest issue is the question of whether nitrogen fixed
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by microbes actually moves from microbe to the plant host. This uncertainty stems from
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the absence of any clearly articulated mechanism for transfer of nitrogen from microbe to
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plant, along with lack of information on the transfer of nitrogen into plant tissues.
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Although, often plants that have been inoculated with non-pathogenic microbes show
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growth stimulation, this effect is often attributed to growth regulator effects where
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microbes increase root growth resulting in increased soil nutrient absorption. It has been
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difficult to attribute enhanced plant growth to increased nitrogen supply from nitrogen
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fixation rather than increased root capacity. However, recently, Pankievics et al. (2015)
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demonstrated that a diazotrophic and endophytic strain of Azospirillum brazilense
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provided nitrogen to its grass host via ammonia excretion.
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White et al. (2012, 2015) proposed a method for transfer of nitrogen from
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microbe to plant that involved transfer of organic nitrogen to plants. In this hypothesized
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mechanism, referred to as oxidative nitrogen scavenging, microbes secrete enzymes to
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degrade secretions or plant associated substances. Plants secrete reactive oxygen onto
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microbes and their secreted enzymes, and enzymes are then denatured; later plants and
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microbes secrete proteases (Godlewski and Adamczyk, 2007) that further degrade
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denatured enzymes into peptides that may be absorbed by plants and bacteria.
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In order to evaluate potential transfer of nutrients from endophytic bacteria to host plants Beltran-Garcia et al. (2014) conducted isotopic nitrogen tracking experiments. In
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one experiment an endophytic bacterium, Bacillus tequilensis, was labeled with 15N by
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its cultivation in a medium that contained a 15N-labeled nitrogen. The 15-N-labeled
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bacteria were then watered onto plants of Agave tequilana over several months. 15N-
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labeled nitrogen was detected in the chlorophyll molecules in the plant after mass-spec
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analysis. Detection of the 15N label in plant molecules demonstrated conclusively that
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nitrogen in the bacteria passed to the plant. In another experiment comparing absorption
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of 15N-labeled live bacteria to absorption of nitrogen in 15N-labeled heat-killed bacteria,
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it was shown that significantly more nitrogen moved into the plants when bacteria were
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alive than when heat-killed bacteria were used. This result demonstrated that efficient
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movement of nitrogen from microbe to plant was a function of a living endophytic
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microbe and not simply the result of mineralization of bacterial proteins in soils around
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plant roots.
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Another mechanism of transfer of nutrients from microbe to plant was proposed
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by Paungfoo-Lonhienne et al. (2010) who showed that microbes were internalized into
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root cells and degraded over time. These authors denominated the microbe consumption
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process ‘rhizophagy’ because roots consumed microbes (Paungfoo-Lonhienne et al.,
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2013). Similar internalization and degradation of bacterial cells in seedling roots was
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also described by Beltran-Garcia et al. (2014) and White et al. (2014). Thomas and
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Sekhar (2014) observed similar intracellular colonization by bacteria in tissue cultures of
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banana. There is a growing body of evidence that plants may obtain some nutrients by
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direct consumption of microbes or their secreted proteins. However, it is still not clear
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whether direct consumption of microbes or the scavenging of nutrients from microbes is
23
a significant source of nutrients for plants.
18 1
It has been shown that some roots of seedlings do not show gravitropic responses
2
unless microbes colonize roots. White et al. (2015) demonstrated that grass roots do not
3
show gravitropic response unless seed-vectored bacteria are present. We have also
4
observed that Smilax spp. (greenbrier) seedling roots also lose the gravitropic response
5
without the presence of microbes; fungal colonization of seedling roots seemed to restore
6
the gravitropic response of roots (Zambell C. and White JF, Unpublished). The tendency
7
of roots to show anomalous growth suggests that microbes must be present to provide a
8
nutrient or other signal of their presence and readiness to participate in the symbiosis.
9
Proteins or vitamins needed by the plant could easily be a signal that the right microbial
10 11
partners are present. Since Godlewski and Adamczyk (2007) first reported protease activity in roots it
12
has become clear that plants have more options for acquiring nutrients from microbes
13
than was previously believed. It became clear that nutrient transfer to plants did not need
14
to be comparable to that seen in rhizobia or actinorhizae, but instead could involve
15
organic degradation processes. It currently seems evident that plants may acquire
16
nutrients by direct consumption of microbes, but also by consumption of microbial
17
proteins or from ammonia secreted by microbes. Future research will need to evaluate
18
how important these sources of nutrients are for plant development.
19 20 21 22 23
12 Phosphate solubilizing microbes Phosphate is the second most important nutrient for plant growth after nitrogen. Although phosphorus is generally abundant in soils, it is often unavailable for absorption
19 1
in an insoluble mineral or organic complex (Rengel and Marschner, 2005). The
2
phosphorus solubilizing features of microbes include: 1) release of organic acids, protons,
3
siderophores, hydroxyl ions, or carbon dioxide, 2) release of enzymes (e.g. phosphatase),
4
3) organic complex degradation, and phosphate mineralization (Behera et al., 2014;
5
Sharma et al., 2013). In order to identify microbes (fungi and bacteria) that are capable
6
of phosphate solubilization generally a pre-screen is used that consists of tricalcium
7
phosphate (TCP) agar. If a microbe is able to clear TCP it is generally considered to be
8
capable of solubilizing phosphate; however, there are many instances of false positives
9
using this technique, and because of these Bashan et al. (2013) advocated testing
10
potential phosphate solubilizer microbes more rigorously using model plants. Some
11
endophytic and rhizobacterial microbes have been shown to secrete organic acids that
12
degrade rocks and release nutrients that host plants may absorb (Puente et al., 2009). The
13
capacity to degrade rocks to liberate nutrients may be critical for plants that grow on rock
14
surfaces.
15 16 17 18
13 Regulation of the plant endophyte community We hypothesize that plants produce compounds whose function is to regulate
19
endophyte communities by suppressing unrestrained growth and pathogenicity traits of
20
the microbes. An endophytic species, either bacterial or fungal, is housed intercellularly
21
or intracellularly, as groups of cells (or hyphae). The endophyte community typically
22
consists of multiple species of microbes occupying the same habitat (e.g., roots, stems,
23
leaves, seeds, etc.) (Zambell and White, 2014). We propose that individual microbes of
20 1
endophyte communities are under some degree of host control (Persoh, 2015). Important
2
questions in this regard include: How does the host control endophytic communities?
3
And, how are pathogenic members of the community suppressed?
4
We hypothesize that plant-produced secondary metabolites (typically phenolics)
5
are among the compounds that impact on the growth and behavior of endophytic
6
microbes in plants. Supporting this hypothesis is the fact that phenolics in particular are
7
up-regulated in grass plants that contain Epichloë endophytes, when compared to plants
8
that are free of the endophytes (White and Torres, 2009). It seems more than logical that
9
a host would evolve ways to suppress behaviors of endophytic microbial community
10
members that could lead to destruction the host. Such compounds would likely alter
11
behaviors of microbial symbionts with pathogenic capabilities to keep them in a non-
12
pathogenic mode of behavior. Further, virulence suppression is a sustainable way that
13
hosts could deal with microbial symbionts that could revert to pathogenicity, since
14
changing behavior of microbes is less likely than microbe-killing strategies to place
15
evolutionary selective pressure on microbial genomes to evolve resistance to the
16
virulence suppression mechanism.
17
Roots of plants are often highly colonized by microbes that enter into root tissues
18
endophytically (Schulz and Boyle, 2006). Roots also contain high concentrations of
19
phenolic acids (Carvalhais et al., 2011). We suggest a regulatory function for the phenolic
20
organic acids in roots. In nodules of rhizobial plants, host-produced phenolic organic
21
acids are in especially high concentration and have been shown to stimulate production of
22
auxin by the bacteria. Thus phenolic acids produced by hosts change microbe behavior,
23
inducing microbes to produce auxin that likely functions to trigger release of nutrients
21 1
from host cells (Mandal et al., 2010). Tadych et al. (2015) demonstrated that cranberry
2
fruits produced phenolic acids, quinic and benzoic acids, shown to inhibit both fungal
3
growth rates and reactive oxygen secretion by several fruit rot-inducing fungi. Thus,
4
phenolic acids effectively suppressed fungal virulence and cranberry rot disease.
5
An examination of the scientific literature regarding organic acid effects on
6
pathogenic microbes resulted in additional support for a pathogenic suppression role for
7
organic acids (Spratt et al., 2012; Koike et al., 1979; Cowan, 1999). In an examination of
8
bacteria that cause dental caries and gingivitis, phenolic acids from plants, including
9
oxalic, shikimic and quinic acids, were all shown to interfere with pathogenicity related
10
traits of the bacteria (Spratt et al., 2012). Another phenolic acid, caffeic acid (obtained
11
from the consumption of mulberry leaves) in the digestive juice of silkworm larvae was
12
found to suppress pathogenicity of Streptococcus faecalis, a major pathogen and normal
13
component of the gut microbiome of silkworms (Koike et al., 1979). Streptococcus
14
faecalis only becomes a problem when silkworms are fed on an artificial diet free of the
15
precursors that give rise to caffeic acid. Polyphenols (including, quercitin, coumarin and
16
tannins) produced by plants have been found to have anti-microbial activities and have
17
been hypothesized to protect plants from microbial aggression (Cowan, 1999; Jimenez et
18
al., 2015).
19 20
14 Quorum sensing and quorum quenching
21
14.1 Function
22
With regard to regulation of endophytic communities, we propose that phenomena such
23
as quorum sensing are at play in regulating all levels within endophytic and epiphytic
22 1
communities. Quorum sensing is a mechanism based on microbial cell density often
2
expressed in biofilms that regulate the behavior patterns of the contributing cells
3
including production and secretion of virulence factors, development of protective
4
membranes or biofilms, developmental competence, morphological expression, motility,
5
sporulation, and communications system such as bioluminescence. Biofilms are used as
6
predictors of quorum mechanisms, which in most part is based on the historic discovery
7
in bacteria (Fuqua et al., 1994), although such structures are also produced in
8
multicellular organisms such as fungi (Harding et al., 2009; Hogan 2006). Because of
9
quorum sensing, gene expression is regulated, resulting in an array of physiological
10
expressions within a microbiome. Animal pathogens display a variety of quorum sensing
11
systems that control the expression of dozens of specific genes that represent loci for
12
virulence control (Parsek and Greenberg, 2000). It is possible that within the intercellular
13
spaces microbial cell behavior is regulated by quorum sensing similar to the regulation
14
observed in biofilms. Demonstration that biofilms are also produced by both cellular
15
and multicellular fungi suggest the importance of this structure for quorum sensing as it is
16
in bacteria (Harding et al., 2009; Westwater et al., 2005).
17 18
14.2 Occurrence
19
Quorum sensing was discovered in Vibrio fischeri, a Gram-negative marine bacterium
20
(Fuqua et al., 1994; Miller and Bassler, 2001; Bassler, 2002) and this mechanism was
21
later shown to exist in other Gram-negative species (Hornby et al., 2001; Gonzalez and
22
Marketon, 2003; Chen et al., 2004; Raina et al., 2010). The occurrence of quorum
23
sensing is now known to occur in Gram-positive bacteria, yeasts, and other genera of
23 1
fungi (see Albuquerque and Casadevall, 2012; von Bodman, 2003; Hogan, 2006; and
2
Braeken et al., 2008 for reviews). An important component of quorum sensing is
3
signaling and there is evidence that interspecies signaling also occurs (Shank and Kolter
4
2006). It is used to regulate a complex assortment of physiological activities, including
5
symbiosis and pathogenicity (Miller and Bassler 2001; Uroz et al., 2009). For instance,
6
species of Pseudomonas, Agrobacterium, and other genera exhibit quorum sensing-
7
regulated pathogenicity or determinants (von Bodman et al., 2003; Uroz et al., 2009).
8
This suggests that such sociomicrobiological interactions are necessary for maintenance
9
and function within the intercellular milieu. Further, Miller and Bassler (2001) have
10
argued that quorum sensing was one of the earlier steps in the development of complex
11
organisms. Others have attempted, although casually, to indicate and identify quorum
12
sensing in social insects such as ant, and bees (Pratt, 2005; Seeley and Visscher, 2006).
13
The fact that density-dependent regulation in microorganisms serves to regulate behavior
14
of the microbes suggests that quorum sensing is important to survival of the microbes.
15
There are convenient laboratory bioassays and biosensor organisms that are used
16
to detect quorum sensing and quorum-quenching compounds, and these, along with
17
numerous compounds identified from the use of such bioassays and techniques have been
18
previously reviewed (McLean et al., 2004; Kalia, 2013; Koh et al., 2013; Rasmussen and
19
Givskov, 2006; Rasmussen, 2005a,b; Uroz et al., 2009).
20 21
14.3 Quorum sensing and relations to microbes and plant performance
22
Quorum sensing is also interactive with other resistance mechanisms, including oxidative
23
stress resistance (Westwater et al., 2005). Studies using mutants devoid of key
24 1
components of quorum sensing have demonstrated the breakdown in resistance to
2
antibiotics, rapid cell death, and the demise of the population when it is disrupted.
3
Applications of quorum sensing principals have led to the reverse application for quorum
4
sensing: the disruptions of pathogenic organisms for use in plant and human medicines.
5
Further, there are intense searches for quorum quenching or inhibitory compounds with
6
the resulting anticipation of using these for therapeutic control of pathogenic organisms
7
to both plants and animals (von Bodman et al., 2003; Rice et al., 2005; Uroz et al., 2009).
8 9
The mechanism through which quorum sensing occurs employs hormone-like compounds referred to as autoinducers or quorum-sensing metabolites. Thus, it is these
10
substances whose concentrations are directly related to the population density. When this
11
density is exceeded a cascading series of regulatory responses are triggered leading
12
specific genes that are either repressed or derepressed. These expressions lead to events
13
such as antibiotic production, biofilm formation, and production of virulence factors that
14
tend to modulate host reaction.
15 16
14. 4 Quorum sensing metabolites
17
The sensing metabolites or autoinducers responsible for quorum sensing differ in various
18
groups of organisms. Gram-negative bacteria use acylated homoserine lactones as
19
autoinducers, while Gram-positive bacteria use oligopeptides. A universal autoinducer
20
has not yet been identified in fungi. Several studies have indicated that in yeast, and
21
other fungi the autoinducer appears to be the isoprenoid farnesol. Farnesol, which was
22
discovered in Candida albicans, was found to be an effective signaling molecule (Hornby
23
et al., 2001). Farnesoic acid is equally effective. Additional species identified as having
25 1
quorum sensing include Histoplasma capsulatum, Ceratocystis ulmi, Saccharomyces
2
cerevisiae, Crytococcus neoformans, Neurospora crassa and Fusarium species (Severin
3
et al., 2008; Roca et al., 2005; Hornby et al., 2004; Lee et al., 2007).
4
It is speculated that farnesol and farnesoic acid are parts of the system that serves
5
to potentiate the levels of reactive oxygen species (ROS), which has several roles in
6
cellular functions, both negative and positive. Both farnesol and farnesoic acid are
7
stimulatory compounds that produce lag phase during the growth of yeast and function to
8
control ROS-dependent signaling, reducing the deleterious effects produced by ROS.
9 10
14. 5 Microbial quorum quenching metabolites
11
The activity of farnesol in some fungi is best described as quorum inhibiting or
12
quenching, indirectly implying that perhaps there is quorum sensing, and these include
13
Fusarium graminearum, Penicillium spp., Aspergillus fumigatus, A. nidulans, and A.
14
niger (Albuquerque and Casadevall, 2012; Garcia-Contreras et al., 2013; Semighini et al.,
15
2008; Lorek et al., 2008; Dichtl et al., 2010; Rasmussen and Givskov, 2006). Presently,
16
there are well over thirty species of Penicillium that produce penicillic acid and patulin,
17
two quorum-quenching compounds. Most fusaria tested produce the fusaric acids known
18
to be phytotoxins, but which based on structures might also be quorum-quenching
19
compounds, since they are produced in planta in such small amounts that do not produce
20
a phytotoxic response.
21
Other mycotoxins produced by most strains of Fusarium verticillioides include
22
the fumonisins that are sphingolipids and may in fact be quorum signals or quenching
23
metabolites useful for specific metabolic antagonisms during their endophytic life in
26 1
plants. Pathogens such as the fusaria are not obligate biotrophs and probably have a
2
variety of ways of regulating their host defenses during their transient existence with
3
plants. Lipids are reported as being signaling molecules for pathogenic fungi (Shea and
4
Poeta, 2006).
5 6
14.6 Plant quorum quenching metabolites
7
In addition to quorum sensing and quenching metabolites produced by microbes, there
8
are several instances of plant species producing quorum-sensing inhibitors, as well as
9
green and brown algae (Braeken et al., 2008; Uroz et al., 2009; Rasmussen et al., 2005a;
10
Rasmussen and Givskov, 2006; Kalia, 2013). Such quorum quenching compounds are
11
considered to serve as defense against quorum sensing pathogenic species. Mycotoxins
12
such as fusaric acid, penicillic acid, and patulin are now viewed as quorum quenching
13
compounds produced by various fungi that disrupt quorum-sensing regulation induced by
14
pathogenic bacteria (Rasmussen et al., 2005b). Quorum sensing metabolites are sought
15
out for the development of novel compounds for use in preventing infections of plants
16
and animals by pathogens. The chemical structures identified as quorum quenching
17
substances are diverse and most do not have any similarity to the structures identified as
18
quorum sensing or quenching metabolites in microorganisms (Rasmussen et al., 2005a;
19
Rasmussen and Givskov, 2006).
20
Studies of quorum quenching are anticipated to provide alternative ways to
21
control the interactions of microbes with their hosts. New disease control methods will
22
develop from a better understanding of microbiome functioning and regulation, both of
23
which should prove useful in manipulating all plant microbiomes with tremendous
27 1
benefits to agriculture. Additional studies are required to accomplish this goal, which we
2
believe is possible, especially with the use of novel endophytic microbes.
3 4
15.1 Conclusions
5
Plants are colonized by microbial communities that inhabit exterior and interior parts of
6
roots, rhizomes and aerial structures. Many of these non-pathogenic microbes have been
7
shown to modify the way that plants grow and respond to biotic and abiotic stresses. The
8
biological functions of these microbes appear to be enhancement of the ability of host
9
plants to grow, survive and thrive in their particular habitats. Our knowledge of the
10
mechanisms whereby microbes interact with host plants to enhance growth and stress
11
tolerance is still limited. An increased focus on the metabolic interactions of symbiotic
12
microbes and plant hosts is warranted in order to better understand the symbiosis between
13
microbes and plants, and more effectively utilize these widespread mutualisms in
14
agriculture.
15 16 17
28 1
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