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Is Quorum Signaling by Mycotoxins a New Risk Strategy for Bacterial Biocontrol of Fusarium verticillioides and Other Endophytic Fungal Species? Charles Wilson Bacon, Dorothy M Hinton, and Trevor R. Mitchell J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03861 • Publication Date (Web): 01 Dec 2016 Downloaded from http://pubs.acs.org on December 1, 2016

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Journal of Agricultural and Food Chemistry

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Is Quorum Signaling by Mycotoxins a New Risk Mitigating Strategy for Bacterial

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Biocontrol of Fusarium verticillioides and Other Endophytic Fungal Species?

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Charles W. Bacon*, Dorothy M. Hinton, and Trevor R. Mitchell

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USDA, ARS, US National Poultry Research Center, Toxicology & Mycotoxin Research Unit,

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Russell Research Center, Athens, GA 30605

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*Corresponding author (Tel: 706/546-3142; FAX: 706/546-3116; [email protected] )

ACS Paragon Plus Environment


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Abstract Bacterial endophytes are used as biocontrol organisms for plant pathogens such as the

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maize endophyte Fusarium verticillioides and its production of fumonisin mycotoxins.

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However, such applications are not always predictable and efficient. In this work, we

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hypothesize and review work that quorum-sensing inhibitors are produced either by fungi or by

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pathogenic bacteria for competitive purposes, altering the efficiency of the biocontrol organisms.

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Recently, quorum-sensing inhibitors have been isolated from several fungi, including Fusarium

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species, three of which are mycotoxins. Thus, we further postulate that other mycotoxins are

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inhibitors or quenching metabolites that prevent the protective abilities and activities of

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endophytic biocontrol bacteria within intercellular spaces. To test the aforementioned

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suppositions, we review work detailing the use of bioassay bacteria for several mycotoxins for

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quorum activity. We specifically focus on the quorum use of endophytic bacteria as biocontrols

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for mycotoxic fungal endophytes, such as the Fusarium species and the fumonisin mycotoxins.

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Keywords: Quorum sensing, quorum quenching, Fusarium, F. verticillioides, mycotoxins,

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fumonisins, endophytes, N-acyl homoserine lactone, AHL

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INTRODUCTION The literature review presented here focuses specifically on studies pertaining to the

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fusaria species that thrive within maize, such as Fusarium verticillioides (Sacc.) Nirenberg

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(synonym, F. moniliforme; teleomorph, Gibberella moniliformis; mating population A of the

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Gibberella fujikuroi species complex). This species and other members of this complex produce

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the fumonisin mycotoxins. The nature of toxicity resulting from consuming this mycotoxin has

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been extensively studied in the context of all species of livestock and poultry, while research has

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recently been extended to human toxicity as well. F. verticillioides is a common contaminant of

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most environments across the planet and, according to the available evidence, can infect over a

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hundred species of plants, most of which are agronomically important.1 In maize, this species

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produces the three analogues or fumonisins, as well as their isomers. Other metabolites include

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fusarin C and fusaric acid. Fusaric acid and its related metabolites seem to be particularly

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damaging when present in concentrations that are phytotoxic, and or in concentrations that are

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required for mammalian toxicity, rather than the lesser plant physiological concentration.2-7 The

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fumonisins are produced by the majority of isolates of the F. verticillioides and its related species

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complex. This distinguishes it from other mycotoxic species such as those of the Aspergillus

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flavus/A. parasiticus complex, in which only a small percentage of isolates produce the aflatoxin

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mycotoxins. Further, previously conducted surveys of fumonisin producing strains within a

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Fusarium population indicate the widespread occurrence of these producing strains,2-7 suggesting

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that this class of mycotoxin serves an important biochemical function for the competitiveness of

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this fungus rather than strictly herbivore protection.

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Numerous post- and pre-harvest methods aimed at preventing the accumulation of the fumonisins and other fusaria mycotoxins in grains and other commodities have been proposed.



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In this context, the use of biocontrol agents is of particular importance for the present study, as

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several of these are endophytic bacteria and fungi. Endophytic fungi are well known for their

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ability to produce structurally and biologically diverse novel compounds with a wide range of

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applications. Hundreds of metabolites display antibacterial and antifungal activities, and several

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classes of these have strong pharmacological applications. We postulate that host-produced

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compounds that modify behavior of endophytic microbes exist within the endophytic

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community, often reducing nutrient predations and suppressing pathogenic behaviors. These

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behavior-modifying compounds are proposed to include phenolic acids, a variety of nitrogenous

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bases and indole compounds, likely along with other secondary metabolites that are interactive as

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inhibitors and stimulators for each symbiont.8-11

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Recently, research on endophytism has extended to the concept of quorum sensing or cell

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density dependent gene regulation and its controls, which have been explored at the community

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and species levels of endophytic fungi of grasses.12 Quorum sensing is a mechanism of

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microbial communication that is driven by cell density and results in a cavalcade of cellular

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behaviors reflecting specific metabolic regulation. Quorum mechanisms are found in both

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Gram-negative and Gram-positive bacteria and are accomplished by the production and sensing

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of small highly diffusible molecules produced within the colony by cells when population

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density reaches some critical level. These signaling molecules are highly specific and

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structurally varied. Since the focus of this review is on plant pathogens and their control, in this

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context, quorum sensing relates directly to the regulation and secretion of virulence factors and

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toxin or secondary metabolite production. In addition to bacteria, quorum activities have

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recently been discovered in fungi and higher organisms, suggesting its importance in evolution.


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Quorum sensing was first discovered in Aliivibrio fisheri, a bioluminescent bacterium

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that is a mutualist with the bobtail squid. However, they produce luciferase and the resulting

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luminescence in concentrated populations only.13 The importance of this quorum activity was

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extensively studied in relation to cell density effects, leading to its subsequent discovery in other

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bacterial species.14-16 Quorum sensing and inhibition has recently attracted considerable

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research interest due to its importance in fungi and other multicellular organisms.12,15,17 In this

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work, however, we postulate that fungi, like bacteria, use quorum regulation to maintain or alter

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population-level behaviors, including symptomless infections, pathological and morphological

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expressions, and reproductive challenges, such as sporulation, spore dormancy, and germination.

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Density dependence is central to the mechanism of quorum sensing.13,18 Since cell density is a

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prerequisite for quorum sensing, it is usually associated with morphological structures such as

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biofilms, which may be substituted by other structures where endophytes are in close

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associations, such as the intercellular spaces of plant. Empirical evidence indicates that

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endophytic bacteria occupy such spaces in large quantities, and this formation has been observed

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along the entire plant axis.19 As quorum sensing is critical to several aspects of cellular behavior,

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such as toxin production, sporulation, and morphological development, it has been the subject of

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several reviews.

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resulting in nutrient acquisition and the synthesis of secondary stress relieving and deterring

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metabolites.20, 25 In sum, quorum sensing is a competitive system that assists in the survival and

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communication of the organisms.

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Quorum sensing is also affected by environmental influences

In this work, we propose that coordinated regulatory signaling between host and

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endophytes takes place within any endophytic species population.8,9,26 Further, within a

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particular host, there are many endophytic species that are in direct competition. One aspect of



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quorum sensing pertains to its inhibition or quenching.27 The metabolites that act as quorum

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inhibitors are abundant and can potentially play a valuable role in the survival of pathogenic

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species. As facultative endophytes, the fusaria are also capable of living as saprophytes, making

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their control difficult, as they thrive on dying and dead plant matter. As this category of

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endophytes has not been as extensively studied as the obligate endophytes of grasses were, the

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information concerning their control is limited and does not extend to the endophytic alternative

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lifestyle. Recently, three mycotoxins have been shown to inhibit quorum sensing

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mechanisms.28,29 In the context of the present work, focusing specifically on fungal endophytes,

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it is particularly important to establish how widespread quorum sensing in fungi is, as well as

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elucidate the quorum activities by other mycotoxins. Studies on quorum sensing are facilitated

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by bacterial biosensor systems that have been developed along with associated mechanisms to

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define and recognize quorum inducing and inhibiting metabolites in other organisms.

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The focus of this review is on a specific class of fungal secondary metabolites, the

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mycotoxins that are suspected of having inhibitory, mimicking, or autoinducing activity through

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quenching or the quorum degradation via the production of specific enzymes. In particular,

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research on the concept of quorum inhibiting or quenching is discussed, along with the pertinent

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studies on its diversity among fungi and the identity of well-known mycotoxins with possibility

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of quorum activities. We also review available work based on using quorum inhibiting activity,

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since this aspect is of particular interest for the development of agronomic biocontrol strategies

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utilizing endophytic bacteria.

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DETECTION AND CHARACTERIZATION OF QUORUM METABOLITES In order to detect quorum inhibiting or quenching mechanisms, quorum signaling and


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quenching must be first analyzed, ideally using biosensor bacteria. As these bacteria are very

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sensitive to signaling and quenching, they can detect signals at the physiological concentrations

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below the nanomole range. Their detection limits have been discussed.32 However, analytical

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methods based on their use do not allow quantitation, and thus produce results that are reported

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as confidence intervals. In order to alleviate this issue, an authentic quorum signaling system

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must consist of cognate signal receptors and regulators, along with specific genes that are

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expressed in a population-dependent manner. While this is considered the definition of quorum

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signaling,30 biosensor activity is usually determined based on measurements of specific bacterial

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properties. More specifically, when bacteria do not inhibit a signaling molecule they secrete,

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which results in a large number of such signaling molecules, this leads to quorum activity, thus

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allowing it to be measured indirectly.

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Although signaling molecules are usually specific to particular bacteria, the well-

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characterized N-acyl homoserine lactones (AHLs) (Figure 1) are typically found in Gram-

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negative bacteria, while some Gram-positive bacteria use small peptides and derivatives such as

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the cyclic dipeptides.31,32 Other bacterial species rely on different types of compounds to control

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population densities, such as butyrolactones, cyclic dipeptides, and bradyoxetin, while

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oligopeptides are used in a few Gram-positive bacteria. Some signaling compounds, such as the

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furanones, are characteristic of both Gram-positive and Gram-negative bacteria. However, as

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will be discussed below, an Australian alga, Delisea pulchra, utilizes a specific furanone as a

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quorum sensing inhibitor, suggesting not only use specificity, but also complexity.

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Biosensor Bacteria. The common bacteria used for either detecting quorum quenching

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or quorum signaling are presented in Table 1. In these biosensing bacteria, quorum sensing is

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constitutively expressed by the production of specific signaling molecules, as well as having



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specific receptors that detect these signaling molecules. These biosensor strains have strict

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cultural and storage requirements, as well as media affinities, that must be respected in

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experimental studies and practical usage.33, 35,36

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A commonly used bioassay species is A. tumefaciens A136 (pCF218) (pCF372),

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synonym WCF47 (pCF218) (pCF372), which requires supplementation of culture media with β-

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galactosidase substrate such as X-gal to visualize any acyl HSL activity.35 However, several

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species, such as Pseudomonas chloraraphis (aureofaciens), Serratia marscesens, Pseudomonas

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aureofaciens, and Chromobacterium violaceum, produce their natural pigments in response to

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quorum sensing. The commonly used pigmented species for quorum inhibition is the wild type

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strain C. violaceum 12472 whose quorum sensing is inhibited by the lack of production of the

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deep violet pigment, violacein. Some nonpigmented mutant strains of this wild type are utilized

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to substantiate aspects of quorum inhibition, including their use as experimental controls

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depending on the design of an experiment. P. chloraraphis is another popular pigmented species

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that produces a yellow pigment.

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Several authors have reported the application of plant and fungal extracts, as well as

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whole organisms such as bacteria and plants, for screening quorum inducing and inhibition

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sensing.26, 32,33-37

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ecological materials such as extracts, sediment, liquid and gaseous environments, and gene

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expression measurements in vivo and in vitro.38,39 At present, several procedures also exist for

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identifying genes controlled by quorum sensing, while methods for measuring gene expression

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within cells have also been reported.18, 32,40-45

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Other procedures have been developed for identifying quorum activity in

Chemical Characterization. The chemical identities of suspect quorum metabolites distinguished using the biosensor bacteria can be identified by the traditional chemical analyses,


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such as gas chromatography, HPLC, and mass spectrometric analyses, or a combination of these.

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Some authors have reported employing TLC based on biosensor bacteria to identify known or

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unknown separated compounds.40, 41 However, these approaches must still be combined with

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those based on biosensor bacteria, as they are usually more sensitive than the instrumental

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methods.40,41,45

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QUORUM SENSING AND QUENCHING ACTIVITIES FROM PLANT METABOLITES

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The cell-to-cell communication discussed above enables the coordination of several

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essential activities and behaviors within a population density by altering gene expression. This is

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accomplished by the production and release of signaling molecules that are characterized by

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great structural diversity. Most can be classified into chemical types, namely: N-acyl

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homoserine lactones (AHLs), referred to as autoinducer-1, characteristic of Gram-negative

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bacteria; and autoinducer-2 (AI-2).36,39 However, it should be noted that other chemically

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different compounds are produced by these and other microorganisms, along with molecules that

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are able to antagonize or inhibit quorum metabolites.

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Plant Quorum Metabolites. As microbial endophytes develop close associations with

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their host, it is important to elucidate the contributions of each to the final expression of

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quenching or sensing by metabolites. This, however, requires identification of vascular plants

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producing quorum-regulating compounds. The results yielded by two screenings focusing on

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vascular plants indicate that they are capable of producing quorum metabolites.10, 29 Several

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natural products generated by most plants act as a protection against quorum metabolites, which

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in some associations results in plant resistance to pathogens. However, to date only a few

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quorum metabolites from plants have been structurally determined, and most of the research



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conducted in this field has focused on quorum-quenching compounds.10, 29,46-49

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the flavanone compound naringin, a glycosylated flavonoid, was shown to be one of the active

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compounds in orange extract that were inhibitory to quorum sensing effects.49 In the red marine

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seaweed Delisea pulchra, halogenated furanones inhibits several components of the quorum

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systems preventing bacteria from colonizing the seaweed.50 Grapefruit extracts are also

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inhibitory to quorum activity posited to be due to furocoumarins, limonoids, pectin, and several

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unidentified components.51,52 Other quorum plant metabolites that have been identified thus far

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include the phenolic-derived malabaricone C from nutmeg, and the powerful isothiocyanate,46,53

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sulforaphane, and quercetin found in broccoli.10

For example,

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The plants of interest for the present investigation, namely maize, as well as grasses in

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general, have not been thoroughly examined for quorum activity. Grasses are well known for

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their biosynthetic inability to produce secondary metabolites, but have achieved this function

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over time by cohabitating with microbial endophytes. The endophyte produces metabolites that

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serve to protect and alleviate biotic and abiotic stresses. Indeed, the close associations of grasses

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with endophytic microorganisms that are rich in the variety of secondary metabolites produced

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have been presented as a driving evolutionary strategy for the symbiotic and mutualistic

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associations in plants in general. Thus, it is likely that the quorum activity control in maize and

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other plants relates to the production of degrading enzymes, as discussed below, while it can also

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stem from inactivating metabolites with a dual function, such as salicylic acid, indole acetic acid,

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and γ-amino butyric acid.

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Plant Quorum Mimics. Some higher and lower plants produce compounds that mimic

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quorum-sensing signals, interfering with processes.22,46 Other plants rely on common

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metabolites exhibiting multiple functions, such as salicylic acid that, when present in very small


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amounts, is a regulating plant defense compound. However, it is also considered effective in

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down-regulating pathogenic attachments and other factors.54 Other plant metabolites, while

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structurally similar to quorum signals, possess quenching activity in one or several sensing

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systems.55 Some of the most potent inhibitors of quorum sensing compounds include N-

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(heptylsulfanylacetyl)-L-homoserine lactone, and 4-nitro-pyridine-N-oxide. However, other

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potent but unidentified compounds have been isolated from extracts of garlic.29 The

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halogenated furanones, L-canavanine, and cinnamaldehyde also exhibit quorum inhibition

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activity. Many more plant and higher organism metabolites have been detected, although their

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structures have not been determined.11 Further, regulation of the mimic signal metabolites is

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based on accumulation and secretion patterns, which determine how and when plants respond to

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specific mimics that target quorum sensing disruption in bacteria.

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Enzymatic Degradations. The in planta degradation of quorum metabolites by

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competing endophytic microbes, such as bacteria and Fusarium species, is likely characteristic of

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specific plant hosts. This assertion is made due to the complex existence of phytoanticipins

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found in maize and other cereals.56,57 For example, a class of hydroxamic acids, which consists

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of two benzoxazinoids that are present in corn and serve as the early defense system for corn

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seedling against insects and fungal pathogens. This class further includes 2,4-dihydroxy-7-

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methoxy-2H-1, 4-benzoxazin-3(4H)-one (DIMBOA), and 2-hydroxy-4,7-dimethoxybenzoxazin-

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3-one (DIBOA). Both are very effective antimicrobial compounds, but pparently do not act as

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quenchers.58 Nonetheless, both are readily degraded by F. verticillioides and Bacillus

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mojavensis into products that may be highly active59, and perhaps as quenchers. Further, the

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potency of both DIMBOA and DIBOA is very high, whereby even very small concentrations are

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sufficient to limit transformation in corn and other cereal monocots, which is a process that relies



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on signaling.58 Available evidence indicates that enzymes that degrade signals also occur in the

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rhizosphere,60 even though these soil enzymes presumably originate from bacteria. Transgene

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studies indicate that quorum sensing degrading enzymes are present in plant hosts, indicating that

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such studies might serve as foci of biocontrols of pathogenic organisms.57 We further postulate

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that they might also control some of the more symptomless fusaria infections, reducing their

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mycotoxin content. Finally, the degradation of AHLs and related compounds has been shown to

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occur in the roots endophytically infected with certain fungi.55

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FUNGI AND MYCOTOXINS: QUORUM SENSING AND QUENCHING ACTIVITIES?

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Quorum sensing was first observed in the marine bacterium Vibrio harveyi in 1973,

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referred to as autoinduction, and in 1977, the chemical signal was discovered in the related

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organism V. fischeri. These discoveries have promoted extensive research in this field, which

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has resulted in thousands of reports on this phenomenon in several bacteria and higher

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organisms, primarily as a social system of communications between bacteria. The regulation of

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virulence factors by this mechanism has attracted the attention of mycologists, who initially

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demonstrated this effect in a yeast species, Candida albicans. Subsequent studies have resulted

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in identification of several species of human pathogenic yeast, suggesting the existence of

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quorum sensing in fungi (Tables 2 and 3).61-66

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Fungal Quorum Inducers. A density-dependent and quorum sensing-like activity, as

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well as related molecules, necessary for specific signaling-dependent functions, has been

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detected in Aspergillus flavus and its mutants during the morphological changes from sclerotia to

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conidial development.67 Derivatives of multicolic acid have been proposed as quorum sensing

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molecules in the fungi Penicillium sclerotiorum,68 and A. terreus.69 Although a universal


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autoinducer has not yet been identified in fungi, findings yielded by several studies indicate that

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the oxylipins, 3,7,11-trimethyldodeca-2,6,10-trien-1-ol, commonly called farnesol, might be

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very common in yeast and other fungi. The oxylipins are a group of oxygenated polyunsaturated

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fatty acids that function as inter- and extra-cellular signals in higher organisms. Owing to their

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structural diversity, they exhibit functions not only in quorum sensing but also in sexual

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development and cell aggregation sporulation processes, among many others.34,66 Oxylipins

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associated with a variety of fungi, including A. flavus, have been reported in the literature, while

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Tsitsigiannis and Keller 66 examined those found in plants, and reviewed their role as sensing

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metabolites. Greater understanding of the effects of this metabolite has prompted research

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focusing on A. flavus, as well as its production of the mycotoxin aflatoxin, although most of the

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studies are based on the related and useful model of A. nidulans.26,70 Knowledge related to

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oxylipins is relevant for the process of aflatoxin accumulation, given that the density-dependant

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nature of quorum sensing in a higher mycelial fungus is of considerable agricultural

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importance.26 The synthesis of sterigmatocystin, a precursor to aflatoxin, and the antibiotic

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penicillin has also been shown to be stimulated by oxylipins.70

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Oxylipins and related quorum sensing metabolites appear to be chemically similar to

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multicolic acid and other related compounds.62, 63 Farnesoic acid and tyrosol have also been

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studied and have been shown to be equally effective as signaling molecules.71 The occurrence of

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farnesol and other oxylipins provides strong but indirect evidence for the existence of quorum

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sensing in fungi, including Histoplasma capsulatum, Ceratocystis ulmi, Saccharomyces

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cerevisiae, Crytococcus neoformans, Gaeumannomyce sp., Ustilago maydis, Penicillium spp.,

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Fusarium spp., Pleurotus sp., Leptomitus sp., Achlya sp., Saprolegnia sp., Mucor spp.,

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Aspergillus spp., Dipodascopsis sp., and Neurospora crassa.61,72 The widespread presence of



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oxylipins suggests that this might be a common family of signaling metabolites in fungi. Fungal Quorum Inhibitor and Mycotoxins. Quorum activities in fungal endophytes

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have been determined from only a limited number of hosts and studies of quorum activity from

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fungal endophytes are particularly scarce. The endophytes of red creeper, Ventilago

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madraspatana, an Asian vine with some medicinal applications, which is also employed in the

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tannin and dying industry, have been determined.79 While fifteen endophytes have been isolated,

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only F. graminearum and Lasidiplodia sp. exhibited high quorum inhibiting activity in the C.

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violaceum test.79 Fusarium graminearum is the producer of the mycotoxins deoxynivalenol,

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zearalenone, and zearalanone, and the quorum activities of these mycotoxins are discussed

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below. Quorum sensing inhibitory activity to C. violaceum CV026 has also been identified in

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extracts of unknown endophytic marine species of Fusarium, Sarocladium, Epicoccum, and

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Khuskia.80 These species were isolated from coral reefs off the cost of Mexico and no quorum

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compounds were chemically identified by the authors.

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To date, quorum quenchers or inhibitors have been studied more extensively than

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signaling, given their greater relevance for practical applications of biocontrol relative to the

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actual mechanisms of sensing. Quenchers can serve the purpose of designer metabolites that are

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highly useful for fungicidal or bactericidal applications. Quorum sensing inhibitors have been

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demonstrated in Penicillium species,15,17,65 that include the mycotoxins penicillic acid, fusaric

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acid and patulin.17,73 Therefore, other species of fungi that produce these metabolites,15,17,65

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should also show quorum activity. Similarly, since most Fusarium species produce fusaric

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acid,74-76 quorum quenching activity in this one genus includes numerous species, reflecting

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common synthesis by them. It is thus posited that quorum quenching may be widespread in

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some genera of fungi. There are also chemical modifications of fusaric acid by the endophytic


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state of one species, F. oxysporum.77 The activities of fusaric acid may be either

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increased/decreased or completely altered to act as a quorum inducer rather than quencher.

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A recent survey of 25 Penicillium species for quorum activity included six mycotoxic

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species, whose extracts produced quorum sensing inhibitors (Table 1).17 The mycotoxins

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potentially produced by these Penicillium species include citrinin, cyclopiazonic acid, penicillic

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acid, chaetoglobosin, viridicatins, citreoviridin, patulin, mycophenolic acid, roquefortine C,

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penitrems A-F, and thomitrems A and E.78 With the exception of patulin and penicillic acid, the

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identity of additional mycotoxins responsible for the quorum inhibitions observed has not been

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determined. Other fungi that have been reported as having quorum sensing inhibitors, defined by

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the decrease in the production of violacein pigment in C. violaceum as described above, are

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listed in Table 1. Some of these are well-known mycotoxin producers, although most of the

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well-known quorum producing fungi are primarily pathogenic yeast species (Table 2).

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Mycotoxin as Quorum Inhibitors. Penicillic acid and patulin were the first mycotoxins

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established as quorum sensing inhibitors using the C. violaceum biosensor test. Although

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penicillic acid is bactericidal, it did not affect the growth rate of the biosensor organisms, as the

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quorum inhibiting effects were a thousand-fold lower than those obtained for the bactericidal

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activity.17 Fusaric acid exhibits phytotoxic properties in high concentrations but has similar

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quorum inhibitory signaling activity on AHLs produced by the biocontrol bacterium

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Pseudomonas chlororaphis, used to control fusaria pathogens. In addition, in higher

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concentrations, it was shown to repress the production of the antifungal metabolite, phenazine-1-

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carboxamine.81, 82 Thus, when plants are infected with the fusaria, the production of fusaric acid

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during the infection suggests that the failure of both Gram-positive and Gram-negative

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biocontrol bacteria might be due to the combined antibiotic effect and quorum quenching activity



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exhibited by competing organisms as well as the host plant.82 Several mycotoxins have been

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tested for quorum inhibition, and their diverse structures are depicted in Figure 3. Such marked

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structural differences and activity relationships suggest that the substance might be a quorum

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sensing metabolite, since some signals at high concentrations can have an antibiotic effect either

331

on competing organisms or on the host, as exemplified by farnesol.65,83

332

Using the biosensor bacterium for AHL quenching activity or inactivating processes, we

333

present some of our unpublished data (Table 3) indicating that, in addition to patulin, penicillic

334

acid, and fusaric acid,29 other mycotoxins were tested for quorum inhibitory activity. The

335

additional mycotoxins selected represented agronomically important toxins, and the tabulated

336

results are based on the ability of the bacterium C. violaceum ATCC 12472 to produce the

337

violacein pigment when grown following the procedure of McLean et al.36 using 5 nmol of each

338

mycotoxin (Table 3). The results pertain to three replicated cultures, repeated twice, whereby

339

the percentage purity of each mycotoxin varied from 98.25 to 99.7%. None of the mycotoxins at

340

the concentration used inhibited the growth of the test bacterium (data not shown), which is the

341

key requirement for quorum metabolites. Three of the mycotoxin tested, namely citrinin,

342

zearalenone, and an equimolar mixture of fumonisin B1 and B2, were inhibitory to the C.

343

violaceum bioassay (Table 3). Another group of mycotoxins, the diketopiperazines, has not been

344

previously tested for quorum activity. However, some mycotoxins exhibiting a close structural

345

similarity to diketopiperazines have been isolated from plants that have been identified as

346

possessing quorum signaling activities.84 Thus, the mycotoxins gliotoxin, roquefortine C and E,

347

and macrophominol are included in the diketopiperazines. However, it is essential to note that

348

the similarities and diversity of structures of these mycotoxins shown in Figure 3 are not

349

indicative or predictive of inhibitory quenching activity observed by the three mycotoxins


Page 17 of 43


17 350

citrinin, zearalenone, and the fumonisins (Table 3). Inhibitory activities of aflatoxin B1,

351

alternariol, cyclopiazonic acid, and ochratoxin A were not observed in this test using C.

352

violaceum. Additional tests with other biomarker strains and species should be tried. A more

353

extensive test using additional concentrations of these and other mycotoxins, along with assays

354

using the companion mutants of C. violaceum, described in Table 1, might yield valuable

355

information on additional activity, such as quorum inducing activity of those scoring negative as

356

inhibitors.

357 358

QUORUM QUENCHING MECHANISMS FOR AGRICULTURAL AND

359

BIOTECHNOLOGICAL APPLICATIONS

360

Biological controls employing endophytes are highly desirable due to the uniqueness of

361

the endophytic habit. However, successful endophytic microbe control is presently hindered due

362

to the presence of at least two issues, namely: lack of appropriate management of the biocontrol

363

agent in diverse environments, both externally and internally; and the inability of the biocontrol

364

agent to colonize and protect the host throughout its growth cycle, culminating in the harvest of

365

the host. Quorum sensors produced by competing pathogenic and nonpathogenic microbes have

366

the potential to mitigate both of these concerns. In most instances, such endophytic microbes

367

have been shown to produce a variety of metabolites that express in planta activity.85-87 The

368

assumption that endophytes can control pathogens via the production of specific metabolites

369

lacks empirical support; nonetheless, this hypothesis forms the basis for the development of

370

some general concepts pertinent to host and endophyte associations.11

371

noteworthy that no studies have been conducted to date on the signaling between host and

372

microbe, confirming the need for production of metabolites, such as antibiotics, when needed, by


It is particularly


Page 18 of 43

18 373 374

the host. Quorum Inhibiting Enzymes. Considerable research on two general and naturally

375

occurring quorum-quenching or inhibiting enzymes isolated from bacteria and eukaryotes has

376

been conducted, and the findings yielded may lead to the development of transgenic approaches.

377

These enzymes belong to the family of well-characterized quorum sensing degrading enzymes,

378

the N-acyl-homoserine-lactone lactonohydrolases.57 In addition to lactonases, oxidoreductases

379

and paraoxonases have also attracted research interest recently.57 Expressions of these acyl-

380

homoserine lactonases regulate the expression of a range of important biological functions, such

381

as virulence genes within their quorum sensing domain. Thus, in transgenic plants or biocontrol

382

microbes, these acyl homoserine degrading enzymes quench the action of quenching signals,

383

blocking pathogenicity and other functions of potential pathogens. Discovery of such novel

384

quorum inhibitors or degradative enzymes indicates that the biocontrol microbes whose

385

physiological responses rely on quorum activity could be further supported. This is a new

386

challenge for investigating the roles of secondary metabolites in host organisms and their use for

387

enhancing biocontrol organisms.

388

Nutrients. In recent research, nutritional concerns of both the host and biocontrol agent

389

have been explored, along with their effects on the pathogenic species. Nutrient acquisition by

390

the biocontrol agent from its host is posited to involve complex regulation processes that rely on

391

key metabolites within the microbiome. Communication is an important part of regulation and

392

species. However, while it is relatively well understood in certain bacteria, no research on

393

communication mechanisms in fungi has been conducted. Using bacteria as the model, some

394

parallels with fungi have been proposed, allowing some analogies to be made. The signaling

395

process is interactive and complex, since it occurs both within and between species, indicating


Page 19 of 43


19 396

the importance of quorum sensing compounds as modulators of microbe–plant interactions.88-92

397

However, while some degree of biocontrol specificity might be required in this process, there is

398

very little evidence that such specificity exists. Signaling and/or host recognitions are

399

interactions that likely determine the success or failure of biological controls based on

400

endophytes. Observing and analyzing interactions under field conditions is difficult, since host

401

plant endophytic microbiome consists of a variety of interacting microbes, ranging from viruses

402

to fungi, and of which are biotrophs. If mycotoxins have a dual role of mammalian toxicity and

403

quorum quenching, the biocontrol bacteria utilized as its target in planta must have a means of

404

overcoming quenching to prevent accumulation of mycotoxins. If this hypothesis is valid,

405

biocontrol agent usage in practical applications is even more complex than presently understood,

406

as it also requires consideration of quenching activity.

407 408 409

FUTURE PERSPECTIVES Mycotoxins are a unique group of quorum quenching molecules whose physiological

410

behavior might resemble that exhibited by fusaric acid, penicillic acid, and patulin. These

411

mycotoxins control or prevent quorum sensing expression of essential genes.17,35,43,80 Thus, if

412

mycotoxin synthesis is to be controlled this activity should be prevented. A program based on

413

the release of AHLs not responding to specific mycotoxins and other metabolites is highly

414

desired. Moreover, the release of excessive amounts of AHLs and other sensing metabolites

415

must not be contingent on a cell density dependent system, i.e., the buildup of cells should not be

416

prerequisite for the production of quorum sensing molecules. However, timing of AHL

417

production is expected to be critical for the overall biocontrol response.

418

In particular, in order to realize fully the benefits from quorum methodology in practice,



Page 20 of 43

20 419

additional knowledge of control points is required, along with the means of increasing or

420

producing quorum inducers within plants, which could potentially be achieved via the

421

application of transgenic technology. In addition the physiological roles, if any, quorum

422

stimulus by the fungus regarding in planta mycotoxin production must also be better understood.

423

Furthermore, inhibitory or quenching systems based on AHLs or AIPs and similar quorum

424

metabolites that are highly competitive with fungal pathogens and their mycotoxins are required.

425

Finally, better understanding of the role of mycotoxins concerning the colonization and infection

426

of plants is needed.

427

An alternative approach might rely on the application of transgenic AHL mimic

428

compounds for host plant transformations that will augment or restore biocontrol activity against

429

mycotoxic-inhibited system with the impact of overwhelming the fungus or pathogen. Such

430

AHL transgenic plants might modify the behavior of pathogenic and or mycotoxic fungi by

431

altering the performance of other bacteria within mixed populations. Further, by producing AHL

432

transgenic hosts, the need for a specific population density for an appropriate signaling response

433

may be overcome. This development might also result in a constant production of AHL, and its

434

regulated behaviors. In addition, an early production of AHL might allow hosts to avoid

435

infection, whereby presence of AHL would be sufficient to prevent host colonization and

436

mycotoxin synthesis. The production of synthetic AHL mimic compounds may prove effective

437

in deterring the fungus inhibitory activity exhibited by mycotoxins, while also prohibiting fungal

438

degradative enzymes from destroying quorum proteins.

439

Current applications of quorum activity, and attempts to prevent both quenching and

440

sensing, are based on their use for control of pathogens and mycotoxic fungi either indirectly

441

with microbes or directly via transgenic techniques. However, in order for quorum sensing


Page 21 of 43


21 442

inhibitors to be useful, they must be tested for each anticipated use to ascertain their

443

effectiveness.73 In other words, positive results in one context cannot be generalized to other

444

mycotoxins. A class of bioactive proteins from the solanaceous plants has been shown to be

445

active as quorum sensing inhibitors against the virulence of one plant pathogenic strain of P.

446

aeruginosa, which can serve as a starting material for the development of novel plant transgene

447

approaches.93

448

Evidence presented in this review suggests that quorum activities, and quorum quenching

449

metabolites in particular, are important candidates for the successful control of pathogenic and

450

mycotoxin organisms.95,96 As discussed in this work, while research on this subject is limited,

451

available data indicate that endophytic microbes have the potential to affect a biocontrol

452

endophytic microorganism’s performance via quorum sensing mechanisms. While further

453

developments in this field require additional knowledge, research reviewed in this work points to

454

the likelihood of developing resistance to the use of quorum suppressing or inhibiting

455

metabolites. Resistance to one quenching metabolite, furanone, was rapidly developed by

456

several organisms due to the emergence of mutations.10,94 Nonetheless, the presently available

457

information on quorum quenching and related activity is insufficient to understand fully the role

458

that mycotoxins and other fungal compounds play in quorum communications. Still, empirical

459

evidence does suggest that gaining such knowledge is vital for better biocontrol exploitation, as

460

well as for a more effective biotechnological uses of quorum mechanisms.

461 462

ACKNOWLEDGMENT

463

We are grateful to Robert J.C. McLean, Department of Biology, Texas State University-San

464

Marcos, San Marcos, TX, for the cultures of the biosensor bacteria, as well as for the insightful



Page 22 of 43

22 465

discussions pertaining to their use and preservation. We recognize and express special thanks to

466

Clay Fuqua, Department of Biology, Indiana University, Bloomington, IN, for the two

467

overproducers of AHL strains of Agrobacterium tumefaciens he developed, while also

468

acknowledging his generosity in supplying us with strains that are providing the thrust for our

469

research in this area.


Page 23 of 43


23

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Figure Captions Figure 1. Structure of quorum sensors. N-acylhomoserine lactone (AHL), 1, from a Gramnegative bacterium, as an example of very active synthetic quorum inducers, N-(3Oxohexanoyl)-L-homoserine lactone, where R = CH3(CH2)COCH2. AIP-1, 2, an example of cyclic peptide signaling molecule from a Gram-positive bacterium.

Figure 2. Structures of some quorum inhibiting mycotoxins. Fumonisin tested as equimolar mixture of fumonisin B1 and B2.


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Table 1. Typical Strains used to Screen for Quorum Signaling and Quenching Activities35, a Strainsb

Purposes

Chromobacterium violaceum ATCC 12472

C. violaceum CV026

AHSL biosensor and wild type; used to detect QSI (quorum sensing inhibitors) by loss of pigments A C6-HSL overproducer that can detect either C4 or C6-HSLs Used to detect either C4 or C6 HSLs

Pseudomonas chloraraphis (aureofaciens)b 30-84

QSI indicator and type strain

Pseudomonas aeruginosa PA01

P. putida pA5-C8 Agrobacterium tumefaciens 12472

Positive control for QSI for C. violaceum 12472 as it produces both C4-, and 3-oxoC12 HSLs ASI use to detect C8-HSLs QSI indicator strain; wild type

A. tumefaciens NTL4 (pCF218)(jpCF372)

Used to detect a range of AHLs

A. tumefaciens A136 (pCF218)(pCF372)

Biosensor for range of acyl HSLs

A. tumefaciens KYC6 A. tumefaciens KYC55 (pJZ372) (pJZ384) (pJZ410)

Positive control for acyl HSL assay, an overproducer of 3-oxo-C8 HSL For detection of acyl HSLs type quorum alternate highly sensitive biosensing strain

Escherichia coli JM109

Used to detect C8-HSLs

Vibrio harveyi BB170

AI-2 quorum signals

C. violaceum ATCC 31532

Serratia marscesensb Used to detect C8-HSLs a All bacteria were obtained from Dr. Robert McLean, Dept. of Biology, Texas State UniversitySan Marcos, TX, that are designed for specific uses.35,36 b

Other strains that produce pigments in response to quorum sensing bioassays. Abbreviations:

AI-2, autoinducer 2; AHLs, N-acylhomoserine lactones; 3-oxo-C6- or C12-HSLs, N-3-oxohexanoyl homoserine lactone; HSLs, homoserine lactones.



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Table 2. Fungal Species Tested For Quorum Sensing or Inhibiting Activity Fungi

Quorum-sensing

Quorum-inhibiting

Penicillium expansum, P. hirsutum, P. italicum, P. olsonii, P. roqueforti

Reference

+

68

+

60, 65,17

a

Fusarium graminearum, F. sporotrichioides, b F. oxysporum and 21 other species known to produce fusaric acid 74 a

Candida albicans, Histoplasma capsulatum Ceratocystis ulmi Saccharomyces cerevisiae Crytococcus neoformans Neurospora crassa a

Aspergillus flavus, A. nidulans

+

43,62

+

43,62

+

43,62

+

62

+

43,62

+

62

+

26

Auricularia auricular Tremella fuciformis Lasidiplodia sp. a

+

+

42

+

42 64

Denotes species known to produce mycotoxins.

b

There are 21 other Fusarium species also known to produce fusaric acid 74; therefore, this

number is expected to increase, reflecting the universal occurrence within the genus and its effect on quorum activity.


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Table 3. Quorum sensing inhibition by Mycotoxins measured with Chromobacterium violaceum 12472.79

Fungus

Mycotoxin

Mammalian toxicity

Quorum activity (Ref) NDa,b

Aflatoxin B1 Aspergillus flavus/A. parasiticus Alternaria spp

Hepatic necrosis, cirrhosis, or carcinoma of the liver.

Alternariol

Cytotoxic, fetotoxic, teratogenic ND b mutagenic, and genotoxic

Citrinin

Citrinin

Nephrotoxic, hepatotoxic, and cytotoxic effects

Inhibitorb

Penicillium spp., Aspergillus flavus, and A. versicolor

Cyclopiazonic acid

Sarcoplasmic reticulum inhibitor, and toxic in high dosages

NDb

Aspergillus ochraceus, A. carbonarius, and Penicillium verrucosum

Ochratoxin A

Carcinogenic, hapatotoxicity, and neurotoxicity

NDb

Penicillium spp, and A. ochraceus

Penicillic acid

Antibiotic, and carcinogenic activities

Inhibitor (17)

Fusarium species

Fusaric acid

Inhibitor(80)

Fusarium species

Zearalenone

Fusarium species

Inhibitory to cell proliferation, DNA synthesis, and phytotoxic. Infertility, abortion, and sexual disorders hepatotoxic and nephrotoxic

Fumonisins (mixture of B1, and B2 analogues) Patulin Genotoxic

Aspergillus spp., Penicillium spp., and Byssochlamys spp. a ND, no inhibition of violaceum production detected compared to controls. b

Inhibitorb Inhibitorb Inhibitor (17)

These mycotoxins were obtained from Romer Laboratories (Saint Louis, MO); the inhibitory

mycotoxins citrinin, zearalenone, and fumonisin had a purity of 99.6%, 99.7%, and 98.3%,



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respectively. Purity of others ranged from 99.9% to 98.7%. All mycotoxins were in solutions of acetonitrile, whereby appropriate amounts were placed in 96-well microtiter plates, and the acetonitrile was evaporated over an 8 h-period under a hood. The resulting residue was taken up in 0.1 ml Luria-Bertani (LB) medium, and each well received 0.1 ml of inoculum, diluted to 1:100 ratio with LB medium, from an overnight culture of Chromobacterium violaceum ATCC 12472. The inhibition of violacein production in a well of each treatment group was based on 48−72 h cultures of the bacterium incubated at 30 °C following the modified procedure of McLean et al.36 Each well received 5 nM of each mycotoxin. Control groups consisted of LB medium and bacterium, and acetonitrile (unpublished).


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Figure 1.

O

1 O

NH

R

O

AHL S H N

2 O

AIP-1

NH S

HO O

NH2

O

O NH

NH HO

O

O

NH

NH OH

NH

O

O O OH



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Figure 2. HO O

O

O

HO

O

OH OH

OH HO

O

O O HO

O

O

Fusaric acid Fumonisin B1

O

OH

N

NH2

O

O

OH

O O

OH

HO O

O O

O

Patulin

Penicillic acid

OH O O O

O OH

HO

OH O

Citrinin

O

Zearalenone


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Bacon et al. Table of Content Graphic


Is Quorum Signaling by Mycotoxins a New Risk ... - ACS Publications

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