Deciphering endophyte behaviour

FEMS Microbiology Ecology, 92, 2016, fiw114

doi: 10.1093/femsec/fiw114Advance Access Publication Date: 23 May 2016Perspective

PERSPECTIVE

Deciphering endophyte behaviour: the link betweenendophyte biology and efficacious biological controlagentsStuart Card1,∗, Linda Johnson1, Suliana Teasdale1 and John Caradus2

1AgResearch Limited, Grasslands Research Centre, Private Bag 11008, Palmerston North 4442, New Zealandand 2Grasslanz Technology Limited, Private Bag 11008, Palmerston North 4442, New Zealand∗Corresponding author: AgResearch Limited, Grasslands Research Centre, Private Bag 11008, Palmerston North 4442, New Zealand. Tel: +64-6-356-8019;Fax: +64-6-351-8032; E-mail: [email protected] sentence summary: Deciphering endophyte behaviour—the link between endophyte biology and efficacious biological control agents.Editor: Gerard Muyzer

ABSTRACT

Endophytes associate with the majority of plant species found in natural and managed ecosystems. They are regarded asextremely important plant partners that provide improved stress tolerance to the host compared with plants that lack thissymbiosis. Fossil records of endophytes date back more than 400 million years, implicating these microorganisms in hostplant adaptation to habitat transitions. However, it is only recently that endophytes, and their bioactive products, havereceived meaningful attention from the scientific community. The benefits some endophytes can confer on their hostsinclude plant growth promotion and survival through the inhibition of pathogenic microorganisms and invertebrate pests,the removal of soil contaminants, improved tolerance of low fertility soils, and increased tolerance of extremetemperatures and low water availability. Endophytes are extremely diverse and can exhibit many different biologicalbehaviours. Not all endophyte technologies have been successfully commercialised. Of interest in the development of thenext generation of plant protection products is how much of this is due to the biology of the particular endophyticmicroorganism. In this review, we highlight selected case studies of endophytes and discuss their lifestyles and behaviouraltraits, and discuss how these factors contribute towards their effectiveness as biological control agents.

Keywords: Plant–microbe interactions; symbiosis; life cycle; stress tolerance

INTRODUCTION

Microorganisms have been administered as biological controlagents (BCAs) for many decades to manage disease and pestpressure on crop plants. Recent advancements within this dis-cipline have demonstrated that the host genome interacts withthe BCA suggesting that new opportunities exist to exploit natu-ral genetic variation in host species to enhance our understand-ing of beneficial plant–microbe interactions and develop ecolog-ically sound strategies for disease control in agriculture (Smith,Handelsman and Goodman 1999). Glare et al. (2012) concluded

that while significant increases inmarket penetration have beenmade by bio-pesticides they still only make up a small percent-age of all pest control products. These authors considered thatthis was due to paucity of bio-pesticide technologies that aretruly transformational with respect to activity spectra, deliveryoptions and persistence of effect and implementation, and as aresult can consistently deliver the desired results. In order forthis to change, an awareness of the importance of these fac-tors coupled with a greater understanding of the interaction be-tween the host plant and the BCA is required. This can vary fromsituations where the host plant offers little or no support to the

Received: 22 February 2016; Accepted: 19 May 2016C© FEMS 2016. All rights reserved. For permissions, please e-mail: [email protected]

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BCA towhere they operate almost as one organism. The future ofbiocontrol may lie in investigations surrounding the latest theo-ries regarding plant–microbe interactionswhereby the plant andall of its associatedmicroorganisms are described as a unit of se-lection in evolution. Variation in this unit, termed the holobiont,can be brought about by changes in either the host or the micro-biota genomes (Zilber-Rosenberg and Rosenberg 2008).

Microbial endophytes associate with the majority of plantspecies found in natural ecosystems and are regarded as ex-tremely important plant partners, with the low stress toleranceexhibited by axenic plants partly due to the absence of thesemicroorganisms (Hallmann et al. 1997; Arnold et al. 2000). Fossilrecords of microbial endophytes date back more than 400 mil-lion years, implicating these organisms in early host plant habi-tat transitions (Remy et al. 1994; Schardl et al. 2008). It is onlyrecently, however, that these endophytes, including their bioac-tive products, have received attention from the scientific com-munity as knowledge is gained surrounding the beneficial traitsthat many of these microorganisms can confer upon their hosts(Azevedo et al. 2000; Schulz et al. 2002; Aly et al. 2010). Traits in-clude promotion of plant growth and survival through the in-hibition of pathogenic organisms and invertebrate pests, theremoval of soil contaminants including heavy metals, and in-creased tolerance to extreme temperatures and low water avail-ability. These beneficial plant symbionts are expected by manyto play an important role in the future of integrated pest anddisease management schemes (Kauppinen et al. 2016).

It is important to ensure that definitions of key conceptsare clear at the start of any scientific publication. Wilson (1995)and Wennstrom (1994) discuss how the term endophyte, origi-nally coined by the German plant pathologist de Bary in 1866,simply means inside the plant, being derived from the Greekwords endon (within) and phyton (plant), and that in this orig-inal form it is vague and meaningless. The scientific commu-nity generally agrees that the organisms in question are com-monly bacteria or fungi, although it should include other plants(such as mistletoe) and viruses (Calvin 1967; Chanway 1996),with the term also encompassing algal, bryophyte and lichenhosts (Stone, Bacon and White 2000). Some authors argue thatthe definition should be more descriptive and encompass infor-mation regarding the type of association between the microor-ganism and its plant host (Carroll 1988;Wennstrom 1994;Wilson1995; Hyde and Soytong 2008; Rodriguez et al. 2009; Card et al.2015; Hardoim et al. 2015; Mercado-Blanco 2015). More specifi-cally, it is argued that thesemicroorganisms, as well as spendingall or part of their life cycle within the plant, should cause noapparent disease symptoms, with the term ‘mutualistic endo-phyte’ used to describe amicroorganism that has been identifiedas also displaying a mutualistic behaviour, at least for part of itslife cycle, and in doing so conferring a benefit on its host. Thisdefinition then makes the term ‘endophyte’ more informativeand excludes many microorganisms that are latent or dormantpathogens or saprophytes (read Sinclair and Cerkauskas 1996for a detailed discussion) and consequently requires researchersthat survey various plant species to acquire more knowledge,such as using microscopic evidence and applying Koch’s postu-lates (Hyde and Soytong 2008). This approach, if followed, woulddiscourage researchers from claiming that all the organismsisolated from surface-disinfected plant tissues are endophytes(Bent and Chanway 2002; Mercado-Blanco 2015) and these or-ganisms should be more appropriately termed leaf or root ‘as-sociated’ microorganisms.

Endophytes are a class of microorganisms that are extremelydiverse, with many forming localised infections in host tissues

such as bark, flowers, roots, stems, leaves and seed. These or-ganisms also exhibit many different biological behaviours withrespect to the type of symbiotic relationship formed with theirhosts and a range of biological lifestyles linked to their life cycles(e.g. their reproductive propagation). This paper reviews thesebiological behaviours and lifestyles and provides case studies ofparticular endophyte genera and/or species in order to demon-strate the advantages and disadvantages of these traits in thedevelopment of BCAs for use in agriculture. Our objective herewas not to produce a list of all the endophytic species found todate (for comprehensive lists read Jumpponen and Trappe 1998;Sturz, Christie andNowak 2000; SanchezMarquez et al. 2012; Sunet al. 2012; Card et al. 2015; Truyens et al. 2015) or to review theevolutionary paths of endophytes (see informative discussionsby Carroll 1988; Saikkonen et al. 1998; Denison 2000; Schulz andBoyle 2005; Zilber-Rosenberg and Rosenberg 2008; Hardoim et al.2015; Saikkonen et al. 2015), but to highlight particular biologi-cal behavioural traits exhibited by certain endophyte species orgroups that underpin specific biological control abilities that ul-timately make these organisms highly suitable as plant protec-tion agents. Many of these endophytes have at some point intime been promoted as commercial products, but not all havebeenmarket-place successes. This reviewwill identify microbesthat in the literature have been shown to have a beneficial ef-fect on plant growth (directly or indirectly) and then determine ifthey have been successfully commercialised. Does commercialsuccess of BCAs rely on certain biological behaviours of thesemicroorganisms? If this is the case, can these behavioural traitsguide future research to identify new microbial endophytes formajor economically important crops and in doing so reducethe need for synthetic chemistry to manage topical pests anddiseases?

ANALYSING THE ENDOPHYTIC LIFESTYLE

The design and development of suitable techniques for screen-ing of potential BCAs is a crucial factor in the detection of bioac-tive microorganisms (Swadling and Jeffries 1996). However, fordevelopment of a commercially successful BCA many other fac-tors have to be taken into consideration. These are generallynot associated with the exhibited bioactivity per se but linked tothe marketability of a product and can include factors such asstability, predictability, storage and application (see Lewis andPapavizas 1991 for an in-depth discussion). Many researchersandmicrobial companies already select specific antagonists thatpossess traits thatwill aid product development andmarketabil-ity. For example, species like Bacillus subtilis are often targetedin biocontrol programmes and selected over other bacteria dueto their endospore producing capability, which removes the sig-nificant formulation challenges associated with BCA stability(Bunt and Swaminathan 2010). Unlike many other biocontrolfungi, many Trichoderma species also produce a secondary sur-vival structure, the chlamydospore. Unlike the short lived coni-dia produced by these and other fungal genera, chlamydosporesare less dependent on exogenous nutrients for germination andmore adapted to withstand unfavourable environmental con-ditions. Many researchers and companies have taken this se-lection process a step further when developing BCAs from en-dophytic microorganisms. Endophytes can overcome many ofthe dilemmas faced by traditional biological control microor-ganisms by being totally encapsulated within the crop, and forthose species that are seed transmitted there is an extra advan-tage for commercialisation because there is no need to develop

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Card et al. 3

complicated formulations and delivery techniques. In addition,endophytic species that remain within the plant tissues for longperiods of their life cycle, protected by their host, also avoid po-tential environmental changes that could threaten and disrupttheir survival and biocontrol efficacy.

It has been a natural extension from these findings to tryand harness endophytes in crops and pastures to improve yieldand persistence. However, relatively few endophytes have beensuccessfully commercialised. Here we determine if this variablesuccess is related to specific traits of endophytes. Endophyticmicrobes exhibit many different biological behaviours and wehave highlighted these within the case studies presented in thismanuscript. We have focused on six main behavioural traits, orlifestyles, that we believe are important for developing a suc-cessful endophytic BCA product. These are (i) the class of biolog-ical interaction (mutualistic vs. commensalistic); (ii) the level ofsymbiotic intimacy (obligate vs. facultative); (iii) in planta coloni-sation patterns (systemic vs. localised); (iv) degree of host speci-ficity (low vs. high); (v) themeans of propagation (vertical vs. hor-izontal); and (vi) the mechanism of biological control by whichthey operate (i.e. antibiosis vs. competition vs. direct parasitismvs. host-induced resistance).

Class of biological interaction

Plants do not live in isolation and instead develop within an ag-gregation of interacting organisms, with the majority of plantspecies associating with diverse groups of microorganisms,ranging from pathogenic to mutualistic (Andrews and Harris2000; Rodriguez and Redman 2008; Yuan, Zhang and Lin 2010;Mercado-Blanco 2015). The term symbiosis is used to describea close and often long-term interaction between two differentbiological species, and is derived from the Greek words simplymeaning ‘living together’ and originally coined by de Bary (1879).However, there still remains some debate on the exact defini-tion of the term (Schulz et al. 1998; Martin and Schwab 2012). Forthe purpose of this review we have accepted the broader defini-tion that incorporates the spectrum of possible persistent rela-tionships (parasitic, amensalistic, commensalistic and mutual-istic) between all species. As this paper is concerned with ben-eficial endophytes we will obviously not discuss relationshipsthat are detrimental to the host (i.e. amensalistic or parasiticrelationships).

Commensalistic symbiosis is a class of relationship betweentwo organisms where one organism benefits from the otherwithout affecting it (i.e. no detrimental effects) whilemutualisticrelationships are relationships between two organisms whereboth organisms benefit. Many of our case studies can be classi-fied as mutualistic endophytes; for example, rhizobia form rela-tionships with legumes, whereby the plant gains ammonia oramino acids from the bacteria and in return the bacteria re-ceive organic acids (principally as the dicarboxylic acids malateand succinate) as a carbon and energy source. Another clas-sic mutualistic association, especially important for plants lo-cated in low-fertility soils, comprises the highly evolved sym-biosis between fungi in the division Glomeromycota and theroots of many species of vascular plant, together termed ar-buscular mycorrhizae (derived from Greek: mykos, ‘fungus’, andriza, ‘roots’), formerly known as vesicular–arbuscular mycor-rhizae (VAM) (Schußler, Schwarzott and Walker 2001; Walkerand Schußler 2004). Arbuscular mycorrhizal fungi help plantsto capture water and nutrients such as nitrogen, phosphorus,sulphur and certain micronutrients from the soil. In return, thefungi acquire their carbon from the plant as they are unable to

assimilate this themselves. The sebacinoid root-associated fun-gus Piriformospora indica forms non-mycorrhizal mutualistic in-teractions with a diversity of plant hosts and confers a multi-tude of host benefits that have been extensively researched andreviewed (Baltruschat et al. 2008; Oelmuller et al. 2009; Franken2012; Qiang et al. 2012; Ansari et al. 2013; Oberwinkler et al. 2013;Johnson, Alex andOelmuller 2014). Asexual Epichloe species formpermanent mutualistic relationships with certain grass speciesfound in the sub-family Pooideae. This symbiosis is thought tohave co-evolved over 40 million years (Schardl et al. 2008), withthe fungus imparting beneficial attributes to their host as theyenhance the fitness of the grass when under biotic and abioticstresses (Malinowski and Belesky 2000; Popay and Bonos 2005;He et al. 2013). In return, the fungus obtains a suitable habitat,nutrition from the plant and an efficient means of dissemina-tion to successive host generations as it colonises newly formedtillers and seed (Card et al. 2014).

The importance of each type of symbiotic interaction (mutu-alistic vs. commensalistic) is not fully understood with respectto producing successful endophytic BCAs, probably as the rela-tionship betweenmany endophytes and their hosts has yet to befully elucidated. We can, however, deduce from our case stud-ies that certain behavioural traits do interlink. For example themajority of mutualistic associations tend to be more long-termas each partner benefits from the symbiosis, and many are alsopermanent as each partner is largely dependent on the other inone way or another for survival. Mutualistic asexual Epichloe en-dophytes are completely dependent on their grass partners asthey cannot survive outside of their host plant (except for artifi-cial laboratory conditions) due in part to their slow growth ratescompared with faster growing foliar and soil saprophytes. Theyalso lack an initial infectionmechanism required in order to gainentry into a plant host and also possess nomechanism of propa-gation (these fungi are exclusively vertically transmitted via hostseed) (Christensen andVoisey 2007; Christensen et al. 2008; John-son et al. 2013). These fungi then impart beneficial attributes totheir host as they enhance the fitness of the grass when un-der biotic and abiotic stresses and under certain circumstances,such as under high pest pressure, these fungi would be a neces-sary component of survival for a particular grass population.

Level of symbiotic intimacy

Plant–microbial symbiotic relationships can also range from ob-ligate to facultative. Obligate endophytes are those that displayno free-living stage and depend entirely on their host for all oftheir needs, spending the majority or all of their life within theplant. The best example from our case studies of obligate endo-phyte associations are the asexual Epichloe species that formmu-tualistic symbioses with cool season grasses. These endophyteshave been extensively researched and the genus contains strainsthat form the basis for many commercially successful biologi-cal control products (Saikkonen et al. 1998; Schardl, Leuchtmannand Spiering 2004; Rodriguez et al. 2009; Schardl 2009; Johnsonet al. 2013; Pennell et al. 2016). In addition, some of the mostnotable products from obligate fungi are found in the divisionGlomeromycota, which form symptomless associations with ca.two-thirds of all land plant species (Hodge and Storer 2014). Al-though many authors do not regard these fungi as endophytes,they colonise the intracellular regions of plant roots, produceno host symptoms and provide the plant with beneficial traits,the most notable being the transfer of nutrients from the soil tothe host in exchange for photosynthetically fixed carbon (Azcon-Aguilar and Barea 1996; Smith and Read 2008). Another group of

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root-associated facultative endophytes that are becoming moreintensely studied are the dark septate endophytes (DSEs), socalled due to their characteristic melanised hyphae. Unlike my-corrhizal associations, however, little is known about the taxon-omy, host range and level of symbiotic intimacy of these dema-tiaceous fungi (Jumpponen and Trappe 1998). DSEs include anunknown number of fungal taxa, conidial as well as sterile, thatcolonise living plant roots either intracellularly or intercellularly(Jumpponen 2001). Some species have potential biocontrol abil-ities when forming symbiotic associations with a range of planthosts (Narisawa, Tokumasu and Hashiba 1998; Narisawa, Usukiand Hashiba 2004; Lahlali et al. 2014); however, no DSE has yet tobe commercially developed for the purpose of plant protection.

Alternatively, the life cycle of a facultative endophyte does in-clude a free living stage outside of the plant host. This stage cantake place in a variety of locations: still in association with thehost plant, such as the rhizosphere or phyllosphere; in associa-tionwith the rhizosphere or phyllosphere of an alternative plantof the same or different species; or within a separate host, suchas an insect. This group comprises themajority of our listed casestudies that are entomopathogens and require an insect host tofulfil their life cycle, e.g. Beauveria bassiana, those species thatassociate primarily with the rhizosphere of many plant species,e.g. B. subtilis, P. indica, and Trichoderma species, and rhizobia thatassociate exclusively with species of legume (family Fabaceae).In the case of some facultative endophytes, particular environ-mental variables can impact on the ability of the endophyte toprovide a benefit. In comparison, obligate endophytes are pro-tected from many environmental variables including damag-ing ultraviolet light, extreme temperatures, water deficits andcompetition for space and nutrients from other faster growing,antagonistic microorganisms. However, some commercial chal-lenges still exist to produce marketable products from obligateseed-transmitted endophytes. For instance delivering a qualityseed product containing asexual Epichloe endophytes requiresconstant maintenance to ensure a high level of endophyte via-bility and a chemical profile that is true to type (Easton et al. 2001;Rolston and Agee 2007). In addition, there is currently a lack ofprediction regarding compatibility when developing novel or ar-tificial grass–endophyte associations (Easton 2007), with someof these associations resulting in a less than anticipated alka-loid expression profile, and the worst case scenario being totalloss of endophyte from the host or complete death of the plant.

Many facultative fungal mutualistic endophytes that displaybioactivity have been identified. However, as with the encom-passing discipline of biological control, there are few commer-cial developments of facultative fungal endophytes for use inagriculture. At this stage in our scientific understanding, we be-lieve that facultative endophytes may pose greater challengescompared with obligate endophytes from a commercial devel-opmental and marketing viewpoint. These include the require-ment for:

� strategies to bring the endophyte and host plant together sothe symbiosis can occur;

� survival of the endophyte outside the plant, often in a com-plex soil environment;

� often, an appropriate matrix of environmental and geneticfactors to be aligned for successful colonisation and estab-lishment of a fungal microbe as an endophyte.

In planta colonisation patternsThe growth and development of endophytic microorganismswithin their plant hosts can generally be either systemic or lo-

calised (termed by some researchers as simply non-systemic orcausing ‘point infections’) and may have varied influences onthe endophyte–host interaction (Boyle et al. 2001). These coloni-sation patterns can be further classified depending on the planttissue(s) involved in the symbiosis. For examplemany species ofsymbiotic rhizobia and Glomeromycota are located only withinlocalisedmembrane compartments formed on their plant host’sroot systems (Parniske 2000). Rhizobia form populations withinthese specialised compartments, or nodules, only after succes-sive processes induced by the bacteria (Denison 2000). In con-trast, many endophytic members of the tribe Balansieae (fam-ily Clavicipitaceae) systemically infect plant hosts in the fam-ilies Cyperaceae, Juncaceae and Poaceae (Clay 1989, 1990). Forexample, asexual Epichloe species systemically colonise all theabove-ground tissues of their grass (sub-family Pooideae) hostsfrom the seed germination phase to host flowering, fertilisa-tion and seed set and are seen by some to behave more likea plant tissue, being highly regulated and synchronised withplant growth, than an invading microorganism (Tan et al. 2001;Christensen and Voisey 2007; Christensen et al. 2008). Thesefungi exhibit little hyphal branching and grow parallel with thelongitudinal leaf axis within the intercellular spaces of theirgrass hosts where they actually adhere to the plant cell walls(Christensen and Voisey 2007; Christensen et al. 2008). Addi-tionally, there are certain endophytes that neither grow in spe-cialised compartments nor grow systemically, i.e. the many en-dophytes that have limited growth in planta, which can be inter-and/or intracellular, e.g. cryptic associations (Stergiopoulos andGordon 2014).

From the literature and our institute’s (AgResearch Limited,New Zealand) experience with a wide range of fungal endo-phytes, particularly Epichloe (Johnson et al. 2013), the questionremains, how important is the in planta colonisation pattern ofan endophyte with respect to its biological control efficacy? Al-though the mechanisms of action of many endophytic microor-ganisms have yet to be elucidated (discussed further in this sec-tion), it is clear that many do not come into direct contact withtheir target pest or pathogen. Instead they operate by more in-direct methods of biological control. Yan et al. (2015) have ar-gued that systemic colonisation is not a requirement for antago-nistic interactions between endophytes and potential pests andpathogens. Their rationale is thatmany endophyticmicroorgan-isms capable of biological control rely largely on antibiosis astheir dominant mechanism of action, and therefore it is morelogical to understand the mobility, quantity and quality of thesesecondary metabolites within the plant. Studying these com-pounds in situwill then determine the endophyte’s effectivenessfor inhibiting, deterring or killing an invading pest or pathogen.For example, selected grasses infected with asexual Epichloe en-dophytes deter many leaf-, stem- and phloem-feeding insectsby relying on the production of alkaloids that are concentratedin the stems, leaves and seed of their host. These are the sameareas that the fungus systemically colonises. However, high con-centrations of some of the bioactive alkaloids produced by thisassociation, for example up to 1937 μg/g total loline, occur inthe roots of the grasses (Patchett et al. 2008), a region within theplant where Epichloe hyphae are not found (Hinton and Bacon1985). For those endophytes that have been shown to elicit aplant host response (induced systemic resistance; ISR), such asP. indica (Stein et al. 2008; Molitor and Kogel 2009), Heteroconiumchaetospira (Morita et al. 2003) and myriad endophytic bacterialspecies (reviewed by Kloepper and Ryu 2006), the colonisationpatterns and distribution of these endophytes may be even lessimportant with respect to their biocontrol efficacy.

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The majority of endophyte classes and species describedin the literature have been documented as non-systemic andthis is probably related to their ubiquitous nature (Canals et al.2014). This includes fungal species isolated from every plantspecies studied to date, e.g. from bean, barley, sedges, grassesand Silene, plus numerous tropical trees (Lodge, Fisher and Sut-ton 1996; Boyle et al. 2001; Arnold and Herre 2003; Yuan, Zhangand Lin 2010; Sanchez Marquez et al. 2012; Canals et al. 2014;Yan et al. 2015), and bacterial species isolated from cocoa, cot-ton, legumes, rice and wheat (Quadt-Hallmann, Hallmann andKloepper 1997; Brewin 1998; Roncato-Maccari et al. 2003; Melnicket al. 2008). Formany endophytes, however, there is a lack of con-clusive evidence regarding their in planta colonisation behaviour.For instance, for B. subtilis, the only evidence indicating a degreeof systemic colonisation is the isolation of strains from the vas-cular transport systems ofmultiple plant species (Hall, Schreiberand Leben 1986; Put, Clerkx and Boekestein 1992; Wilhelm et al.1998; Zhang et al. 2009). However, to conclusively determine thein planta colonisation pattern of a specific endophyte, techniquessuch as fluorescence microscopy and/or direct isolation into ax-enic culture from a series of specific plant tissues are requiredand this evidence is still lacking for a number of plant–microbialsystems (Reinhold-Hurek and Hurek 2011).

Some authors have suggested classification systems for en-dophytes based on various criteria including in planta colonisa-tion patterns and host specificity factors. Rodriguez et al. (2009)separated fungal endophytes into twomain groups, the Clavicip-itaceous endophytes (including Balansia spp. and Epichloe spp.)and the non-clavicipitaceous types, and further separated thelatter group into three sub-classes based on distinct functionalgroups linked to their ecological significance and lifestyle char-acteristics, including in planta colonisation patterns and trans-mission routes. Rodriguez and Redman (1997) suggest fourclasses based on the endophytes behaviour in plant tissues:(1) those fungi that extensively colonise plant tissues; (2) thosethat actively colonise a small portion of their host tissues;(3) metabolically quiescent endophytes that are ‘walled off’ orinhibited within the host; and (4) metabolically active endo-phytes that are ‘walled off’ from certain tissues or regions ofthe host. While knowledge on the location and growth patternsof certain endophytes may have implications for those creat-ing/developing novel associations in terms of inoculation tech-niques (especially around sites for inoculation) and developingendophyte detection techniques (i.e. tracking hyphal growth orbacterial populations in situ), we would propose that a numberof other lifestyle characteristics need to be also understood toensure effective use of endophytes in crop protection.

Degree of host specificity

In the context of this review host specificity can be defined asthe number and identity of host species with which a partic-ular endophytic microorganism forms a symbiotic association.As withmany plant saprophytes and pathogens, endophyticmi-croorganisms display various degrees of host specificity rangingfrom highly evolved permanent associations with a single plantspecies to more superficial relationships involving a wide rangeof hosts (Petrini 1996). The degree of host specificity in animal–parasite associations is viewed by some as the most importantproperty as it governs the spread of a particular pathogenic or-ganism following introduction into a new geographic area anddetermines whether a symbiont can survive after extinction ofits host (Poulin, Krasnov and Morand 2006). Some researchersbelieve that the general principles of host specificity apply to

most microorganisms that have adapted to life in a particularhabitat, regardless of their taxonomic position or the ecologicalniche they occupy (Petrini 1996). For certain groups of organisms,alternative, more informative terminology has been suggestedsuch as ‘host exclusivity’ and ‘host preference’ (Zhou and Hyde2001). Regardless of the terminology, it is clear from any symbio-sis that an adaption has occurred of the host and endophyte toone another (Schulz and Boyle 2006).

Higgins et al. (2014) using genotypic and phylogenetic anal-yses have shown that some endophytic fungi are found in bothgrasses (family Poaceae) and non-grass hosts within the sametropical forest. The authors showed that at least 90% of theoperational taxonomic units identified from sequence data oc-curred in more than one genus of Poaceae and that these for-est grasses harbour diverse Class 3 endophytes (as describedby Rodriguez et al. 2009) that are predominantly host general-ists. From our listed case studies, the most host-specific en-dophytic microorganisms are clearly the rhizobia and Epichloespp., bacterial and fungal endophytes, respectively, while theopposite is true for the fungal endophyte P. indica that seemsto lack any host specificity. To date, one strain of this fun-gus, originally isolated from an orchid plant in the Indian TharDesert, has been documented to colonise the roots of a widerange of monocot and dicot plant species, including Arabidop-sis thaliana, Chinese cabbage, barley, maize, onion, sugarcaneand tomato (Verma et al. 1998; Bagde, Ram and Ajit 2010; Leeet al. 2011), after artificial inoculation. Some rhizobia form ex-clusive associations with leguminous plants and, furthermore,only locate in specialised organs on the host’s root systemwherethey fix nitrogen. However, this host specificity is more com-plex than originally announced and although many species ofrhizobia exhibit associations with only a single host species oflegume, other rhizobia species are more promiscuous (Rocheet al. 1991; Pueppke and Broughton 1999; Denison 2000). Strainsin the genus Frankia, can be classified into major host speci-ficity groups, although many exhibit overlap between the hoststhat each strain can nodulate (Baker 1987). As with these bac-teria, the relationship between Epichloe fungal endophytes andtheir hosts is not always clear-cut. Many taxonomic groupingsof Epichloe are confined to one host species in nature. For ex-ample, Epichloe coenophialum, E. festucae var. lolii and E. uncina-tum only associate with the grasses Festuca arundinacea, Loliumperenne and F. pratensis, respectively. However artificial or novelassociations can be createdwith different combinations of theseendophytes and grass species in the laboratory (Christensen1995). In addition, strains of E. typhina, a sexual species, are quitepromiscuous and naturally associate with a number of hostgrass species (Leuchtmann and Clay 1993). Piriformospora indicais the most promiscuous endophyte species listed in our casestudies.

It is apparent that the host specificity exhibited by endo-phytic microbes can be complex, and although many strict as-sociations seem to be formed in nature, this can be manipu-lated, to a degree, when developing novel endophyte–host asso-ciations. Additional experiments are therefore required to un-derstand host range, host specificity, host compatibility andrecognition mechanisms in the development of BCAs for cropprotection (Petrini 1996). As with many pathogen–host sys-tems, we expect that further research into endophyte–hostinteractions will uncover a greater degree of host specificityfrom certain genera that will require further formal and infor-mal taxonomic groupings (i.e. formae specialis (ff. spp.), patho-types, pathovars, physiological races and/or variants) to beapplied.

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Means of propagation

Facultative endophytes can exhibit a biphasic reproductivelifestyle, with some alternating between their plant host andtheir environment, i.e. the soil (Hardoim, van Overbeek and vanElsas 2008), while obligate endophytes are totally dependent onthe host plant for their growth, survival and propagation. Trans-mission of endophytes between host plants can fall into twomain categories, vertical or horizontal. Vertical transmission re-lies on the plant’s reproductive strategy and generally involvescolonisation of the plant’s reproductive tissues, i.e. pollen orseed, or vegetative propagules by vegetative structures such asendophytic hyphae or bacterial cells. Horizontal transmissiongenerally relies on the reproductive structures of the endophyte,i.e. spores, which can be wind dispersed or vectored by a partic-ular agent, such as an insect. For example, it has been demon-strated that propagules of the endophytic fungus Lecanicilliumlecanii can be transferred between its host coffee plants by anant (Azteca instabilis). This system is further complicated by thefact that the ant itself exhibits amutualistic associationwith thecoffee green scale (Coccus viridis), a pest of coffee that the BCA istargeted towards and eventually parasitises (Vandermeer, Per-fecto and Liere 2009; Jackson, Skillman and Vandermeer 2012).Those microorganisms that display only vertical transmissionare regarded as having developed further down the evolution-ary time-line as the endophyte has lost all powers of contagion.Ewald (1987) suggested that modes of transmission are relatedto the severity of parasitism and that the presence of verticaltransmission favours the evolution of mutualism.

The transmission of fungal endophytes to successive gen-erations in woody plants has been hypothesised to occur hori-zontally (Ganley and Newcombe 2006) a situation common withthe majority of the endophytes highlighted in our case stud-ies. For example, Phialocephala scopiformis, a foliar endophyte ofwhite spruce (Picea glauca), is spread from infected mature treesto seedlings of the same tree species growing in close proxim-ity to the adult plants (Frasz et al. 2014). This endophyte pro-duces a number of anti-insect toxins, including rugulosin, whichreduce the growth and development of the insect pest Choris-toneura fumiferana (Miller et al. 2009; Sumarah and Miller 2009).However, a number of endophytic bacterial species are trans-mitted vertically via seed and these have been shown to bene-fit the host’s germination and seedling establishment as well asthemature plant (read Truyens et al. 2015 for an in-depth discus-sion). Without a doubt, the most studied vertically transmittedendophytes are the asexual Epichloe species. These fungi infectthe floret of their Pooideae grass hosts at the base of the ovaryand surround themegagametophyte. After fertilisation, the fun-gus grows with a high degree of hyphal branching, and at seedmaturity the endophyte is found within the embryo tissues. Onseed germination, the fungus breaks fromdormancy in synchro-nisation with the plant and systemically invades the seedling’sapical meristem, and the clonal vertical transmission cycle iscomplete (Philipson and Christey 1986). This is an example ofseed transmission where the microorganism has infected cer-tain tissues of the seed, i.e. the embryo, and uses this as a domi-nant or solemeans of transference fromplant to offspring. Manymicroorganisms, including many endophytic bacteria, may beseed-borne,whereby they are commonly carried on orwith seed,either on the seed surface or associated with the seed coat tis-sues, but they are not necessarily seed-transmitted (as describedby Neergaard (1977) when discussing seed pathogens).

The transmission routes of many endophytes remain to beelucidated, and although evidence exists showing the isolation

of many species from seed tissues, this does not guarantee thatthese organisms are seed-transmitted. For many economicallyimportant crops, deciphering transmission routes of particularendophytic BCAs is crucially important not only to understandhow they might benefit the plant at various parts of its life cy-cle (i.e. seed) but also from a commercial viewpoint to identifyhow best to manage the endophyte as technology. For an en-dophytic BCA that is encapsulated in the plant’s reproductivetissues, companies can take full advantage of this naturally oc-curring phenomenon and will not need to develop complicatedand expensive seed formulations to captivate and preserve theparticular beneficial microbial entity within the encompassingproduct, the elite plant cultivar. However the production anddistribution of endophyte-infected seed products is not com-pletely free of technical and biological difficulties (Rolston andAgee 2007).

Mechanism(s) of action

Obviously if an endophytic microbe is to be commercialised andmarketed then it must display one or more bioactive propertiesthat provide that benefit to its host and as a result make the fi-nancial investment associated with its development as a BCAworthwhile. As with the broader discipline of biological con-trol, endophytic microorganisms can display one or moremech-anisms of action towards a specific pest or pathogen. These areclassified into four main categories: (1) antibiosis, (2) compe-tition, (3) host-induced resistance, and (4) direct parasitism. ABCA that displays multiple mechanisms of action would there-fore be advantageous as this could potentially increase theBCA’s efficiency, delay or stop resistance developing in the tar-get pest/pathogen population and assist the organism in con-trolling multiple unrelated pests/pathogens on different hostplants, which would be advantageous (Punja 1997). As discussedin the previous section on in planta colonisation patterns, one ofthe key differences between a BCA and an endophytic BCA is thecontact between the BCA and the target pest or pathogen. Themajority of the target pests and pathogens will arrive at the BCAarena (the plant) by some fashion and then try to either gainentry and colonise (as with pathogenic microorganisms) or feedon the plant (as with many invertebrates pests). When the BCAis endophytic it will, by its very definition, have very little to nodirect contact with the target pest or pathogen. Thereforemech-anisms such as direct parasitism and competition are likely tobe less effective than the two indirect mechanisms of antago-nism, namely antibiosis and host-induced resistance for an en-dophytic BCA. Antibiosis has to be the most documented formof antagonism exhibited by endophytic BCAs. Many bacterialand fungal endophytes produce myriad secondary metabolitesthat have antagonistic and inhibitory and/or deterrent proper-ties. These two mechanisms may be highly linked, as many ofthese endophyte-derived compounds can have dual roles andalso function as elicitors of plant-induced systemic resistance(Danielsson, Reva and Meijer 2007), acting as signals mediat-ing cross-talk between the endophyte and its host (Graner et al.2003).

Case studies of endophytic BCAs

The following examples were chosen to highlight specificmicro-bial lifestyles and particular traits that not only make these mi-croorganisms successful in the biocontrol arena but also providea range of successes as commercial products, i.e. they are easyto propagate, package and distribute to the required destination

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Card et al. 7

(see Table 1). By no means is this list inclusive of all microbialendophytes that act as BCAs, nor does the list suggest any formof ranking (themicroorganisms are listed alphabetically) relatedto efficacy or any other comparative measurement.

Bacillus subtilisBacillus species are Gram-positive, rod-shaped, endospore-forming bacteria within the phylum Firmicutes with strains ofthis genus amongst the most common endophytic bacteria (Lil-ley et al. 1996). Bacillus subtilis is exceptionally well known inbiocontrol circles for its antagonism towards a plethora of plantpathogenic fungi and bacteria, including but not limited to Cer-atocystis ulrni (Schreiber et al. 1988), Colletotrichum spp. (He et al.2004; Ruangwong et al. 2012), Fusarium moniliforme (Hinton andBacon 1995), Sclerotinia sclerotiorum (Hu et al. 2011; Gao et al. 2013)and Xanthomonas campestris pv. campestris (Wulff et al. 2002; Di-nesh, Shri and Yadav 2010) as well as its plant-growth promot-ing abilities (Poonguzhali, Madhaiyan and Sa 2006). Strains of B.subtilis can be isolated from one host and inoculated into an-other, where they successfully colonise and provide the hostwith additional beneficial traits (Hinton and Bacon 1995; Baconand Hinton 2002). This characteristic seems common amongstmany Bacillus species (Wulff, van Vuurde and Hockenhull 2003)indicating little or no host specificity; however, this fact doesnot guarantee that the beneficial attributes of these bacteria willalso be transferred across hosts.

Although conclusive evidence is lacking with respect to thecolonisation pattern exhibited by B. subtilis, there is evidencewithin the literature describing the isolation of strains fromnearand inside the vascular transport systems ofmany plant species,indicating systemic growth (Wulff, van Vuurde and Hockenhull2003; Zhang et al. 2009). In addition, vertical transmission mayalso be possible as strains have been recovered from plant re-productive tissues including seed (Mundt and Hinkle 1976; Hall,Schreiber and Leben 1986; Schreiber et al. 1988; Zhang et al. 2009).As with many other species listed as case studies, the symbioticrelationship that B. subtilis strains have with their hosts is notclear and potentially ranges from commensalistic to mutualis-ticwith the relationship, inmany situations, between endophyteand host identified as long-term (Wulff et al. 2002; Fan et al. 2007).

Strains of B. subtilis have been documented to possess mul-tiple mechanisms of biocontrol action including antibiosis (viaantifungal, antibacterial and biosurfactant substances) and in-duced systemic resistance (Loeffler et al. 1986; Schreiber et al.1988; Bacon and Hinton 2002; Cho et al. 2002; Kloepper, Ryuand Zhang 2004; Stein 2005; Hu et al. 2011), which may ex-plain their widespread antagonism towards fungal and bacte-rial pathogens. Strains of B. subtilis exhibit a lack of toxicitytowards mammals and possess the capability to produce ex-tremely tough, dormant resting structures known as endospores(a characteristic of many members of the Firmicute family) (Ba-con and Hinton 2002; Bolstridge et al. 2009). These vegetativestructures ensure the survival of a bacterium through periodsof environmental stress and are highly resistant to desiccation,starvation, and many types of radiation.

Beauveria bassianaThe fungal genus Beauveria (also known as white ento-mopathogenic fungi) belongs to the family Cordycipitaceae andis collectively more commonly found associated with insectsor insect habitats, including soil. However, the most publicisedspecies, B. bassiana, has also been described as a plant symbiont,capable of colonising a wide range of monocot and dicot groupsof plants (see Lohse et al. 2015 for an extensive list) including Ta

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cocoa, coffee, banana, maize and wheat (Bing and Lewis 1992;Posada and Vega 2005; Posada and Vega 2006; Akello et al. 2008;Gurulingappa et al. 2010) indicating the lack of host specificityexpressed by this species. As B. bassiana is commonly recognisedas an entomopathogen, the endophytic lifestyle of the species isclearly facultative as not all of its lifestyle is spent within the tis-sues of a plant host. The fungus also colonises a range of planttissues, across various hosts, including leaves, shoots, roots andseed (Ownley et al. 2008; Vega 2008), although evidence is lackingto clearly define the growth as systemic within a particular planthost. The fungus in many systems seems to form long-term as-sociations with particular plant hosts suggesting a high degreeof mutualism (Ownley et al. 2008; Biswas et al. 2013).

Although numerous reports associate B. bassianawith the re-productive tissues (i.e. seed) of particular plant hosts, transmis-sion of the fungus by this mechanism (Koycu and Ozer 1997;Ganley andNewcombe 2006; Quesada-Moraga et al. 2006) has notbeen established. Therefore, any product based on B. bassianawould have to rely on regular foliar sprays or soil drenches toapply propagules (spores or mycelial fragments) of the fungusto the target plant host. Selected strains of B. bassiana havebeen successfully disseminated in this fashion and commer-cialised for their insect killing traits, especially for controllinga wide range of pests including aphids, beetles, caterpillars,termites, thrips and whitefly (Gurulingappa, McGee and Sword2011; Biswas et al. 2013; Parsa, Ortiz andVega 2013). In addition, B.bassiana has been reported to inhibit disease caused by a num-ber of plant pathogenic fungi (see Ownley et al. 2008 for a re-cent review) increasing the marketability of a potential biocon-trol product substantially due to the dual protection it may offerwith respect to herbivory and disease (Ownley et al. 2008; Parsa,Ortiz and Vega 2013). Linked to this wide bioactivity, B. bassianaachieves this biocontrol efficacy through multiple mechanismsof action including antibiosis via an array of secondary metabo-lites, direct parasitism, competition and induced systemic resis-tance (Bark et al. 1996; Leckie 2002; Griffin et al. 2006; Ownley et al.2008; Xu et al. 2008; Hu et al. 2014).

Asexual EpichloeEpichloe (family Clavicipitaceae) are filamentous fungi thatform close associations with cool-season grasses (sub-familyPooideae). The asexual species, formerly known asNeotyphodiumspp. (Leuchtmann et al. 2014), have been well characterised withrespect to their bioactive metabolite profiles, genetic diversityand growth characteristics (Johnson et al. 2013). These fungi alsoexhibit traits that make them ideally suited asmarketable BCAs.Although it is possible to propagate many Epichloe strains in vitrowith the use of commonly available culture media (Latch andChristensen 1985; Bacon and White 1994), all asexual Epichloespecies display obligate lifestyles and are generally not foundoutside of their grass host’s tissues.

These endophytic fungi are not found in association withplant root tissues after seed germination (Christensen andVoisey 2007; Card et al. 2011) and instead grow only in the fo-liar tissues of their hosts, where they colonise systemically via amechanism termed intercalary hyphal extension (Christensenet al. 2008; Voisey 2010). Furthermore, these fungi rely on theplant’s reproductive system to transmit to the next generationvia seed (vertical transmission) (Freeman 1904; Philipson andChristey 1986; Zhang et al. 2015) and exhibit a high degree of hostspecificity, forming long-term, permanent mutualistic relation-ships with specific grass species (Leuchtmann 1993; Karimi et al.2012; Card et al. 2014). The subject of host specificity is gener-ally approached in detail within the literature when attempts

have been made to form artificial associations between Epichloestrains and alternative grass host species that result in less thansatisfactory partnerships (Koga, Christensen and Bennett 1993;Christensen 1995; Christensen et al. 1997; Easton 2007). SelectedEpichloe strains can deter a multitude of animals, invertebratesand fungi depending on the secondary metabolite profile thatan individual fungal strain possesses as well as conferring otherbeneficial traits on their hosts, such as drought tolerance (Westet al. 1993; He et al. 2013). Although these fungi are only dis-tributed in the host’s above-ground tissues, their bioactivity to-wards certain pests has been documented in the rhizosphere(Popay and Bonos 2005) and is attributed to the mobility of alka-loids (a class of secondarymetabolites produced during the sym-biosis) within the plant’s vascular system. Due to their biologicalfunctionality, these fungi also possess numerous traits makingthem suitable for commercialisation, especially their propaga-tion through host seeds, which allows their bio-protective prop-erties to be delivered at the start of the plant’s life cycle withoutthe need for any expensive or complicated application technol-ogy (Johnson et al. 2013).

The asexual Epichloe species—most notable commercially areE. festucae var. lolii that associates with perennial ryegrass andE. coenophiala that associates with tall fescue—have been suc-cessfully marketed for decades in the New World continents ofAustralasia (New Zealand and Australia) and the Americas (par-ticularly the USA, Argentina, Brazil, Chile and Uruguay) for theirinsect deterrent and wildlife management properties (Caradus,Lovatt and Belgrave 2013; Johnson et al. 2013; Pennell et al. 2016;Finch et al. 2015). These endophytic fungi were the first patentedtechnology to be placed in grass cultivars (Bouton and Hop-kins 2003) and this has led to an expansion in the number ofseed products available frommany pasture seed companies. Thelifestyles exhibited by these fungi and their behavioural traitsdiscussed above make asexual Epichloe species ideal BCAs interms not only of their mechanisms of action via deterrent al-kaloids that are effective against a wide range of invertebratepests, but also of the fungus being captivated, for its entire lifecycle, within the plant.

Lecanicillium lecaniiLecanicillium lecanii, previouslywidely known asVerticillium lecanii(Family, Clavicipitaceae), is a ubiquitous entomopathogenic fun-gus that has been shown to affect survival and reproduction ofseveral aphid species, many other insect pests (Lee et al. 2002;Hong and Kim 2007; Ganassi et al. 2010; Guclu et al. 2010; Shindeet al. 2010; Gurulingappa, McGee and Sword 2011; Banu andGopalakrishanan 2012; Bouhous and Larous 2012; Jackson, Skill-man and Vandermeer 2012; Wang et al. 2013; Gao et al. 2015) andnematodes (Meyer and Meyer 1996). In addition, this fungus hasalso been identified as a horizontally transmitted endophyte ofa number of species including cotton (Gossypium hirsutum; fam-ily Malvaceae) (Anderson et al. 2007), Araceae (rhizomatous ortuberous herbs) (Petrini and Dreyfuss 1981) and American horn-beam (Carpinus caroliniana; family Betulaceae, a small hardwoodtree) (Bills and Polishook 1991). Under laboratory conditions, astrain of the fungus has been transferred from a cotton leaf toan insect (Aphis gossypii) and from the insect back to a leaf, whileclosely related fungal species have been successfully introducedas endophytes into date palm (Phoenix dactylifera) (Gomez-Vidalet al. 2006) and cucumber (Cucumis sativus) roots (Benhamou andBrodeur 2001; Hirano et al. 2008). In certain hosts, such as cot-ton, insects are regarded as important in the establishment ofthe fungus as an endophyte where the fungus forms localisedinfections within host leaves (Anderson et al. 2007).

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Card et al. 9

In numerous independent, comparative experiments, L.lecanii has shown a higher degree of bioactivity towards a num-ber of insect pests. Vu, Hong and Kim (2008) compared 12 strainsof entomopathogenic fungi including L. lecanii, Paecilomyces fari-nosus, B. bassiana, Metarhizium anisopliae, Cordyceps scarabaeicolaand Nomuraea rileyi for aphid control. They concluded that L.lecanii strain 41185 showed the highest virulent pathogenicity forboth Myzus persicae (green peach aphid) and A. gossypii. Similarcomparative assays performed by Abd El-Salam and El-Hawary(2011) found that L. lecanii showed a higher virulent pathogenic-ity for both adult and nymph stages of Aphis craccivora whencompared with a strain of B. bassiana.

Under laboratory conditions Mahmoud (2009) used con-tact bioassays and oral bioassays to test the pathogenicity ofthree commercial products of the entomopathogenic fungi, B.bassiana, M. anisopilae and L. lecanii, against adults of olive fly(Bactrocera oleae). The virulence of L. lecanii was higher than thevirulence of the other two fungal species tested, although Mah-moud (2009) considered that field trial datawere required to con-firm the commercial efficacy of L. lecanii.

As well as possessing anti-insect qualities, L. lecanii hasalso been shown to antagonise fungal pathogens includingSphaerotheca macularis (strawberry powdery mildew) (Miller et al.2004) and Hemileia vastatrix (coffee rust) (Vandermeer, Perfectoand Liere 2009), with the primary mechanism displayed by theBCA suggested to be mycoparasitism and not induced systemicresistance (Ownley, Gwinn and Vega 2010) when targeting thesephylloplane pathogens. However, the BCA has also shown an-tagonism in cucumber roots towards soil-borne pathogens likePythium ultimum, where the mechanism of action is proposed tobe host-induced resistance and the production of structural andbiochemical barriers in the root tissues (Benhamou and Brodeur2001).

Verhaar, Hijwegen andZadoks (1996) considered that L. lecaniiwould onlymake a successful BCAwithin glasshouse situations,most likely due to a requirement of the fungus for high relativehumidity for growth, sporulation and its biological control abil-ity of certain pathogens via direct parasitism (Hall 1980; Verhaar,Hijwegen and Zadoks 1996, 1998, 1999). However, more recenttrials have indicated that repeated sprays of the BCA can resultin reduced disease in a field situation (Miller et al. 2004). Com-pared with many biotrophic fungi, L. lecanii is easily grown onsynthetic media, and from a commercial perspective the funguscan be grown on cheap, solid ingredients such as steamed rice,where a high conidial concentration can be produced (up to 18.2× 109 conidia/g) (Vu, Hong and Kim 2008).

Piriformospora indicaPiriformospora indica is a cultivatable root-colonising endophyticfungus of the recently defined order Sebacinales (Basidiomy-cota) (Weiss et al. 2004) originally isolated from the root systemof several xerophytic plants located in the Indian Thar dessert(Verma et al. 1998). The endophyte has a broad host range,colonising bothmono- and dicotyledonous plants, including theagriculturally important barley (Hordeum vulgare), and themodelplants tobacco (Nicotiana tabacum) and Arabidopsis thaliana (forreviews see Oelmuller et al. 2009; Franken 2012; Qiang et al. 2012;Ansari et al. 2013; Oberwinkler et al. 2013; Johnson, Alex andOelmuller 2014). All plant species tested so far have been foundto be colonised by P. indica (Oelmuller et al. 2009); however, onlyone strain of this species has ever been isolated and used ex-perimentally (Franken 2012; Oberwinkler et al. 2013). Previously

reported as a monotypic genus, a second species, P. william-sii, has been recently described that is distinct but phyloge-netically closely related to P. indica (Basiewicz et al. 2012). Thehost benefits conferred by colonisation of P. indica are exten-sive and include resistance/tolerance to abiotic stress (salinity,drought, water, cold, high temperature and heavy metals) andbiotic stress (root and foliar pathogens) and promotion of plantgrowth, including the delivery of phosphate to the plant (Varmaet al. 1999; Waller et al. 2005; Sherameti et al. 2008; Yadav et al.2010; Franken 2012). These attributes suggest that this endo-phyte has great promise as a BCA for achieving sustainable cropproduction.

The promotion of plant growth by P. indica is the most recog-nisable benefit that has been shown repeatedly for a large va-riety of species. The extent of growth promotion is typicallyaround 50%, but a large variation has been observed that is likelydue to experimental and environmental conditions (Franken2012). Piriformospora indica exerts growth promotion by promot-ing the early growth stages of plant development mainly via ac-celerated root development (Waller et al. 2005; Baltruschat et al.2008). The mechanisms of early root development appear com-plex and have been studied in barley, Chinese cabbage and Ara-bidopsis. Not surprisingly, this process involves phytohormones,particularly ethylene, and their signalling networks (see reviewby Franken 2012).

Piriformospora indica is a model system for exploring themechanisms of endophyte–root compatibility due to its genetictractability and broad host range, which includes model plantspecies. Infection experiments have shown that P. indica prefer-entially colonises the maturation zone of roots, where it directlypenetrates living roots cells and triggers the suppression of theplants immunity systems to achieve colonisation (Deshmukhet al. 2006; Jacobs et al. 2011; Qiang et al. 2012). It exhibits a dualor biphasic lifestyle where early colonisation of the root com-mences with a biotrophic growth phase, colonising living cells,followed by a switch to a cell death-dependent phase, where itgrows onmostly dead plantmaterial and secretes hydrolytic en-zymes to degrade plant cell walls and proteins (Deshmukh et al.2006; Zuccaro et al. 2011; Qiang et al. 2012; Lahrmann et al. 2013).However, upon comparing the colonisation strategy of P. indicain Arabidopsis vs barley, a non-destructive long-term biotrophicnutrition approach is described for Arabidopsis compared with aswitch to saprotrophic nutrition in barley (Lahrmann et al. 2013).The host-dependent colonisation strategies employed by P. in-dica are likely to require a specialised set of effectors (Zuccaroet al. 2011; Lahrmann et al. 2013)—proteins and small moleculesthat alter host-cell structure and function, suppressing or cir-cumventing host defences to enhance microbial infection (Elliset al. 2009; Plett et al. 2014). One P. indica candidate effector has sofar been characterised which enhances colonisation ofArabidop-sis and barley roots by suppressing the salicylate-mediated basalresistance response (Akum et al. 2015). Other effectors are likelyrequired for the colonisation process and it is hypothesised thateffectors play a role in how P. indica can interact with such a largeplant host range. Additionally, host-related metabolic cues areproposed to influence the colonisation strategies of the endo-phyte (Lahrmann et al. 2013). The phenotypic plasticity of P. in-dica and the ability to express alternative lifestyles are suggestedto be associated with making this root endophyte a typical gen-eralist that is highly adaptive and responsive to different envi-ronmental and host signals.

One of the advantages of P. indica compared with arbuscu-lar mycorrhizal fungi is that it is not an obligate biotroph, andthus it can be easily cultured under laboratory conditions and

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has the potential for rapid and large-scale fermentation requiredfor field applications (Oelmuller et al. 2009). Piriformospora indicacan also colonise plant roots independently of phosphate avail-ability (also in contrast to arbuscular mycorrhizal fungi) (Varmaet al. 1999), providing a clear benefit for agricultural applicationswhenplant growth promoting benefits are needed for high phos-phorous zones found in anthropogenic ecosystems (Achatz et al.2010).

However, for P. indica to be used as an endophyte productin agriculture, there are some challenges. Sufficient inoculumfor plant infection on a large scale is clearly required. The sub-strate in the media used to cultivate the fungus appears to beone of the most important factors as this can alter the planthost response and even produce a negative outcome for the host(Kaldorf et al. 2005; Franken 2012). Research towards manipulat-ing substrate composition, the environmental conditions (Ku-mar, Sahai and Bisaria 2011), the application of nanomaterials(Suman et al. 2010), and finding a suitable carrier for distribution(Sarma et al. 2011), in addition to considering a number of pa-rameters such as the amount of inoculum, the time point of ap-plication and the choice of plant cultivation materials, have allbeen carefully considered for commercialisation purposes (Ser-fling et al. 2007; Fakhro et al. 2010). The fungus has recently beenformulated as a powder under the trade nameROOTONIC for usein field trials in India (Varma et al. 2013; Shrivastava and Varma2014).

Commercialisation of P. indica may also be delayed until it isunderstood why at times negative consequences have been ob-served for plant growth (Deshmukh et al. 2006; Murphy, Doohanand Hodkinson 2014). As mentioned previously, only one strainof P. indica has ever been found (Franken 2012; Oberwinkler et al.2013) and this absence of genetic diversity is a concern from amarket development viewpoint as this may hinder widespreadutility. In addition, the patent (European Patent Office, Munich,Germany, Patent No. 97121440.8–2105, Nov. 1998) that protectsthe intellectual property (IP) of this endophyte strain indicatesthat any commercial development must be initiated or at leastpermitted by the IP owner.

Rhizobia

Many authors do not consider microorganisms that residewithin a specialised compartment as endophytes, with some la-belling these organisms separately as endosymbionts (Reinhold-Hurek and Hurek 2011). However, for the purpose of this arti-cle we will include them. Rhizobia are soil bacteria that havebeen known for many decades to be essential for the growthand survival of leguminous plants (Peoples, Ladha and Her-ridge 1995). Both the bacteria and the host plant can grow inde-pendently, but nitrogen is only fixed when rhizobia invade theleguminous root and N-fixing nodules are formed, with manyplants forming permanent associations with these bacteria fortheir entire lifespan. These soil bacteria infect through the roothair of their host and are therefore only transferred horizon-tally. In some leguminous species the rhizobia that form nod-ules can be very strain specific, whereas other legumes canbe very promiscuous, while some rhizobium strains can infectmany legume species (Pueppke and Broughton 1999). The sym-biosis between legumes and rhizobia is classed as mutualis-tic (Sprent, Sutherland and Faria 1987), but in some situationswhere nodulation occurswith rhizobial strains that do not fix ni-trogen (Singleton and Stockinger 1983) the relationship could benearer to commensalistic. In addition, most plants are infectedby more than one strain of rhizobia (Dowling and Broughton

1986) and therefore there is no exclusivity expressed in theserelationships.

As well as providing a natural fertiliser for their hosts (in theform of ammonia), certain rhizobia species can also act as BCAsof plant root pathogens, inhibiting disease caused by Fusariumsolani and Macrophomina phaseolina (Al-Ani et al. 2012). They se-crete secondary metabolites such as antibiotics and HCN, andproduce siderophores, which have been shown to result in theexclusion of pathogens due to iron starvation (Deshwal et al.2003). Under controlled conditions, Hemissi et al. (2011) showedthat some specific rhizobia strains were effective in controllingRhizoctonia solani in dual culture in vitro and under greenhouseconditions. The biocontrol strain Rhizobium etliG12 has also beenshown to be an antagonist towards the potato cyst nematode,Globodera pallida, and the root-knot nematode,Meloidogyne incog-nita (Hallmann et al. 2001). In the field, the majority of the docu-mented reports for rhizobia are focused on the level of nitrogenfixation associated with commercialised strains (Roughley andPulsford 1982), but one commercial BCA strain, Rhizobium rhizo-genes strain K84, is used worldwide to control crown gall disease(Abarca-Grau et al. 2012).

Trichoderma speciesCommonly associated with the soil, Trichoderma species (fam-ily Hypocreaceae) are often found in opportunistic symbioseswith a variety of plant species (Harman et al. 2004). These rela-tionships range from pathogenic on hosts such as mushrooms,to saprophytic on a range of hosts (Samuels 1996). Particularspecies within the genus, including T. asperellum, T. atroviride, T.hamatum, T. harzianum, T. polysporum and T. viride, are also im-portant biocontrol fungi, particularly utilised in the rhizosphereto control plant diseases where they are considered avirulentplant symbionts (Howell 2003; Harman et al. 2004; Woo et al.2006; Shoresh, Harman and Mastouri 2010). Many Trichodermastrains have been commercialised into products that are mar-keted and soldworldwide to particularly target soil-borne fungalpathogens, i.e. Pythium, Phytophthora, Rhizoctonia, Sclerotinia andVerticillium, and a few foliar fungal pathogens, i.e. Alternaria andBotrytis (Papavizas 1985; Elad 2000; Altintas and Bal 2008; Wooet al. 2014).

The advantages of Trichoderma as BCAs are their excellentadaptability to a wide range of environmental conditions, theirtolerance to certain fungicides, diversified mechanisms of ac-tion, fast growth (in vitro and in situ), and simple nutritional re-quirements (Tang, Yang and Ryder 2001). Likemycorrhizal fungi,Trichoderma species are dominant fungal species amongst thesoil microflora and are able to colonise plant roots and growinto the rhizosphere, residing in and interacting with both en-vironments simultaneously (Smith and Read 2008). Unlike my-corrhizal fungi, however, Trichoderma root symbionts have notbeen found to successfully colonise living plant roots, eitherintercellularly or intracellularly. Although recent research hasdescribed certain strains of T. hamatum as foliar endophytes ofTheobroma cacao (Bailey et al. 2006, 2008; Bailey, Strem and Wood2009), conclusive evidence is currently lacking of its endophyticnature and therefore we have to accept that these fungi aremore likely to form commensalistic symbioses with their Mal-vaceae hosts rather than mutualistic endophytic associations.Thus we consider the majority of Trichoderma–plant interactionscurrently described in the literature to be those of success-ful BCAs that associate closely with plants and not endophyticassociations.

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Card et al. 11

LIFESTYLE CHARACTERISTICS OFSUCCESSFUL ENDOPHYTIC BCAS USEDIN COMMERCE

The initial screening of BCAs often occurs under controlled envi-ronmental conditions and in these situations thesemicroorgan-isms generally provide a benefit to the host plant. Consequently,an optimal environment is selected that is conducive to the de-velopment and impact of the BCA. These benefits can be man-ifold and the literature provides numerous examples of fungaland bacterial endophytes affording improved pest and diseaseresistance and improved plant performance under extreme con-ditions of temperature and water availability, as well as manyother benefits. Lugtenberg and Kamilova (2009) have noted thatmany bacterial strains exert their beneficial effects in culture,but a much lower number are successful in the greenhouse, andan even lower number function effectively under practical con-ditions, i.e. in a commercial greenhouse or in the field. They con-sidered that understanding the reasons for the failures in situmay lead to the isolation of improved strains. Facultative endo-phytes will clearly be impacted by many environmental condi-tions to a greater extent than obligate types. For example, the vi-ability of B. bassiana conidia used in foliar sprays can be reducedby unfavourable temperatures, low rainfall (Bing and Lewis 1991)and exposure to ultraviolet light (Copping and Menn 2000).

As concluded earlier the type of colonisation exhibited by aparticular BCA may not be important from a commercial prod-uct viewpoint. BCA products based on endophytes that displaysystemic as well as localised in planta colonisation patterns havebeen, and continue to be, developed andmarketed. For example,there are many products based on rhizobia and asexual Epichloespp., BCAs that display localised and systemic in planta coloni-sation patterns, respectively. The more important factor is themechanism of action exhibited by the BCA within the particu-lar host towards the target pest or pathogen and whether bio-logical control can be achieved. For example, if antibiosis is themajor mechanism exhibited by a BCA, can these particular sec-ondary metabolites move around the plant from the site of pro-duction to the location where the plant requires them for pestor pathogen protection? In addition, in order for a particularBCA product to be commercially successful, a broad spectrumof bioactivity, or the addition of multiple host beneficial traits,is ideally required for marketability. BCAs such as B. bassiana, E.festucae var. lolii and E. coenophiala gained their commercial suc-cess through their broad bioactive abilities. Beauveria bassianaexhibits dual entomopathogenic and plant pathogen inhibitoryabilities (Ownley et al. 2008) while selected strains of Epichloe spp.display proven broad insect deterrent qualities, coupled withheightened host drought tolerance and increased seed produc-tion in certain hosts (Johnson et al. 2013).

The level of a BCA’s host specificity may also not be as im-portant in commerce as other lifestyle traits. An advantage ofhaving a BCA that displays strong host specificity is that it maybe easier to protect the IP surrounding the product, as a finitenumber of hosts can be well researched, understood and devel-oped (i.e. E. festucae var. lolii and its association with perennialryegrass). The same information can then also be used to sellthe product into well-defined market niches and, if successful,a strong market-place reputation can be achieved. However aBCA that has little to no host specificity could potentially forman infinite number of associations with different host speciesproviding an opportunity for many plant-based products to beproduced for various cropping situations. For example, B. subtilisforms symbiotic associations with many economically impor-

tant plants including cabbage, cauliflower, chestnut, citrus andoilseed rape (Wilhelm et al. 1998; Wulff, van Vuurde and Hock-enhull 2003; Jiang et al. 2007; Zhang et al. 2009; Dinesh, Shri andYadav 2010; Gao et al. 2013); this has led to a great number ofcommercially available BCA products based on this species thatare available in many countries (Kabaluk et al. 2010). Althoughthe commercialisation of P. indica is in its infancy, ROOTONIC,which is the trade name for a powder formulation of this rootendophyte, is being extensively trialled in India on a broad rangeof economically important plants (Shrivastava and Varma 2014).In this case, one product may have endless utilities as a BCA indiverse plant species.

Another key to the commercial success of BCAs is under-standing the location where biological control will ultimatelytake place and therefore development of appropriate applica-tion or dissemination mechanisms, e.g. via seed transmission,and/or techniques that will be required for the end-user toachieve success. Vidal and Jaber (2015) reviewed a number of en-tomopathogenic fungi that were also able to colonise plant tis-sues as symptomless endophytes (e.g. B. bassiana, M. anisopliaeand L. lecanii). The authors conclude that while these specieshave shown reasonable detrimental effects on herbivorous in-sects feeding on plants containing these fungi as endophytes,the efficacy data are highly variable and as a result do not eluci-date the underlying mechanisms that would explain these ef-fects. They state that ‘An optimal and probably the most ef-fective option for making use of the endophytic growth of en-tomopathogenic fungi would be the inoculation of host plantswith these fungi at the start of the germination of seeds, ei-ther by producing seeds already containing these fungi, or bycoating the seeds with spores and protecting against adverseenvironmental conditions for their survival in the soil’. Jaron-ski (2010) states further ‘Unlike other insect pathogens, ento-mopathogenic fungi are percutaneously infectious agents, i.e.they act by contact’. So to be effective, spores from the fungusmust come into direct contact with an insect either from a sprayor from a fungus-contaminated surface (Jaronski 2010).

We have concentrated on the major behavioural traits of en-dophytic BCAs while there are also minor traits and/or otherrequirements that may be needed in a particular country,context or host to meet biosecurity, containment or exportlaws/requirements (such as toxicity testing). For instance, inFrance, Epichloe fungal endophytes are prohibited from beingmarketed in commercial grass seeds intended for forage andonly legally permitted for use in grass varieties intended for turfapplications (Huyghe 2012). This is largely a historical issue asEuropean law prohibits their inclusion in grass seeds due to po-tential animal toxicity problems (Bri 2005) even though researchconducted more than a decade ago clearly identified the toxinsresponsible and many countries have adopted selected endo-phyte strains that confer beneficial traits with no or little ani-mal toxicity problems (Caradus, Lovatt and Belgrave 2013; John-son et al. 2013). Inmany other countries, including New Zealand,Australia and the USA, the use of selected endophytes are ofgreat economic importance primarily due to their beneficial in-sect deterrent properties (Johnson et al. 2013; Young, Hume andMcCulley 2013). As for all BCAs, including those that are endo-phytic, it is an imperative that suitable techniques are designedfor the development and screening to ensure that results ob-tained are a reliable predictor of BCA performance in situ, i.e.within the host located in the field or greenhouse (Andrews 1992;Card et al. 2009).

In conclusion, for a microbial endophyte to be commerciallysuccessful as a BCAwe suggest that the following criteria bemet.

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� The BCAmust be easily applied/disseminated to crops (i.e. nospecialised equipment is required) but have restricted trans-mission routes such that it does not transmit to neighbouringcrops/plants (this has implications for IP as well as poten-tial incompatibility issues surrounding the biology of novelassociations).

� The BCA must harbour one or more effective mechanismsof action (e.g. bioactive compounds that can be mobilised tothe biocontrol arena), but the antagonistic properties of theBCA must have restricted bioactivity so as not to influencebeneficial non-target species such as pollinators (honey beesand bumble bees) and earthworms.

� The BCA can be suitably protected such that an individualstrain can be easily identified and commercialised withoutconcern for it becoming commoditised.

� The BCA must not under any circumstance (environmentalor otherwise) cause symptoms or create a significant biolog-ical cost to its host.

CONCLUSIONS AND FUTURE DIRECTIONS

Fungicide-tolerant strains of pathogens and loss of efficacioussynthetic chemicals—through their rejection by consumers andthrough regulatory bodies banning those that cause environ-mental pollution and human toxicity—have led to the searchfor alternative disease control practices in agriculture. Over 100years of biological research has demonstrated the importanceof microorganisms in maintaining the health of higher organ-isms, with some researchers ranking microorganisms amongstour best allies in plant disease control (Sutton and Peng 1993).Perhaps in part due to the difficulties experienced by traditionalbiological control programmes, and the advent of sophisticatedscientific methods leading to increased biological knowledge,endophytic BCAs are regarded by many as the emerging tool forcontrolling plant pests and diseases (Backman and Sikora 2008).This sub-category of BCAs have clear advantages over their tra-ditional phylloplane or rhizosphere counterparts. In many tra-ditional biological control systems, not enough consideration isgiven to formulation and delivery systems since on applicationthe BCA can experience sub-optimal environmental conditions,leaving the organism fighting for survival rather than flourish-ing and able to control the target pest or pathogen. An additionalobstacle for post-harvest BCAs is that they are generally unableto control established or latent pathogenic infections (Ippolitoand Nigro 2000). Attempts to overcome this issue have been cen-tred on applying the BCA early on in the field before the ini-tial pathogen infection. However, the use of an endophytic BCAthat remains within the host, potentially throughout its entirelife cycle, means that timing considerations of BCA applicationwould become irrelevant. For example, endophytes are not ex-posed to the same degree of ultra-violet radiation and fluctua-tions of temperature andmoisture experienced bymany phyllo-plane bacteria (Chen et al. 1995). But these organisms are notsynthetic chemicals, and educational workshops for the end-user must be provided by research teams and commercial com-panies when selling a live BCA to ensure users understand notjust its advantages but also its limitations. This review has high-lighted both advantages and limitations of the diverse and in-trinsic relationships that endophytic BCAs have with their hostplants. In some instances these endophytes are regarded as anextension to the plant’s own defence response in view of the factthatmany of the bioactivemetabolites produced are truly a com-bined effort with neither partner able to synthesise them alone.

An increased understanding ofmolecular mechanisms suggeststhat this is the evolutionary direction in which many of thesemicroorganisms are moving.

This review has illustrated the advantages and challengesposed by the use of endophytic BCAs from both a biologicaland commercial perspective. It has identified attributes and be-havioural traits of the endophytic BCAs that can be successfullyexploited and applied in agricultural systems to not only pro-vide an efficacious solution to plant and disease control but alsomake economic sense. Our intention was not to develop an ex-act checklist of behavioural qualities but to make researchersand companies aware of the important lifestyle characteristicsto be considered in these symbiotic associations and that maydetermine whether a BCA will be successfully delivered into themarket place. The main factor that limits the commercial de-velopment of microbial biological control products is a lack ofbiological understanding regarding the endophyte-plant asso-ciation, especially their dissemination, limitations surroundingefficacy and their compatibility within a novel host. This reviewhas highlighted a clear distinction between those that will re-main as interestingmodel systems for research and educationalpurposes and those that will form commercial products beingable to be marketed to make real differences in an agriculturalcontext as well as returning a profit for investors and end-users.

With the advent of molecular biology and the new ‘omics’approaches, valuable tools are now available for exploring,identifying and characterising the contributions of genetic andmetabolic elements involved in the interactions between hostplants and endophytic microorganisms (Hardoim et al. 2015).These avenues for research have already proposed valuable in-sights into these symbiotic relationships with the latest pro-posals suggesting many endophytes should be regarded as ‘dis-armed pathogens’ (Krause et al. 2006). For example, this has beensuggested after the complete genome analysis of Azoarcus, anendophytic bacteriumofmany crops including sugarcane, as theendophyte lacks many of the toxins and plant cell wall degrad-ing enzymes possessed by its pathogenic relatives (Krause et al.2006).

Endophyte research is still an exceptionally young scientificfield and there are potentially thousands, perhaps many mag-nitudes higher, of endophytic microorganisms still to be dis-covered, investigated and understood. In addition, new knowl-edge continues to be gained and used with respect to ‘estab-lished’ endophytic BCA species in terms of their behaviouraltraits and lifestyles. For example, fungi generally regarded as en-tomopathogens or labelled as saprophytes have been observedcolonising the intracellular tissues of plants in symptomlesssymbiosis. As many plants can harbour more than one endo-phyte, indirect competition between species is expected (Muller2003; Liu et al. 2011), as well as direct competition from thosespecies and strains occupying the same ecological niche. Onthe other hand, many endophytic species may work together inunison to bring about wider benefits for themselves and theirshared host. For example the fascinating interaction between P.indica and rhizobia is only just being realised (Molitor and Ko-gel 2009). Little is known about these more complicated trophicinteractions (Mercado-Blanco 2015). Additionally other avenuesof plant–microbial interactions are now being investigated in-dicating that a complete change in plant microbial flora can beinfluenced by the endophytic community (Roberts and Lindow2014), or the allelopathic potential of the symbiosis (Vazquez-de-Aldana et al. 2011).

Not so surprisingly, the presence of some endophytes, andthe difference between endophyte strains, can affect some plant

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Card et al. 13

properties more than genetic variation intrinsic to the host (Eas-ton 2007). The challenge for the endophyte research commu-nity is to better understand the biology that surrounds specificendophyte–plant interactions including their beneficial traitsand lifestyle characteristics, i.e. their biology, and in doing sopropel agricultural microbiology forward as a commercially vi-able technology, rather than spending excessive time on tax-onomy and nomenclature. As scientists we like to be organ-ised and understand taxonomic relatedness between microor-ganisms but this surely should not be our main priority.

ACKNOWLEDGEMENTS

We thank COST action FA 1103 for the initial opportunity topresent the first rendition of this article at the Microbe-assistedcrop production – opportunities, challenges and needs (mi-CROPe), 23–25 November 2015, Vienna, Austria. We thank WeiZhang for supplying the micrograph of Epichloe sp. in planta usedfor the graphical abstract.

Conflict of interest. None declared.

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