Endophytic Actinobacteria and the Interaction of Micromonospora and Nitrogen Fixing Plants

15 Pages·2015·1.27 MB·English

by  Martha E. Trujillo

REVIEW
published:01December2015
doi:10.3389/fmicb.2015.01341
Endophytic Actinobacteria and the
Interaction of Micromonospora and
Nitrogen Fixing Plants
MarthaE.Trujillo*,RaúlRiesco,PatriciaBenitoandLorenaCarro
DepartamentodeMicrobiologíayGenética,UniversidaddeSalamanca,Salamanca,Spain
For along time,itwasbelievedthata healthyplantdid not harbor anymicroorganisms
withinitstissues,asthesewereoftenconsidereddetrimentalfortheplant.Inthelastthree
decades, the numbers of studies on plant microbe-interactions has led to a change in
our view and we now know that many of these invisible partners are essential for the
overall welfare of the plant. The application of Next Generation Sequencing techniques
is a powerful tool that has permitted the detection and identification of microbial
communities in healthy plants. Among the new plant microbe interactions recently
reportedseveralactinobacteriasuchasMicromonosporaareincluded.Micromonospora
is a Gram-positive bacterium with a wide geographical distribution; it can be found
in the soil, mangrove sediments, and freshwater and marine ecosistems. In the last
years our group has focused on the isolation of Micromonospora strains from nitrogen
fixing nodules of both leguminous and actinorhizalplants and reportedfor the first time
Editedby:
its wide distribution in nitrogen fixing nodules of both types of plants. These studies
ShengQin,
JiangsuNormalUniversity,China have shown how this microoganism had been largely overlooked in this niche due to
Reviewedby: its slow growth. Surprisingly, the genetic diversity of Micromonospora strains isolated
JamesA.Coker,
from nodules is very high and several new species have been described. The current
UniversityofMarylandUniversity
College,USA dataindicatethatMicromonosporasaelicesensisisthemostfrequentlyisolatedspecies
JuliaMaresca, from the nodular tissues of both leguminous and actinorhizal plants. Further studies
UniversityofDelaware,USA
have also been carried out to confirm the presence of Micromonospora inside the
*Correspondence:
noduletissues,mainlybyspecificinsituhybridization.Theinformationderivedfromthe
MarthaE.Trujillo
[email protected] genome of the model strain, Micromonospora lupini, Lupac 08, has provided useful
information as to how this bacterium may relate with its host plant. Several strategies
Specialtysection:
potentially necessary for Micromonospora to thrive in the soil, a highly competitive,
Thisarticlewassubmittedto
ExtremeMicrobiology, and rough environment, and as an endophytic bacterium with the capacity to colonize
asectionofthejournal
the internal plant tissues which are protected from the invasion of other soil microbes
FrontiersinMicrobiology
were identified. The genome data also revealed the potential of M. lupini Lupac 08 as
Received:21July2015
Accepted:16November2015 a plant growth promoting bacterium. Several loci involved in plant growth promotion
Published:01December2015 features such as the production of siderophores, phytohormones, and the degradation
Citation: of chitin (biocontrol) were also located on the genome and the functionality of these
TrujilloME,RiescoR,BenitoPand
genes was confirmed in the laboratory. In addition, when several host plants species
CarroL(2015)Endophytic
ActinobacteriaandtheInteractionof were inoculated with Micromonospora strains, the plant growth enhancing effect was
MicromonosporaandNitrogenFixing
evident under greenhouse conditions. Unexpectedly, a high number of plant-cell wall
Plants.Front.Microbiol.6:1341.
doi:10.3389/fmicb.2015.01341 degradingenzymeswerealsodetected,atraitusuallyfoundonlyinpathogenicbacteria.
FrontiersinMicrobiology|www.frontiersin.org 1 December2015|Volume6|Article1341
Trujilloetal. EndophyticActinobacteriaandMicromonospora
Thus, Micromonospora can be added to the list of new plant-microbe interactions.
The currentdataindicatethatthismicroorganism mayhaveanimportantapplicationin
agricultureandotherbiotechnologicalprocesses.Theavailableinformationispromising
but limited, much research is still needed to determine which is the ecological function
ofMicromonosporaininteractionwithnitrogenfixingplants.
Keywords:Micromonospora,legumes,PGPB,actinorhizal,endophytic,nodule
INTRODUCTION In this review, the diversity and interaction between
actinobacteria and plants will be discussed, focusing on their
Bacteria,archaea,andvirusesarepresentineverynichepresent ecologicalaspectsandpotentialapplicationsinagriculture.The
in our planet and have a great impact on other forms of life. secondpartofthisrevisionwillfocusonthespecificinteraction
SincetheappearanceofplantsonEarth,theircapacitytoadapt ofthegenusMicromonosporawithnitrogenfixingplants.
to different ecosystems and their evolutionary process have
inherentlybeenassociatedtomicroorganisms(ReidandGreene,
2012). PLANT-ASSOCIATED ACTINOBACTERIA
Microbialcommunitiespresentinsoilaccountfortherichest
reservoir of biological diversity in our planet (Berendsen et al., Actinobacteria represent approximately 20–30% of the
2012). Microorganisms that live in the rhizosphere, the soil rhizospheric microbial community (Bouizgarne and Ben
regioninfluencedbyplantroots,areofgreatimportanceasthis Aouamar, 2014). They are Gram-positive and show a wide
is where most plant-microbe interactions occur (Schenk et al., morphological spectrum ranging from unicellular organisms
2012) and this complex plant-associated microbial community to branching filaments that form a mycelium. A unique
is for the most part beneficial to the plant (Berendsen et al., feature is their high guanine plus cytosine content (>50%)
2012). Despite the importance of microorganisms for plants, in their genome. These microorganisms are for the most part
theseextremelycomplexmicrobialcommunitieshaveremained saprophytic, soil-dwelling organisms with an important role
largely uncharacterized mainly due to our lack of culturing in the turnover of organic matter. In addition, many species
most microorganisms under laboratory conditions (Schenk are sporulated and spend the majority of their life cycles as
et al., 2012). Fortunately, our awareness of mutually beneficial semidormantspores(CoombsandFranco,2003a).
relationshipsandtheirpotentialapplicationinbiotechnological Severaltaxaarewell-knowntointeractwithplantsandthese
processes is expanding, in part due to the new sequencing include examples of both endophytic and plant-pathogenic
technologiesandinformationderivedfromtheiruse. species. The first actinobacterial endophyte isolated, Frankia
Microbes that interact with plants are termed rhizospheric (Callahametal.,1978),isanitrogen-fixingmicroorganismthat
or endophytic depending on their localization outside or inducesnodulationonseveralangiospermplantfamiliesandhas
inside the plant, respectively, and many endophytes originate receivedalotofattentionduetoitsroleinthenitrogeneconomy
from the rhizosphere or phyllosphere (Dudeja et al., 2012). of its hosts (Verma et al., 2009). Several plant-pathogenic taxa
These organisms can accelerate seed germination, promote include Streptomyces acidiscabies, Streptomyces europaeiscabiei,
plant establishment under adverse conditions, enhance plant Streptomycesscabies,andStreptomycesturgidiscabieswhichcause
growth or prevent pathogen infections (Hurek et al., 2002; potato scab (Loria et al., 2006; Bignell et al., 2010); Clavibacter
Ryan et al., 2008). Thus, a complex and invisible ecosystem michiganensis with several subspecies and pathogen for alfalfa
sustains plant growth and health (Reid and Greene, 2012). (C. michiganensis subsp. insidiosus), maize (C. michiganensis
The potential application of beneficial microbes in different subsp. nebraskrensis), potato (C. michiganensis subsp.
fields(e.g.,agriculture,biotechnology,medicine,etc.)isimmense michiganensis) and wheat (C. michiganensis subsp. tessellarius);
providedprogressismadeinunderstandingthesecomplexplant- Leifsoniaxylisubsp.xyliwhichcausesratoonstuntingdiseaseof
microbeinteractionsinaglobalcontext. sugarcane (Young et al., 2006); Curtobacterium flaccumfaciens
Hitherto,plantassociatedGram-negativebacteriaarethebest which affects several Phaseolus and Vigna species, Beta vulgaris
studiedgiventheirrelativefacilitytoberecoveredfrominternal species(redandsugarbeet),Ilexopaca(Americanholly),Tulipa
plant tissues and also because mutants can be easily generated species(tulips),andEuphorbiapulcherrima(poinsettia)(Saddler
for interaction studies (Francis et al., 2010). However, many and Messenber-Guimaraes, 2012); Rathayibacter iranicus and
Gram-positive bacteria included in the phyla Firmicutes and Rathayibactertriticiwhichcausegumminginseveralgrassesand
Actinobacteria (e.g., Bacillus, Micromonospora, Streptomyces, wheat(EvtushenkoandDorofeeva,2012).
etc.) have excellent biocontrol, plant growth-promoting and Inthelastdecade,manyreportsontheisolationanddiversity
bioremediation activities. In addition, several characteristics of plant-associated and endophytic actinobacteria from wild
observedincludingpigmentandsporeproduction,biosynthesis plantsandcropshavebeenpublished.Inmanyofthesestudies,
of secondary metabolites and unique lifestyles present in aneutraloraplantgrowthpromotioneffectwasobserved.The
these microorganisms can be advantageous for different isolationandidentificationofactinobacteriainhealthyinternal
biotechnologicalapplications,includingagriculture. root tissues of wheat was reported by Coombs and Franco
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Trujilloetal. EndophyticActinobacteriaandMicromonospora
(2003a);theseauthorsfurtherdemonstratedthecolonizationof Again, the genus Streptomyces accounted for >60% of the
germinating wheat by one of the isolated strains, Streptomyces isolates.
sp. EN27 (Coombs and Franco, 2003b). A Streptomyces strain, Althoughthepercentageofplantspeciessampledatpresent
WYEC108, isolated from linseed rhizosphere soil in Great is very low, medicinal plants have received special attention
Britain(Crawfordetal.,1993)wasabletocolonizetherootsof giventheirimportanceaspotentialreservoirsofactinobacterial
Pisum sativum, increased the number and size of root nodules, communities that produce compounds with biotechnological
and enhanced the assimilation of iron and other nutrients by application. Qin et al. (2009, 2012) conducted a thorough
the plant (Tokala et al., 2002). Several actinobacterial strains study screening medicinal plants growing in the tropical
recovered from wild plants adapted to poor soil and severe rain forests in Xishuangbanna, China. These authors focused
climateconditionsoftheAlgerianSaharadesertwerereportedby on the isolation of non-streptomycetes and found that the
Goudjaletal.(2013).Someofthesestrainsproducedtheauxin genus Pseudonocardia was the predominant taxon, followed
indol acetic acid (IAA), which promoted seed germination and byNocardiopsis,Micromonospora,andStreptosporangiumwhile
root elongation when tomato seeds were treated with bacterial almost 25% of the strains could not be identified at the
supernatants. genus level. An in depth analysis of the plant Maytenus
Thesearchofendophyticactinobacteriaasbiologicalcontrol austroyunnanensisapplyingculture-dependentandindependent
agents of plant disease is also of interest given their ability to methods revealed an immense diversity reporting genera
colonize healthy plant tissues and produce antibiotics in situ such as Actinostreptospora, Amnibacterium, Catenuloplanes,
(Kunoh,2002;Caoetal.,2004).Maize(Zeamays),animportant Quadrisphaera, and Pseudokineococcus which were previously
crop cultivated in many countries, especially in tropical areas, unknowntoresideinsideplanttissues(Qinetal.,2012).
was also screened for the presence of bioactive actinobacteria A list of endophytic and plant-associated actinobacteria
(deAraújoetal.,2000).Endophyticstreptomycetesisolatedfrom recovered from different plant species and their potential
healthy banana plants (Musa sp.), were studied for the ability applicationinagricultureispresentedinTable1.
to produce antifungal molecules that inhibited the growth of In recent years, metagenomic analyses have been used to
Fusarium oxysporum, which causes fusarium wilt (Cao et al., determine the bacterial communities of several agriculturally
2005).Similarly,Streptomycesstrainswereisolatedfromtomato important crops. These studies have shown that actinobacteria
and native plants of the Algerian Sahara and screened for are present in many of these plant microbiomes. Okubo
biocontrol activity against Rhizotocnia solani (Goudjal et al., et al. (2014) demonstrated that while the shoots of two field-
2014). grownricecultivarscollectedinNipponbareandKasalathwere
Severalstudieshavefocusedonthediversityanddistribution dominated by Alphaproteobacteria (approximately 52%), the
of actinobacterial communities in plants, these works have actinobacterial populations made up to 15% of the bacterial
provided information about the most common taxa found, community structure. The characterization of the natural
e.g., the genus Streptomyces, but have also discovered new microbiome of Vitis vinifera leaves in Portugal reported a
plant-actinobacteria associations as those represented by the high diversity of proteobacteria, firmicutes, and actinobacteria,
interactionMicromonospora-nitrogenfixingplants. wherethelattergroupaccountedforapproximately19%ofthe
Members of the genera Microbispora, Micromonospora, microbialcommunitycompositionandmembersofthefamilies
Nocardia, Streptosporangium, and Streptoverticillium were Corynebacteriaceae,Microbacteriaceae,andKineosporiaceaewere
recovered from the surface of sterilized roots of different plant identified(Pintoetal.,2014).
species in Italy (Sardi et al., 1992) and of maize in Brazil (de A recent study to determine the bacterial communities of
Araújo et al., 2000). Interestingly, the genus Microbispora Olea europaea L. cultivars collected from different regions
was the most abundant genus recovered in maize (44%), in the Mediterranean basin also confirmed the presence of
followed by Streptomyces and Streptosporangium. A diverse actinobacterial populations on the olive leaf endosphere. An
collection of 11 native Korean plants were screened for the interesting conclusion of this work was that soil, climate
presence of endophytic actinobacteria. Streptomyces was conditions, and geographical distances had little effect on the
the most common taxon accounting for almost 50% of the endophytic microbial community composition (Müller et al.,
strains isolated and followed by the genera Microbacterium, 2015). In another study, the root microbiota of Lactuca sativa
Microbispora, Micrococcus, Micromonospora, Rhodococcus, cultivars and its wild ancestor Lactuca serriola were analyzed,
and Streptacidiphilus. Single isolates representing the the lettuce microbiota was dominated by Proteobacteria and
genera Arthrobacter, Dietzia, Herbiconiux, Kitasatospora, Bacteriodetes, but Chloroflexi and Actinobacteria were also
Mycobacterium,Nocardia,Rathayibacter,andTsukamurellawere abundant (Cardinale et al., 2015). The composition of the
alsorecovered(Kimetal.,2012). actinobacterial population included members of the families
Kaewkla and Franco (2013) demonstrated the high diversity Micromonosporaceae and Nocardioaceae but also the genera
of actinobacterial strains distributed in native Australian Actinoplanes, Aeromicrobium, Arthrobacter, Demequina, and
plants using highly designed isolation protocols which Streptomyces.Interestingly,thedomesticatedcultivar(L.sativa)
included low concentration isolation media, plating larger wasricherinspeciesdiversitythanitswildcounterpartL.serriola.
quantities of plant sample and long incubation times (up Unfortunately for most of the above studies, the function of
to 16 weeks). These authors reported the isolation of >500 these microorganisms on their host plants is unknown. In the
actinobacterialstrainsthatwereidentifiedin16differentgenera. caseoflettuce,whichisoneoftherawfoodswidelyconsumed,
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Trujilloetal. EndophyticActinobacteriaandMicromonospora
TABLE1|Endophyticandplant-associatedactinobacteriareportedintheliterature.
Genus Hostplant Isolationsource References Potentialuse
Frankia* Comptonia Rootnodule Callahametal.,1978 Nitrogenfixation
Actinosynnema Grassblade – Hasegawaetal.,1978 Notdetermined
Streptomyces Alliumporrum,Amaryllisbelladona,Betula Roots Sardietal.,1992 Notdetermined
pendula,Brassicaoleracea,Callunavulgaris,
ChelidoniummajusCichonumintybus,
Euphorbiasp.,Fragariavesca,Lactuca
scariola,Quercussp.,Rubusidaeus
Streptomyces Linumusitatissimum Rhizospheresoil Crawfordetal.,1993 Growthpromotion
Microbispora,Streptomyces,Streptosporangium Zeamays Roots deAraújoetal.,2000 Biocontrol
Microbispora,Micromonospora,Nocardioides, Triticumaestivum Rootsandleaves CoombsandFranco, Biocontrolagent
Streptomyces 2003a
Streptomyces Licopersiconesculentum Roots Caoetal.,2004 Biocontrol
Streptomyces,Streptoverticillium, Musasp. Roots Caoetal.,2005 Biocontrolof
Streptosporangium Fusarium
oxysporum
Agromyces,Microbacterium Retamataetam,Ononisnatrix,Argyrolobium Rootnodules Zakhiaetal.,2006 Notdetermined
uniflorum,Astragalusarmatus
Actinoplanes,Micromonospora,Streptomyces Cucumissativus Roots El-Tarabilyetal.,2009 Biocontrol;growth
promotion
Microbispora,NocardiaSacchromonospora, Azadirachtaindica Leaves,stems, Vermaetal.,2009, Siderophore
Streptomyces,Streptosporangium, roots 2011 production;
Streptoverticillium biocontrol
Pseudonocardia,Nocardiopsis, Phyllanthusurinaria,Kadsuraheteroclita, Leaves,stems, Qinetal.,2009 Secondary
Micromonospora,Streptosporangium Maesaindica,Rauvolfiaverticillata,Paris roots metabolites
yunnanensis,Maytenusaustroyunnanensis,
Gloriosasuperba,Scopariadulcis,Tadehagi
triquetrum,Goniothalamussp.,Cephalotaxus
sp.,andAzadirachtasp.
Arthrobacter,DietziaHerbiconiux, Artemisiaprinceps,Capsellabursa-pastoris, Roots Kimetal.,2012 Growthpromotion,
Intrasporangium,Kitasatospora,Microbacterium, Chelidoniummajus,Conyzacanadensis, biocontrol
Microbispora,MicrococcusMicromonospora Erigeronannuus,Irisrossii,Lamium
Mycobacterium,NocardiaRathayibacter, purpureum,Physostegiavirginiana,Rudbeckia
Rhodococcus,Streptacidiphilus,Streptomyces, bicolor,Setariaviridis,Violamandshurica
Tsukamurella
Actinomadura,Amycolatopsis, Maytenusaustroyunnanensis Root,stem,leaves Qinetal.,2012 Notdetermined
Cellulosimicrobium,Gordonia,Glycomyces,
Janibacter,Jiangella,Microbacterium,
Micromonospora,Mycobacterium,Nocardia,
Nocardiopsis,Nonomuraea,Plantactinospora,
Polymorphospora,Promicromonospora,
Pseudonocardia,Streptosporangium,
Streptomyces,Saccharopolyspora,Tsukamurella
Actinomadura,Actinomycetospora, Callitrispreissii,Eucalyptuscamaldulensis, Leaves,stems, KaewklaandFranco, Notdetermined
Actinopolymorpha,Amycolatopsis,Gordonia, Eucalyptusmicrocarpa,Pittosporum roots 2013
Kribbella,Micromonospora,Nocardia, phylliraeoides
Nocardioides,Nocardiopsis,Nonomuraea,
Polymorphospora,Promicromonospora,
Pseudonocardia,Streptomyces,Williamsia
(Continued)
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Trujilloetal. EndophyticActinobacteriaandMicromonospora
TABLE1|Continued
Genus Hostplant Isolationsource References Potentialuse
Actinomadura,Kibdelosporangium, Achilleafragrantissima,Artemisiajudaica, Notspecified El-Shatouryetal.,2013 Growthpromotion
Kitasatospora,Micromonospora, Centaureascoparia,Chiliadenusmontanus,
Microtetraspora,Nocardia,Nocardioides, Echinopsspinosus,Iphionamucronata,
Nocardiopsis,Promicromonospora, Pulicariacrispa,Scariolaorientalis,Seriphidium
Pseudonocardia,Saccharopolyspora, herba-album,Tanacetumsinaicum
Streptoalloteichus,Streptomyces
Streptomyces Cleomearabica,Solanumnigrum,Astragallus Roots Goudjaletal.,2013, Biocontrol,IAA
armatus,Aristidapungens,Panicumturgidum 2014 production,
growthpromotion
Amycolatopsis,Isoptericola,Micromonospora, Acaciaauriculiformis,Bauhiniapurpurea, Roots,rhizosphere Mingmaetal.,2014 Biocontrol
Microbispora,Nocardia,Nonomuraea, Canavaliagladiate,Cassiafistula,Clitoria
Promicromonospora,Pseudonocardia, ternatea,Erythrinavariegata,Leucaena
Streptomyces leucocephala,Mimosapudica,Peltophorum
pterocarpum,Pithecellobiumdulce,Poinciana
pulcherrima,Pterocarpusmacrocarpus,
Samaneasaman,Sesbaniagrandiflora,
Tamarindusindica
Microbacterium Trichiliaelegans Leaves Rhodenetal.,2015 Notdetermined
Thedatapresentedisbasedonthereferencesprovidedincolumn4.
*Frankiaisknowntoinducerootnodulesonadiversegroupofangiospermplantstermedactinorhizals.
it has been suggested that bacteria present in the plant’s root et al., 2014). However, our knowledge about these new plant-
such as Streptomyces, may serve as biological control agents by microbe interactions is still very poor given the limited data
producing antibiotics to eliminate potential human pathogens currentlyavailable.
(e.g.,enterobacteria)(Cardinaleetal.,2015). Inlightoftheirecologicalimportance,Frankiaasaprovider
Several soil microbiomes related to Andropogon gerardii, of nitrogen to actinorhizal plants, and Streptomyces as a plant
Schizachyrium scoparium, Lespedeza capitata, and Lupinus pathogen for important crops such as potato, these bacteria
perennis grown in communities which varied in plant richness have been under research for many decades, but this is
(1–16 species) were determined (Bakker et al., 2014). In this not the case for most of other reported plant-actinobacteria
study the antagonistic activity and community structure of interactions. However, in the last 10 years the interaction
Streptomycespopulationswasassessedinrelationtothespecies Micromonospora-nitrogen fixing plants is gaining attention
plant richness. The authors reported that the diversity and due its potential application in downstream biotechnological
richness of bacterial and Streptomyces communities displayed applications,especiallyintheareaofagriculture.Inthefollowing
differentrelationshipswithbioticandabioticsoilcharacteristics, sections we will provide a general overview on the past and
thereforeinfluencingbacterialcommunities. presentstatusofMicromonosporaanditscloseinteractionwith
The roots, leaves, and stems are the main plant tissues legumesandactinorhizalplants.
that have been screened for the presence of bacteria, however,
nitrogen fixing nodules produced by legumes and actinorhizal
MICROMONOSPORA AND NITROGEN
plants are also an important reservoir of microorganisms.
Nodulesarerichinnutrientsandthereforecanalsobecolonized FIXING NODULES: A UNIVERSAL
bybacteriaunrelatedtorhizobialorFrankiasymbioticnitrogen PLANT-MICROBE INTERACTION?
fixation.
Actinobacterial strains identified in the genera Agromyces, TheactinobacteriumMicromonosporawasfirstdescribedin1923
Curtobacterium, Microbacterium, Micromonospora, and (Ørskov,1923).ThefirststrainsoriginatedfromsoilandJensen
Streptomyces have been reported from nodule tissues (Sturz (1932)pointedouttheimportanceofthismicroorganisminthis
etal.,1997;Trujilloetal.,2006,2007,2010;Zakhiaetal.,2006; niche.ThisbacteriumbelongstothefamilyMicromonosporaceae
Muresu et al., 2008; Stajkoviæ et al., 2009; Deng et al., 2011; and includes aerobic, filamentous, spore-producing and
Hoqueetal.,2011;Lietal.,2011;Carroetal.,2012a).Ofthese, mesophilic microorganisms. Micromonospora colonies are
thegeneraMicrobacteriumandMicromonosporawerethemost usually pigmented and range in color from orange, red, or
frequently isolated. Host plants inoculated with some of these brown. In many old cultures a brown-black, or black mucous
strains showed better growth and development in comparison massofsporesisobserved.Theformationofsinglesporesisthe
with non-inoculated controls suggesting a beneficial effect mainmorphologicalcharacteristicofthegenusMicromonospora;
(Trujilloetal.,2010,2014b;Dengetal.,2011;Martínez-Hidalgo however, spores are also produced in dense clusters on the
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Trujilloetal. EndophyticActinobacteriaandMicromonospora
surface or completely embedded in the substrate mycelium microorganismisalsoanormaloccupantofactinorhizalnodules.
(Figure1)(Genilloud,2012;Trujilloetal.,2014a). Thus, the systematic recovery of Micromonospora populations
The presence of Micromonospora has been reported from strongly suggests that this bacterium closely interacts with the
many geographical sites worldwide and although soil is the host plant and nitrogen-fixing bacteria occupying the same
most frequent source of isolation, marine, aquatic sediments niche.
and mangrove environments are also inhabited by this The biogeographical and species distribution of
microorganism(Maldonadoetal.,2009;Genilloud,2012;Trujillo Micromonosporae isolated from nitrogen fixing nodules of
et al., 2014a). In recent years Micromonosporae have been legumesandactinorhizalplantssampledhithertoispresentedin
reported as major components of nitrogen fixing root nodules Table2.
ofbothleguminousandactinorhizalplants(Valdésetal.,2005;
Trujilloetal.,2006,2007,2010;Garciaetal.,2010;Carroetal.,
DISTRIBUTION, LOCALIZATION AND
2012a,2013a).IsolationofMicromonosporastrainsfrominternal
GENETIC DIVERSITY OF
nodular tissues has been reported from the legumes Arachis
hypogaea,Cicerarietinum,Glycinemax,Lensculinaris,Lupinus MICROMONOSPORA IN NITROGEN
angustifolius, Lupinus gredensis, Medicago sativa, Melilotus sp., FIXING NODULES
Mucunasp.,Ononissp.,Ornithopussp.,Phaseolussp.,Trifolium
sp.,andViciasp.TheisolationofMicromonosporastrainsusually ThedistributionofMicromonosporastrainsinthenitrogenfixing
requires selective isolation procedures to favor its slow growth, nodules sampled so far indicate that its distribution is not
however,inalltheaboveexamples,thesameisolationprotocolas homogeneousanditvariesfromnoduletonoduleandplantto
thatusedfortheisolationofrhizobiawasapplied(Cerda,2008; plant(Trujilloetal.,2010;Carroetal.,2012a).
Rodríguez,2008;Carro,2009;AlonsodelaVega,2010;Trujillo ThedistributionpatternofMicromonosporainLupinusspp.is
etal.,2010). highlyvariablewithnoisolatesforsomenodulestoasmanyas
ActinorhizalplantsthathavebeensampledtodateinMexico, approximately30(AlonsodelaVega,2010;Trujilloetal.,2010).
Spain, Canada, and France include the species Alnus viridis, Variationisalsoreportedfromplanttoplantandfromdifferent
Casuarinaequisetifolia,Coriariamyrtifolia,Elaeagnusxebbingei, nodulesofthesameplant(Trujilloetal.,2010).Acomparisonof
Hippophae rhamnoides, Myrica gale, and Morella pensylvanica thespeciesLupinusangustifoliusandLupinusgredensiscollected
(Valdés et al., 2005; Trujillo et al., 2006; Carro et al., 2013a). in the same geographical area in Spain, indicated that 67 and
Except for the study of Valdés et al. (2005), the isolation of 60% of the plant samples screened (17 in total) contained the
Micromonospora from actinorhizal nodules also followed the targetmicroorganism,respectively.Outofthe45noduleschosen
sameisolationprotocolsasthatoflegumes,usingyeast-mannitol forisolation,95Micromonosporastrainswererecovered,74from
agar as isolation medium (Vincent, 1970). Currently our group L.angustifoliusand21fromL.gredensis.Interestingly,48%ofthe
maintainsacollectionof∼2000isolatesrecoveredfromdiverse nodules did not appear to contain any Micromonospora strains
legumeandactinorhizalplantsspeciescollectedinSpain,France, (AlonsodelaVega,2010).
Germany,Ecuador,Nicaragua,andAustraliabutourhypothesis Intermsofthebacterialspeciesdistribution,Micromonospora
is that Micromonospora is also present in those plant species saelicesensisandMicromonosporalupiniwerethemostabundant,
which have not been sampled to date. In the case of legumes, neverthelessthediversitydeterminedonthebasisof16SrRNA
the above examples indicate how Micromonospora had been genesequencingwasveryhigh(AlonsodelaVega,2010;Trujillo
largely overlooked in this niche due to its slow growth as etal.,2010).Theseauthorsalsoscreenedlupineplantsatdifferent
compared to rhizobial strains which can be readily recovered growthstageswhichcorrespondedtoyoung,maximumgrowth,
fromisolationplatesafter3–5dayswhileMicromonosporastrains and flowering plants. In this case, the number of bacteria
usually appear after 7–10 days on the same plates. While the increased in parallel to the plant growth and decreased as the
work carried by Carro et al. (2013a) strongly suggests that this plantsbecameold.
FIGURE1|MorphologicalfeaturesofMicromonospora.(A)Micromonosporaeisolatesrecoveredfromanitrogenfixingnodule.(B)14dayoldcolonyproducing
brown-blackspores.(C)Scanningelectronmicrographofamucousmassofspores.Bar,1µm(Carro,2009;AlonsodelaVega,2010).
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Trujilloetal. EndophyticActinobacteriaandMicromonospora
TABLE2|BiogeographicalandspeciesdistributionofMicromonosporaeinnitrogenfixingnodulesoflegumesandactinorhizalplantssampled.
Hostplant(Legumes) Commonname Geographicalorigin Closestspeciesidentification(16SrRNAgene) References
Arachyssp. Peanut Nicaragua M.chaiyapumensis,M.endolithica Cerda,2008
Cicerarietinum Chickpea Spain ND Trujilloetal.,2010
Glycinemax Soy Nicaragua ND Trujilloetal.,2010
Lensculinarium Lentil Spain ND Trujilloetal.,2010
Lupinusangustifolius Bluelupine Spain M.aurantiaca,M.auratinigra,M.chaiyapumensis,M. Trujilloetal.,2007;
coriariae,M.coxensis,M.echinospora,M.fulviviridis,M. Rodríguez,2008;Alonsode
lupini,M.matsumotoense,M.narathiwatensis,M. laVega,2010
olivasterospora,M.sagamiensis,M.saelicesensis
Lupinusgredensis Lupine Spain M.chaiyapumensis,M.chersina,M.coxensis,M. AlonsodelaVega,2010
echinofusca,M.echinospora,M.lupini,M.olivasterospora,
M.saelicesensis,M.viridifaciens
Lupinussp. Lupine Germany M.saelicesensis Trujilloetal.,2010
Medicagosp. Alfalfa Australia,Spain M.aurantiaca,M.chokoriensis,M.lupini,M.saelicesensis, Martínez-Hidalgoetal.,
M.schwarzwaldensis,M.tulbaghiae,M.viridifaciens 2014
Mucunasp. Mucuna Ecuador ND Trujilloetal.,2010
Ononissp. – Spain ND Trujilloetal.,2010
Ornithopussp. – Spain ND Trujilloetal.,2010
Phaseolusvulgaris Bean Nicaragua M.chaiyapumensis,M.chersina,M.endolithica Cerda,2008
Pisumsativum Sweetpea Spain M.aurantica,M.auratinigra,M.chaiyapumensis,M.chersina, Carro,2009;Carroetal.,
M.coerulea,M.coriariae,M.coxensis,M.fulviviridis,M. 2012a
lupini,M.matsumotoense,M.pattaloongensis,M.
saelicesensis,M.sagamiensis„M.siamensis
Trifoliumsp. Clover Spain ND Trujilloetal.,2010
Viciasp. Vetch Spain ND Trujilloetal.,2010
HOSTPLANT(ACTINORHIZALS)
Alnusglutinosa Alder France M.cremea,M.coxensis,M.lupini,M.matsumotoense,M. Carroetal.,2013a
olivasterospora,M.saelicesensis,M.siamensis
Alnusviridis Alder France M.chokoriensis,M.coriariae,M.lupini,M.matsumotoense, Carroetal.,2013a
M.pisi,M.rifamycinica,M.saelicesensis
Casuarinaequisetifolia Coastsheoak Mexico M.aurantiaca Valdésetal.,2005
Coriariamyrtifolia Redoul Spain,France M.coriarie,M.saelicesensis,M.peucetia Trujilloetal.,2006;Carro
etal.,2013a
Elaeagnusxebbingei – France M.aurantiaca,M.auratinigra,M.chaiyaphumensis,M. Carroetal.,2013a
coriariae,M.coerulea,M.cremea,M.coxensis,M.equina,
M.lupini,M.matsumotoense,M.mirobrigensis,M.peucetia,
M.saelicesensis,M.siamensis
Hippophaerhamnoides Sandthorne France M.chaiyapumensis,M.chersina,M.coxensis,M.equina,M. Carroetal.,2013a
lupini,Mnarathiwatensis,M.saelicesensis,M.siamensis,M.
viridifaciens
Morellapensylvanica – France M.coriariae,M.cremea,M.olivasteraspora,M.peucetia,M. Carroetal.,2013a
saelicesensis
Myricagale Canada M.lupini,M.tulbaghiae Carroetal.,2013a
As for the legume Pisum sativum, a similar pattern of the number of isolates also varied significantly. High numbers
distribution was observed. However, for this plant, at least ofMicromonosporastrainswereisolatedfromAlnus,Elaeagnus,
one Micromonospora strain was recovered from every nodule andHippophaenodules,whilethenumberofisolateswasmuch
sampled (Carro et al., 2012a). It is also important to note that lower in Myrica, Morella, and Coriaria nodules. Similarly to
whilelupineplantswerecollectedinthefield,allPisumsativum legumes, most isolates were related to M. saelicesensis and M.
samples originated from cultivation fields where chemical lupini but M. coriariae was also isolated in high numbers. The
fertilizersareappliedperiodically(Carroetal.,2012a). latterspecieswasfirstreportedfromCoriariamyrtifolianodules
In a recent study, Carro et al. (2013a) screened several (Trujilloetal.,2006).
actinorhizalplantsandrecordedthenumberofMicromonospora The first Micromonospora strains isolated from nitrogen
strains and species found. Micromonospora strains were fixing nodules were considered contaminants because it was
recoveredfromallplantssampled,and,asinthecaseoflegumes, assumed that the spores produced by this microorganism were
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Trujilloetal. EndophyticActinobacteriaandMicromonospora
soil contaminants that had resisted the sterilization protocols. that many of these bacterial strains represented new species
However, the absence of other fast-growing sporulating and include Micromonospora coriariae (Trujillo et al., 2006);
microorganisms, e.g., fungi or Streptomyces strongly indicated Micromonospora lupini and Micromonospora saelicesensis
that the strains had originated from the internal plant tissues (Trujilloetal.,2007);Micromonosporapisi(Garciaetal.,2010);
(Trujilloetal.,2010).Applyingfluorescentinsituhybridization Micromonospora cremea, Micromonospora zamorensis, and
(FISH) and transmission electronic microscopy (TEM), Micromonosporahalotolerans(Figure2).Thelatterthreestrains
Micromonospora lupini Lupac 08 was localized inside the were isolated from the rhizospheric soil of the sampled plants
nodular tissues of lupin suggesting a close interaction between (Carroetal.,2012b,2013b).
the host plant and the bacterium (Rodríguez, 2008; Trujillo The species M. saelicesensis is the most frequently isolated
etal.,2010).FurtherexperimentsusingaMicromonosporastrain fromthenoduletissuesinbothlegumeandactinorhizalplants,
taggedwithgreenfluorescentproteintotracethemicroorganism followed by the species M. lupini (Cerda, 2008; Carro, 2009;
inplantaareintheprocessofcompletion. Alonso de la Vega, 2010; Trujillo et al., 2010; Carro et al.,
The degree of genetic variation of Micromonospora strains 2012a). Furthermore, the number of new species found in this
recovered from the nitrogen-fixing nodules of various plants niche also appears to be very high as commented above. To
was analyzed using several molecular typing techniques (e.g., expand the taxonomic studies of the genus Micromonospora,
BOX–PCR,ARDRA,RFLP,RAPDS)(Cerda,2008;Carro,2009; Carro et al. (2012a) carried out a multilocus sequence analysis
AlonsodelaVega,2010;Trujilloetal.,2010;Carroetal.,2012a; study based on five loci and over 90 Micromonospora isolates
Martínez-Hidalgoetal.,2014).Highlydiversegeneticfingerprint recovered from the rhizosphere and plant tissues (nodules)
profiles were found among the isolates studied, indicating that of P. sativum. These studies were complemented with DNA-
theywerenotclones;thediversityfoundwasunexpectedlyhigh DNA hybridization analyses to confirm the high diversity at
consideringthatinsomecases,thestrainsanalyzedwereisolated the species level (Carro et al., 2012a) and revealed that many
fromthesamenodule(AlonsodelaVega,2010).Subsequently, of the new isolates represent new species (Carro et al., 2012b,
taxonomic studies carried for some of these isolates confirmed 2013b).
FIGURE2|Maximum-likelihoodphylogenetictreebasedon16SrRNAgenesequencesofMicromonosporaspeciesisolatedfromplantmaterialand
rhizosphericsoil.Therewere1408nucleotidesinthefinaldataset.AnalyseswerecarriedinMEGA6software.Barindicates0.005substitutionspernucleotide
position(BasedonreferencesprovidedinTable2).
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GENOME FEATURES OF foractinobacteriasequencedtodate.ThenumberofrRNAand
MICROMONOSPORA ISOLATED FROM tRNA genes in a genome appear to be correlated and is an
NODULES indicationofpositiveselectionrelatedtothetimeofresponseofa
bacteriumtoadapttoitsenvironment(DethlefsenandSchmidt,
Very few Micromonospora strains have been sequenced. At 2007;Yanoetal.,2013).
present, only five Micromonospora genomes are available The core genome of the strains M. lupini Lupac 08,
in the public databases: Micromonospora sp. strain L5 and M. aurantiaca ATCC 27029T and Micromonospora sp. L5 was
M. lupini Lupac 08 and isolated from nodules of Casuarina determined and the results indicated that the strains shared a
equisetifolia and Lupinus angustifolius, respectively (Alonso- commongenepoolofonlyapproximately32%suggestingahigh
Vega et al., 2012; Hirsch et al., 2013). The remaining are the degreeofgenomicdiversity(Trujilloetal.,2014b).Asexpected,
soil isolates Micromonospora aurantiaca ATCC 27029T (Hirsch the strains M. aurantiaca and Micromonospora L5 with 85%
et al., 2013), Micromonospora sp. ATCC 39149 (Accession No. genomesimilarityconfirmtheircloserelationship.M.lupinion
GCF_000158815.1) and Micromonospora carbonacea JXNU-1 the other hand appears to be very different, with 66.6% of its
(Jiangetal.,2015).Severalgenomiccharacteristicsofthestrains genomebeingstrainspecific.AsmoreMicromonosporagenomes
are presented in Table3. Actinobacterial genomes are usually aresequencedthecoregenomeshouldbebetterdefined.
larger than those of most other bacteria, e.g., proteobacteria A number of genomic traits that probably participate in
and Micromonospora is no exception, the currently available the plant/soil life style of endophytic Micromonospora include
genomesrangefrom6.9to7.3MbandshareasimilarGCcontent transport and secretion systems. Several genes coding for
(72–74%). transportandsecretionsystemswhichmaybeinvolvedinplant
ThegenomesequenceofstrainLupac08wasdeterminedto colonizationwerealsoidentified.Thenumberoftransportersis
identifygenomictraitspotentiallyinvolvedinthisplant-microbe slightly higher in M. lupini Lupac 08 than in Micromonospora
interaction(Alonso-Vegaetal.,2012;Trujilloetal.,2014b).The L5, and included ATP dependent (mainly of the ABC family
annotated genome disclosed various traits potentially involved type), ion channels, PTS (phosphotransferase) and secondary
in the capacity of this bacterium to alternate a lifestyle as a transporters(Trujilloetal.,2014b).
saprophyte in the soil and as an endophyte inside the root
nodules (Trujillo et al., 2014b). The genome of strain Lupac
MICROMONOSPORA LUPINI LUPAC 08: A
08 has a circular chromosome of 7.3 Mb with a GC content
FRIENDLY BACTERIUM HIGHLY
of 71.9% and lacking plasmids. A total of 10 rRNA genes
were identified, specifically 3 5S rRNA, 4 16S rRNA, and 3 EQUIPPED WITH PLANT CELL WALL
23S rRNA genes. In addition 77 tRNA genes were predicted DEGRADING ENZYMES
(Alonso-Vega et al., 2012). Approximately, 62% (4338 CDSs)
of the genes were assigned a biological function while 38% Micromonosporae are well-known for their capacity to produce
wereannotatedhypotheticalopenreadingframeswithunknown highnumbersofcellulases,theseenzymesverylikelycontribute
biological activities (Alonso-Vega et al., 2012). The genome to the turn-over of decayed material in different habitats (de
of Micromonospora sp. L5 is smaller, 6.9 Mb, a GC content Menezes et al., 2008, 2012). However, the presence of high
of 72.9% and 6332 open reading frames (Hirsch et al., 2013). numbersofthesemoleculesandotherplant-cellwalldegrading
This strain is highly related to M. aurantiaca ATCC 27029T enzymes in beneficial endophytic bacteria is usually very low
and average nucleotide identity values (ANI) of their genomes (Krause et al., 2007; Mastronunzio et al., 2008; Taghavi et al.,
strongly suggest that Micromonospora sp. L5 belongs to this 2010;Pujicetal.,2012).
species. The number of tRNAs identified in Micromonospora The genome of strain Lupac 08 contains a high number
sp. L5 is 52 (Hirsch et al., 2013) which is much lower when of genes encoding enzymes potentially involved in plant cell
comparedtothe77tRNAsidentifiedinM.lupini08.Indeed,the wall degradation. Approximately 10% of the genome codes
latter strain has one of the largest numbers of tRNAs reported for carbohydrate metabolism, and almost 200 out of the 685
TABLE3|GenomicfeaturesofsequencedMicromonosporastrainsavailableinthedatabases.
Feature M.lupini M.aurantiaca Micromonospora Micromonospora Micromonospora
Lupac08 ATCC27029T sp.L5 sp.ATCC39149 carbonaceaJXNU-1
Size(Mb) 7.3 7 6.9 6.8 7.6
GC% 72 73 73 72 74
rRNAOperon 10 9 9 6 7
tRNA 77 52 53 51 50
CDSnumber 7054 6676 6617 5633 6247
GenesinCOGs(%) 70.20% 68.30% 69% nd nd
nd,notdetermined.
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genes have a putative hydrolytic function. Hydrolytic activities of loci involved in carbohydrate transport and metabolism are
for cellulose, pectin, starch, and xylan, were confirmed in slightlylowerinstrainL5(8.9%),ascomparedtostrainLupac08
the laboratory and indicate that this strain could degrade (9.7%)(Trujilloetal.,2014b).
plant cell wall components in a way similar to that of Bacterialendophyticcolonizationisstillapoorlyunderstood
phytopathogen bacteria (Trujillo et al., 2014b). Plant-polymer process,inpartbecauseitisverycomplex.Formicroorganisms
degradingenzymesarethoughttobeinvolvedininternalplant that colonize the roots, plant exudates appear to play a crucial
colonization(Compantetal.,2005).Plantpathogenicfungiand role (Badri et al., 2009). Molecules present in root exudates
bacteria usually enter plant tissues by degrading plant cell wall mayserveascarbonsourcesformicroorganismsandtherefore,
components using several hydrolases which include cellulases these are attracted to the plant roots (Shidore et al., 2012).
and endoglucanases. On the other hand, genome data show Thus, plant exudates may act as signals that influence the
thatnon-pathogenic(endophyticorsymbiotic)microorganisms ability of a bacterium to colonize the root or survive in
contain a low set of plant-polymer degrading enzymes (Krause the rhizosphere. These signals may induce the alteration of
et al., 2007; Mastronunzio et al., 2008; Taghavi et al., 2010). In specific gene expression patterns in the bacterium, which in
thecaseofMlupini,thegenomeofthismicroorganismrevealed turn may influence its interaction with the plant (Morrissey
ahighnumberofhydrolyticenzymes(e.g.,cellulases,xylanases, et al., 2004; Mark et al., 2005; Shidore et al., 2012). While it is
endoglucanases) with the potential to degrade plant tissues consideredthatplantexudatesaffectthebehaviorofrhizospheric
(Figure3). However, green-house experiments show that when microorganisms, our knowledge as to how these molecules
host plants are inoculated with strain Lupac 08 no damage is influence bacterial gene expression is still very limited (Mark
produced.Onthecontrary,M.lupinistimulatesnodulationand et al., 2005). Furthermore, it is not known how these altered
plant growth (Cerda, 2008; Trujillo et al., 2014b). Therefore, if bacterialgenesaffecttheplant-microbeinteractionprocessand
theplantdoesappeartobenegativelyaffectedbytheseenzymes, onlyafewstudiesareavailable(Morrisseyetal.,2004;Marketal.,
whatistheirpotentialfunctionwhenthebacteriuminteractswith 2005;Shidoreetal.,2012).
its host plant? Our group is currently working on this subject, InthecaseoftheMicromonospora-plantinteraction,itcould
someoftheloci,especiallythoserelatedtocellulosemetabolism be that the plant’s root exudates might be involved in the
mayparticipateinotherprocessessuchascellulosebiosynthesis repressionofhydrolyticenzymegenes(e.g.,cellulases,xylanases,
(Robledoetal.,2008,2012;MbaMedieetal.,2012).Severalgenes etc.)fromthebacteriumwhich,ifexpressedduringitsinteraction
coding for plant cell-wall degrading enzymes were also located with the plant would be detrimental upon infection. The effect
in the genome of Micromonospora sp. L5 (Hirsch et al., 2013). on Azoarcus sp. gene expression upon exposure to plant root
Similarly to strain Lupac 08, target substrates include cellulose, exudates was recently reported (Shidore et al., 2012). This
hemicellulose, pectin, starch, and xylan, however, the number study concluded that the genes expressed by Azoarcus strain
FIGURE3|CirculargenomerepresentationofMicromonosporalupini,Lupac08.(A)Distributionofvariousplant-cellwallhydrolyticenzymeloci.Red,
cellulases,andcellulose-bindingsites;blue,pectinases;yellow,xylanases.(B)Invitrocellulasedegradation.(C)Invitrostarchdegradation.(D)Invitroxylanase
degradation(BasedonTrujilloetal.,2014b).
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