Australasian Plant Pathology, 2007, 36, 524–531 www.publish.csiro.au/journals/app

Actinobacterial endophytes for improved crop performance

Christopher FrancoA,C, Philip MichelsenA, Nigel PercyA, Vanessa ConnA, Erna ListianaA,Simon MollA, Rosemary LoriaB and Justin CoombsA

ADepartment of Medical Biotechnology, School of Medicine, Flinders University,Bedford Park, SA 5042, Australia.

BDepartment of Plant Pathology, Cornell University, Ithaca, NY 14853, USA.CCorresponding author. Email: chris.franco@flinders.edu.au

Presented as a Keynote Address at the 16th Biennial Conference of the Australasian Plant PathologySociety, 24–27 September 2007, Adelaide

Abstract. Increasing numbers of endophytic bacteria are being isolated and identified revealing a rich vein of microbialinteraction within a variety of crop plants. In addition, cultivation-independent studies have exposed a broader diversity,with many of the species belonging to culturable genera. Microscopic evidence of endophytic colonisation has been shownin some cases, proving ‘true’ endophytic status and providing an understanding of how these microorganisms gain entryand deploy through their host.

The application of endophytic actinobacteria as inoculants isa new concept in increasing grain yields by controlling thelosses caused by the major fungal root pathogens. Endophyticpopulations, though protected within the plant, are conditionedby biotic and abiotic factors both within the plant and in therhizosphere and phyllosphere environments. In turn, they areable to affect and manipulate plant function.

Actinobacteria are well known as producers of antibiotics andother useful ‘interactive’ metabolites. Their role as endophytesworking inside a plant makes them more effective in promotingplant growth and crop yield via a range of mechanisms includingnutrient acquisition, phytohormone production, removal ofcontaminants, direct suppression of pathogens via antibiosis orcompetition, and induction of plant defence responses.

Effective screening analysis based on the ability to controldiseases in planta, both in the glasshouse and the field hasprovided very effective biocontrol strains. Selected endophyticactinobacteria significantly improved grain yields by between 5and 60% compared with the untreated controls, in the presenceof take-all, rhizoctonia and crown rot diseases.

The use of endophytic actinobacterial inocula provides areliable, non-disruptive solution to enhance grain yields ina manner that uses natural microbial resources to promotesustainability. This technology also has the potential to beapplied to other cropping systems, including pasture production,horticultural crops and floriculture.

Wheat is Australia’s largest crop and its production is ofvital importance to the Australian economy. In the 2003–04growing season, over 13 million hectares (ha) were sownyielding 26 million tonnes, valued at $5.6 billion. Most wheat isexported from Australia, earning $3.6 billion in 2003–04 (Anon.2006), but its susceptibility to diseases results in huge lossesannually. The most damaging are the fungal pathogens, including

Gaeumannomyces graminis var. tritici (Ggt) the causal agent oftake-all (Herdina and Roget 1998) and Pythium, Rhizoctonia andFusarium species (Wallwork 2000).

Wheat diseases are controlled by farming practices suchas the rotation of crops and tillage, although there stillis a strong reliance on the use of chemicals. Pressuresto increase sustainability have promoted the evaluation ofbiological control agents as it is recognised that a major factorinfluencing plant growth and health is the microbial populationliving in the rhizosphere and within the healthy plant tissueas endophytes.

A proportion of microorganisms in the rhizosphere havedisease-controlling and/or growth promoting properties andhave been termed plant growth promoting rhizobacteria (PGPR).Research into PGPR biocontrol agents of fungal root diseaseshas mostly focussed on the use of Pseudomonas spp. (Wellerand Cook 1983; Mazzola et al. 1995), Bacillus spp. to controltake-all and rhizoctonia root rot (Ryder et al. 1998), andTrichoderma spp. (Papavizas 1985).

Most of these bacterial and fungal biocontrol agents havebeen isolated from soil and rhizosphere samples and someare available commercially. However, the use of rhizobacteriaas biocontrol agents has been met with varying degrees offailure. This has been attributed mainly to the difficultiesof incorporating non-resident bacteria into established andacclimatised microbial communities. In order for a microbialinoculant to be effective it has to proliferate in the rhizosphereof the plant to a level where it can exert an influence in thepresence of other microbial populations that have adapted tothe plant and the particular soil system. Due to the difficulty ofmanipulating the highly complex rhizosphere environment weinvestigated the use of endophytes as agents to improve grainproduction in a sustainable manner.

© Australasian Plant Pathology Society 2007 10.1071/AP07067 0815-3191/07/060524

Keynote Address CSIRO PUBLISHING

Actinobacterial endophytes and crop performance Australasian Plant Pathology 525

Endophytic microorganisms such as Rhizobium andmycorrhizae have been shown to promote plant growth, andas they are in intimate contact with the plant, they are anattractive choice as agents to introduce beneficial effects. We setout to test the hypothesis that endophytic microorganisms offera tremendous advantage as an endophytic microorganism canovercome the limitations of other rhizosphere microorganism.This is because it is able to colonise the internal tissue of theplant, and play a beneficial role directly within the plant whereconditions are optimum for its survival and effectiveness interms of efficiency of colonisation, which will lead to improvedeffectiveness and reliability (Rosenblueth and Martinez-Romero 2006).

Colonisation of the endorhizosphere is expected to facilitatestability through a steady supply of nutrients and reducedpopulation densities of competing microorganisms.

The choice of filamentous actinobacteria as the endophyticpartner is also an original concept, as this class of microorganismis well known for its ability to produce metabolites, such asantibiotics and growth hormones that can interact withother biota. Actinobacteria offer an additional ecologicaladvantage in their ability to form spores, which surviveadverse conditions, and can produce filamentous mycelia thatimprove colonisation within the plant. This intimate interactionwith plants is also expected to influence the induction ofsystemic resistance in plants, adding to the advantagesof the technology.

Isolation of endophytic actinobacteria

Targeted isolation of filamentous actinobacteria was performedfrom surface-sterilised plant tissue obtained from field-grownwheat plants in multiple locations over at least two growingseasons.

Over 300 endophytic actinobacteria were isolated andidentified and were found to fall within a narrow speciesdistribution. The most common genera were Streptomyces,Microbispora and Micromonospora with Nocardioides,Streptosporangium, Nocardia, Mycobacterium, Tsukamurella,Nonomuraea and Actinopolyspora present to a lesser extent(Coombs and Franco 2003a). For these isolations, surfacesterilisation was validated extensively, but other specialisedisolation protocols were not employed; isolation methodsdeveloped predominantly for soil actinomycetes were used. Ourmost effective actinobacterial strains belong to a new speciesof Streptomyces based on 16S rRNA gene sequence as well aspolyphasic analysis (Coombs and Franco 2003a).

Endophytic actinobacteria have received increasing attentionin recent years, with reports of isolates from a range of planttypes (Taechowisan et al. 2003), including horticultural plantsand cereals, such as wheat (Triticum aestivum L.) (Germida et al.1998; Coombs and Franco 2003a), sweet corn (Zea mays L.)and cotton (Gossypium hirsutum L.) (McInroy and Kloepper1995), potato (Solanum tuberosum L.) (Sturz et al. 1998, 1999;Garbeva et al. 2001; Sessitsch et al. 2001, 2004), carrots (Daucuscarota L.) (Surette et al. 2003), rhododendron (Shimizu et al.2000), tomato (Solanum lycopersicum L.) (Nejad and Johnson2000) and citrus (Citrus sinensis L.) (Araujo et al. 2001, 2002).

Culture-independent methods revealed a higher endophyticdiversity than seen by isolation alone (Garbeva et al. 2001).

In addition to the extensive attempts to isolate a range ofactinobacteria, we studied the diversity of the endophyticactinobacteria in wheat roots using culture-independentmolecular techniques (Conn and Franco 2004a). This revealedthat there was a broader diversity than was achieved by culture-dependent methods and that plants are colonised by a broadrange of culturable genera. At least 20 genera were not isolatedusing conventional isolation protocols, and this underscorestwo factors: (1) that molecular techniques can be used toguide the isolation of a range of actinobacterial genera and(2) that more targeted isolation protocols need to be usedor developed.

Efficiency of colonisation of the plant host

A major test of endophytic colonisation of the plant isvisualisation by microscopy. This was achieved by tagging oneof the endophyte strains with green fluorescent protein andmonitoring the infection process after coating onto a germinatingwheat seed. The results (Coombs and Franco 2003b) provedthat colonisation occurred rapidly and that the strain could bevisualised in the growing plant from the first day.

Green fluorescent protein-labelled Streptomyces sp.EN27 colonisation was observed on seedlings of wheat, in bothhydroponic and sand-based systems using confocal and scanningelectron microscopy (SEM ) imaging. Macroscopically,Streptomyces sp. EN27 inoculated seedlings were identical tonon-inoculated controls and no symptoms of disease, such asnecrosis, chlorosis, stunting or stem cankers, were observed.

Streptomyces sp. EN27 hyphae were not detected on wheatwithin the first 2 weeks after inoculation. After this period,colonisation was observed principally on primary root tips(Fig. 1), lateral root junctions and root hairs. The observedintracellular colonisation was limited to the epidermal cell layerwith no colonisation of the root cortex observed.

Experiments were repeated using a sand system and aswith hydroponically grown wheat plants, hyphal growth wasobserved after the initial 3 weeks following inoculation(Fig. 2). Colonisation occurred in the upper part of theseminal roots, hyphal growth at lateral root junctions wasobserved (Fig. 2G), and intracellular colonisation was observedwithin epidermal cells. However, in these plants, intracellularsporulation of the bacterium was also observed (Fig. 2H–L) andappeared more frequent than was observed for hydroponicallygrown plants.

Surface colonisation of hydroponically grown wheatseedlings was analysed by SEM. This revealed thathyphal growth was extensive throughout root junctionsand colonised the root cracks surrounding lateral roots (Fig. 3).Colonisation of cracks formed at the emergence of lateralroots was also observed for Klebsiella sp. Kp342, in wheatand alfalfa (Dong et al. 2001), and for Glucoacetobacterdiazotrophicus and Herbaspirillum seropedicae (James andOlivares 1998).

Microscopic analysis was reinforced by reisolation of thetagged strain using its apramycin resistance marker. Theendophyte readily colonises the external surface of the roots(rhizoplane) as well as the leaves of wheat plants and can beisolated from the leaves of the treated wheat plant from the

526 Australasian Plant Pathology C. Franco et al.

Root tip

Lateral rootjunction

Root hair

Epidermalcell layer

(A) (B)

(C) (D )

(E ) (F )

(G) (H )

75 μm 75 μm

75 μm

75 μm

50 μm

50 μm

50 μm 20 μm

Fig. 1. Green fluorescent protein-labelled Streptomyces sp. EN27 colonisation of hydroponicallygrown wheat seedlings 2–4 weeks following inoculation. Confocal images showing Streptomyces sp.EN27 colonisation of roots of wheat seedlings as mycelium and spores, 2–4 weeks following inoculation.Colonisation of: (A, B) root tips; (C, D) lateral root junctions; (E, F ) root hairs; (G, H) epidermal cells.Micrographs available in colour in the online version of this paper.

time of germination. Isolation of the tagged strain from theroots, stems and leaves, both from external surfaces and aftersurface sterilisation, was observed until at least 12 weeks aftergermination (Fig. 4).

The impact of added non-tagged microbial inoculaon the ‘normal’ endophytic population of wheat plants wasfurther investigated by the molecular technique terminalrestriction fragment length polymorphism or T-RFLP.

Actinobacterial endophytes and crop performance Australasian Plant Pathology 527

(I )

100 μm

150 μm 15 μm 15 μm

5 μm50 μm

75 μm

50 μm 15 μm

100 μm 150 μm

(A) (B) (C)

(D) (E) (F )

(G) (H)

(L)(J) (K)

Fig. 2. Green fluorescent protein-labelled Streptomyces sp. EN27 colonisation of wheat seedlings, grown in a sandmatrix. Confocal micrographs showing Streptomyces sp. EN27 colonisation of wheat roots grown in a sand matrix.No colonisation was observed in the first 4 weeks following inoculation. (A–C) Colonisation up to 4 weeks followinginoculation. (D, E) Presence of spores on inoculated seedling roots up to 4 weeks following inoculation. (F–L)Colonisation of lateral root junctions (F) and epidermal cells (H–L) 4–5 weeks following inoculation. Image I is aclose-up of image H and image L is a close-up of images J and K. Micrographs available in colour in the online versionof this paper.

The results (Conn and Franco 2004b) indicated that the use of acommercial non-adapted mixed microbial inoculant applied tothe soil disrupts the indigenous endophyte population presentin the wheat roots and dramatically reduces the diversity ofthe normal endophytes and level of colonisation. In contrast,

the use of our (adapted) endophytic actinobacterial inoculantsof strains of Streptomyces, Micromonospora and Nocardioidescould be detected, but did not significantly affect this normalindigenous population (Conn and Franco 2004b). This impliesthat endophyte strains that show a significant physiological

528 Australasian Plant Pathology C. Franco et al.

Fig. 3. Colonisation of the wheat roots by Streptomyces sp. EN27,4 weeks post-inoculation scanning electron microscopy micrographsshowing Streptomyces sp. EN27 colonisation of 28-day-old wheat seedlingsat lateral root cracks. The close-up is of the regions indicated by white arrows.

effect do so without causing major changes to the normalmicrobial population within the wheat plant.

Induced systemic resistance

Endophytic actinobacteria isolated from healthy wheat plantshave been shown to enhance disease resistance in wheat whenapplied as a seed coating (Conn 2005). In concert with antibioticproduction as one method of disease control, the endophyticactinobacteria were found to ‘prime’ both the systemic acquiredresistance (SAR) and jasmonic acid/ethylene (JA/ET) pathwaysthat result in systemic resistance via the upregulation ofpathogenesis related proteins and other anti-microbial proteinseffective against several pathogens.

Using Arabidopsis as a model plant as its defence pathwayshave been studied extensively, Quantitative PCR was used todetermine the level of gene induction in the SAR and JA/ETpathways of Arabidopsis as a result of endophytic actinobacteriainoculation.

Selected endophytic actinobacteria were found to stimulatethe plant defence pathways although the elicitation profile ofthe genes involved was different to that previously reportedusing other plant growth promoting bacteria or biocontrolbacterial strains. Streptomyces sp. EN27 activated the SAR

pathway (PR-1 gene), whereas Streptomyces sp. EN28 triggeredthe JA/ET pathway (PDF1.2 gene), even though these strainsare morphologically very similar (Table 1). These elicitationpatterns were not observed with the non-pathogenic PGPRPseudomonas fluorescens WCS417r (van Wees et al. 1999)and P. fluorescens LSW17S (Ahn et al. 2007), which did notinduce salicylic acid (SA) or JA-responsive genes on their own.The ability of some endophytic actinobacteria to activate theplant defence genes at a low level in the absence of a pathogenindicates that they are detected by the plant as ‘minor’ pathogens,because they do not have pathogenic determinants (Coombs andFranco 2003a).

The priming response was displayed in Arabidopsis plantsinoculated with endophytic actinobacteria at planting whenchallenged after 7 weeks with the non-specific bacterial plantpathogen Erwinia carotovora subsp. carotovora. Priming is thephenomenon mediated by some PGPR where inoculation withthe bacterium alone does not induce the plant defence responsebut when attacked by a pathogen the defence response is mountedquickly inducing systemic resistance (Conrath et al. 2002).

Induced resistance to the soilborne fungus, Fusariumoxysporum, mediated by the endophytic actinobacteria occurredprimarily by the SAR pathway. The endophytic actinobacteria,were found to work via both the SAR pathway and JA/ETpathway, and when both pathways are in operation a strongerprotection was noted. In contrast, defence responses by PGPRP. fluorescens WC417r or LSW17S and Bacillus spp. is SA-independent and associated with JA and ET (Pieterse et al. 1996,1998; Kloepper et al. 2004).

Disease suppression

Antibiotic assays in Petri dishes showed that a high proportionof these actinobacteria strains displayed antifungal activityagainst the major root phytopathogens: the take-all fungusGaeumannomyces and Rhizoctonia, Pythium and Fusarium.

Pot trials in the glasshouse to evaluate growth promotion,as well as disease suppression, was done with a minimum of25 replicate plants for each endophyte with the results analysedstatistically. Selected endophytes that were effective were furthertested using infected field soils in the glasshouse before field

(A) (B)

Fig. 4. Reisolation of inoculum (Green fluorescent protein-labelled Streptomyces sp. EN27) from leaves ofwheat plants at 6 weeks after germination. (A) Isolates from non-sterilised leaves pressed onto agar surface for10 min. (B) Isolates emerging from surface-sterilised leaves placed onto agar surface. The agar contains 10 ug/mLapramycin.

Actinobacterial endophytes and crop performance Australasian Plant Pathology 529

Table 1. Fold induction of the salicylic acid (PR-1, PR-5) and jasmonicacid/ethylene (PDF1.2, Hel) pathway gene transcripts in 7-week-oldArabidopsis thaliana (Col-0) plants preinoculated with endophytic

actinobacteria compared with untreated plants (Conn 2005)

Endophyte PR-1 PR-5 PDF1.2 Heltreatment

Streptomyces sp. 19.3 ± 12.8 1.6 ± 1.0 1.5 ± 1.5 1.7 ± 0.6EN27

Streptomyces sp. 4.1 ± 1.9 2.9 ± 1.3 22.8 ± 10.9 2.4 ± 1.6EN28

Nocardioides albus 6.4 ± 3.4 2.6 ± 2.1 2.0 ± 1.0 2.5 ± 0.8EN46

Micromonospora sp. 1.1 ± 1.0 0.1 ± 0.04 0.1 ± 0.1 1.5 ± 1.3EN43

testing (Coombs et al. 2004). Therefore, a rigorous testingregime was employed to select a shortlist of actinobacteria forfield trials.

Two-thirds of the endophytic actinobacteria isolates testeddisplayed in vitro activity towards at least one of these pathogens,with the novel Streptomyces isolates showing the greatest levelsof activity. Furthermore, when those isolates displaying in vitroactivity against either Ggt or R. solani were tested for in plantaactivity in field soil, six isolates were shown to significantlyreduce Ggt disease severity (Coombs and Franco 2003b). Morerecently, field trials have been conducted with five Streptomycesand Nocardioides isolates applied as seed inoculants, againstGgt, R. solani, Pythium spp. and F. pseudograminearum in wheat(crown rot).

These research field trials were carried out at 10–16 siteseach year for four growing seasons using seed coated withspores of the selected actinobacterial endophytes. There werefour replicate treatments using a randomised block design ateach research trial site with appropriate untreated controlswith management of the research trial sites (preparation,sowing, maintenance, and grain yield analysis) performedby professional agronomists from SARDI or Landmarkorganisations. Trial sites were chosen to test for growthpromotion in the absence of disease, and for disease suppressionagainst the take-all fungus, Rhizoctonia, Pythium and crown rot.Sites were chosen on the basis of soil DNA tests and history ofdisease prevalence, and were selected to include different soiltypes and climatic conditions.

Normal farm management practices were used. For example,before the trials it was confirmed that the normal range of agentsused to ‘pickle’ seed (e.g. Baytan, Raxil, Premis) were ineffectiveagainst the actinobacterial inocula.

Sites were sampled regularly during the growing season, anddisease pressure was scored in the laboratory. It was also decidedthe evaluation of effectiveness was based on grain yield, as thisis a rigorous factor used by farmers. Grain yield results fromthe field trials were analysed statistically by BiometricsSA toremove any operator bias.

In these field trials, the endophytic actinobacteria wereshown to significantly increase plant emergence (Table 2) andthe grain yields of wheat infected with each of the pathogens,in comparison to the untreated control (Table 3). For take-all,the levels were similar to those obtained with a commercial

Table 2. Plant emergence due to treatment with actinomyceteendophyte added as a seed treatment compared with untreated seed

Site Disease Crop Predicted % ofplants/ha untreated

seed

Dimboola, Victoria Crown rot Durum 81.5 109–126.6Haslam, South Rhizoctonia Wheat 37.2 109.1

Australia (SA)Koppio, SA Disease free Wheat 30.9 106.3–124.3Mt Madden, Rhizoctonia Wheat 37.4 106.6–108.8

Western AustraliaPaskeville, SA Pythium Wheat 51.3 105.4–116.1Turretfield, SA Take-all Wheat 61.2 105.6–110.3

Table 3. Grain yield due to treatment with actinomycete endophyteadded as a seed treatment compared with untreated seed

Site Disease Crop Predicted % ofkg/ha untreated

seed

Dimboola, Crown rot Durum 1635 109–113Victoria (Vic.)

Elmore, Vic. Take-all Wheat 2707 106–108.5Hardwicke Bay, South Rhizoctonia Wheat 2309 106–110.6

Australia (SA)Junee, Rhizoctonia Barley 1879 107–115.6

New South WalesMt Madden, Rhizoctonia Wheat 1093 107.4

Western AustraliaPaskeville, SA Pythium Wheat 2609 107.9Pt Kenny, SA Rhizoctonia Wheat 752 106.6–112.9

fungicide added (Jockey) as a comparison in our trials. Themost effective endophytic strains demonstrated consistentresults over three years that were carried out using differentcultivars of wheat, including durum, and barley, in a varietyof soil types and climatic conditions ranging from WesternAustralia, the Eyre and Yorke peninsulas, the mid-north andsouth-east of South Australia, the Mallee region of Victoria andNew South Wales.

In the absence of disease pressure, grain yield increases werenot obtained with the application of the chemical fungicide, butincreases of 5–15% generally, with a maximum of 60%, achievedwith the endophyte application due to growth promotion activity.This allows the farmer to recover costs associated with theapplication of the microbial inoculant, but not with thechemical fungicide, and will assist with the take-up rate ofthis technology.

Actinomycetes have been found to prime defence pathways,leading to enhanced defence gene expression and a resistantphenotype, following pathogen challenge (Conn 2005). Anyreduction in plant fitness due to priming of defence pathways isoutweighed by increased disease resistance in disease affectedareas (van Hulten et al. 2006).

Summary

Our technology can deliver increased yields by making use ofnatural microbial resources to improve plant growth and control

530 Australasian Plant Pathology C. Franco et al.

diseases and by enhancing natural processes that operate inplant ecosystems. The reliable results achieved will enhancesustainability by reducing reliance on chemical fungicides andpesticides.

Plants have developed complex mechanisms to recogniseand respond to pathogen invasion and disease resistance canoccur at different levels, including SAR and induced systemicresistance.

Acknowledgements

The authors wish to acknowledge the investment into this project by theAustralian Grains Research & Development Corporation.

References

Ahn I-P, Lee S-W, Suh S-C (2007) Rhizobacteria-induced priming inArabidopsis is dependent on ethylene, jasmonic acid and NPR1.Molecular Plant-Microbe Interactions 20, 759–768. doi: 10.1094/MPMI-20-7-0759

Anon. (2006) Chapter 14 – Agriculture. In ‘Australian Bureau of Statistics2006. Year Book Australia’. ABS Catalogue No. 1301.0.

Araujo WL, Maccheroni W Jr, Aguilar-Vildoso CI, Barroso PA,Saridakis HO, Azevedo JL (2001) Variability and interactionsbetween endophytic bacteria and fungi isolated from leaf tissues ofcitrus rootstocks. Canadian Journal of Microbiology 47, 229–236.doi: 10.1139/cjm-47-3-229

Araujo WL, Marcon J, Maccheroni W Jr, Van Elsas JD, Van Vuurde JW,Azevedo JL (2002) Diversity of endophytic bacterial populationsand their interaction with Xylella fastidiosa in citrus plants.Applied and Environmental Microbiology 68, 4906–4914. doi: 10.1128/AEM.68.10.4906-4914.2002

Conn VM (2005) Molecular interaction of Endophytic Actinobacteriain wheat and Arabidopsis. PhD Thesis, Department of MedicalBiotechnology, Flinders University, Adelaide.

Conn VM, Franco CMM (2004a) Analysis of the endophytic actinobacterialpopulation in the roots of wheat (Triticum aestivum L.) by TerminalRestriction Fragment Length Polymorphism (T-RFLP) and sequencingof 16S rRNA clones. Applied and Environmental Microbiology 70,1787–1794. doi: 10.1128/AEM.70.3.1787-1794.2004

Conn VM, Franco CMM (2004b) The effect of microbial inoculants onthe indigenous actinobacterial endophyte population present in theroots of wheat as determined by Terminal Restriction Fragment LengthPolymorphism (T-RFLP). Applied and Environmental Microbiology 70,6407–6413. doi: 10.1128/AEM.70.11.6407-6413.2004

Conrath U, Pieterse CM, Mauch-Mani B (2002) Priming in plant-pathogeninteractions. Trends in Plant Science 7, 210–216. doi: 10.1016/S1360-1385(02)02244-6

Coombs JT, Franco CMM (2003a) Isolation and identification ofactinobacteria isolated from surface-sterilized wheat roots. Appliedand Environmental Microbiology 69, 5603–5608. doi: 10.1128/AEM.69.9.5603-5608.2003

Coombs JT, Franco CMM (2003b) Visualisation of an endophyticStreptomyces sp. in wheat seed. Applied and EnvironmentalMicrobiology 69, 4260–4262. doi: 10.1128/AEM.69.7.4260-4262.2003

Coombs JT, Michelsen PP, Franco CMM (2004) Evaluation of endophyticactinobacteria as antagonists of Gaeumannomyces graminis var.tritici in wheat. Biological Control 29, 359–366. doi: 10.1016/j.biocontrol.2003.08.001

Dong Y, Glasner JD, Blattner FR, Triplett EW (2001) Genomicinterspecies microarray hybridization: rapid discovery of three thousandgenes in the maize endophyte, Klebsiella pneumoniae 342, bymicroarray hybridization with Escherichia coli K-12 open readingframes. Applied and Environmental Microbiology 67, 1911–1921.doi: 10.1128/AEM.67.4.1911-1921.2001

Garbeva P, Van Overbeek LS, Van Vuurde JWL, Van Elsas JD (2001)Analysis of endophytic bacterial communities of potato by plating andDenaturing Gradient Gel Electrophoresis (DGGE) of 16S rDNA basedPCR fragments. Microbial Ecology 41, 369–383.

Germida JJ, Sicilliano SD, De Freitas JR, Seib AM (1998) Diversity ofroot-associated bacteria associated with field-grown canola (Brassicanapus L.) and wheat (Triticum aestivum L.). FEMS MicrobiologyEcology 26, 43–49. doi: 10.1111/j.1574-6941.1998.tb01560.x

Herdina, Roget DK (1998) Investigation of soil suppression to thetake-all fungus using a DNA-based assay. In ‘7th internationalcongress of plant pathology, Edinburgh, Scotland, August 1998’.Available at http://www.bspp.org.uk/icpp98/2.7/8.html [Verified26 September 2007].

James EK, Olivares FL (1998) Infection and colonization of sugar cane andother graminaceous plants by endophytic diazotrophs. Critical Reviewsin Plant Sciences 17, 77–119. doi: 10.1016/S0735-2689(98)00357-8

Kloepper JW, Ryu C, Zhang S (2004) Induced systemic resistanceand promotion of plant growth by Bacillus spp. Phytopathology 94,1259–1266. doi: 10.1094/PHYTO.2004.94.11.1259

Mazzola M, Fujimoto DK, Thomashow S, Cook RJ (1995) Variation insensitivity of Gaeumannomyces graminis to antibiotics produced byfluorescent Pseudomonas spp. and effect on biological control of take-allof wheat. Applied and Environmental Microbiology 61, 2554–2559.

McInroy JA, Kloepper JW (1995) Population dynamics of endophyticbacteria in field-grown sweet corn and cotton. Canadian Journal ofMicrobiology 41, 895–901.

Nejad P, Johnson PA (2000) Endophytic bacteria induce growth promotionand wilt disease suppression in oilseed rape and tomato. BiologicalControl 18, 208–215. doi: 10.1006/bcon.2000.0837

Papavizas GC (1985) Trichoderma and Gliocladium: biology, ecology, andpotential for biocontrol. Annual Review of Phytopathology 23, 23–54.doi: 10.1146/annurev.py.23.090185.000323

Pieterse CM, Van Wees SC, Hoffland E, Van Pelt JA, Van Loon LC (1996)Systemic resistance in Arabidopsis induced by biocontrol bacteria isindependent of salicylic acid accumulation and pathogenesis-relatedgene expression. Plant Cell 8, 1225–1237. doi: 10.2307/3870297

Pieterse CM, Van Wees SC, Van Pelt JA, Knoester M, Laan R, Gerrits H,Weisbeek PJ, Van Loon LC (1998) A novel signaling pathway controllinginduced systemic resistance in Arabidopsis. Plant Cell 10, 1571–1580.doi: 10.2307/3870620

Rosenblueth M, Martinez-Romero E (2006) Bacterial endophytes andtheir interactions with hosts. Molecular Plant-Microbe Interactions 19,827–837. doi: 10.1094/MPMI-19-0827

Ryder MH, Yan Z, Terrace TE, Rovira AD, Tang W, Correll RL (1998)Use of strains of Bacillus isolated in China to suppress take-all andrhizoctonia root rot, and promote seedling growth of glasshouse-grownwheat in Australian soils. Soil Biology & Biochemistry 31, 19–29.doi: 10.1016/S0038-0717(98)00095-9

Sessitsch A, Reiter B, Pfeifer U, Wilhelm E (2001) Cultivation-independentpopulation analysis of bacterial endophytes in three potato varietiesbased on eubacterial and Actinomycetes-specific PCR of 16S rRNAgenes. FEMS Microbiology Ecology 1305, 1–10.

Sessitsch A, Reiter B, Berg G (2004) Endophytic bacterial communitiesof field-grown potato plants and their plant-growth promoting andantagonistic abilities. Canadian Journal of Microbiology 50, 239–249.doi: 10.1139/w03-118

Shimizu M, Nakagawa Y, Sato Y, Furumai T, Igarashi Y, Onaka H,Yoshida R, Kunoh H (2000) Studies on endophytic actinomycetes (I)Streptomyces sp. isolated from Rhododendron and its antifungalactivity. Journal of General Plant Pathology 66, 360–366.doi: 10.1007/PL00012978

Sturz AV, Christie BR, Matheson BG (1998) Associations of bacterialendophyte populations from red clover and potato crops with potentialfor beneficial allelopathy. Canadian Journal of Microbiology 44,162–167. doi: 10.1139/cjm-44-2-162

Actinobacterial endophytes and crop performance Australasian Plant Pathology 531

Sturz AV, Christie BR, Matheson BG, Arsenault WJ, Buchanan NA (1999)Endophytic bacterial communities in the periderm of potato tubers andtheir potential to improve resistance to soil-borne plant pathogens. PlantPathology 48, 360–369. doi: 10.1046/j.1365-3059.1999.00351.x

Surette M-A, Sturz AV, Lada RR, Nowak J (2003) Bacterial endophytes inprocessing carrots (Daucus carota L. var. sativus): their localization,population density, biodiversity and their effects on plant growth. Plantand Soil 253, 381–390. doi: 10.1023/A:1024835208421

Taechowisan T, Peberdy JF, Lumyong S (2003) Isolation of endophyticactinomycetes from selected plants and their antifungal activity.World Journal of Microbiology & Biotechnology 19, 381–385.doi: 10.1023/A:1023901107182

Van Hulten M, Pelser M, Van Loon LC, Pieterse CMJ, Ton J (2006) Costsand benefits of priming for defense in Arabidopsis. Proceedings of theNational Academy of Sciences of the United States of America 103,5602–5607. doi: 10.1073/pnas.0510213103

Van Wees SC, Luijendijk M, Smoorenburg I, Van Loon LC, Pieterse CM(1999) Rhizobacteria-mediated induced systemic resistance (ISR) inArabidopsis is not associated with a direct effect on expression of knowndefense-related genes but stimulates the expression of the jasmonate-inducible gene Atvsp upon challenge. Plant Molecular Biology 41,537–549. doi: 10.1023/A:1006319216982

Wallwork H (2000) ‘Cereal root and crown diseases.’ (SARDI: Adelaide,SA; GRDC: Canberra)

Weller DM, Cook RJ (1983) Suppression of take-all of wheat by seedtreatments with fluorescent Pseudomonads. Phytopathology 73,463–469.

Manuscript received 15 August 2007, accepted 29 August 2007

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