Wheat seeds harbour bacterial endophytes with potential as plant growth promoters and biocontrol agents of Fusarium graminearum

Wheat seeds harbour bacterial endophytes with potential as plant growth promoters and biocontrol agents of Fusarium graminearum

Accepted Manuscript Title: Wheat seeds harbour bacterial endophytes with potential as plant growth promoters and biocontrol agents of Fusarium gramine...

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Accepted Manuscript Title: Wheat seeds harbour bacterial endophytes with potential as plant growth promoters and biocontrol agents of Fusarium graminearum Author: Silvana D´ıaz Herrera Cecilia Grossi Myriam Zawoznik Mar´ıa Daniela Groppa PII: DOI: Reference:

S0944-5013(16)30013-1 http://dx.doi.org/doi:10.1016/j.micres.2016.03.002 MICRES 25866

To appear in: Received date: Revised date: Accepted date:

28-12-2015 3-3-2016 6-3-2016

Please cite this article as: D´iaz Herrera Silvana, Grossi Cecilia, Zawoznik Myriam, Groppa Mar´ia Daniela.Wheat seeds harbour bacterial endophytes with potential as plant growth promoters and biocontrol agents of Fusarium graminearum.Microbiological Research http://dx.doi.org/10.1016/j.micres.2016.03.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

*Manuscript

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Wheat seeds harbour bacterial endophytes with potential as plant growth promoters and

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biocontrol agents of Fusarium graminearum

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*

4

*

5

a

6

Universidad de Buenos Aires, Junín 956, Buenos Aires, Argentina.

7

b

Silvana Díaz Herreraa,b, Cecilia Grossia, Myriam Zawoznika, María Daniela Groppaa,b

All authors contributed equally to this work and should be considered co-first authors

Cátedra de Química Biológica Vegetal, Departamento de Química Biológica,

IQUIFIB, CONICET

8 9 10 11 12 13

Corresponding author:

14

Myriam S. Zawoznik

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Cátedra de Química Biológica Vegetal, Facultad de Farmacia y Bioquímica, Universidad de Buenos

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Aires.

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Junín 956, (1113) Ciudad Autónoma de Buenos Aires, Argentina.

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E-mail: [email protected]

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Abstract:

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The role of endophytic communities of seeds is still poorly characterised. The purpose of this work

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was to survey the presence of bacterial endophytes in the seeds of a commercial wheat cultivar

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widely sown in Argentina and to look for plant growth promotion features and biocontrol abilities

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against F. graminearum among them. Six isolates were obtained from wheat seeds following a

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culture-dependent protocol. Four isolates were assignated to Paenibacillus genus according to their

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16S rRNA sequencing. The only gammaproteobacteria isolated, presumably an Enterobactereaceae

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of Pantoea genus, was particularly active as IAA and siderophore producer, and also solubilised

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phosphate and was the only one that grew on N-free medium. Several of these isolates demonstrated

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ability to restrain F. graminearum growth on dual culture and in a bioassay using barley and wheat

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kernels. An outstanding ability to form biofilm on an inert surface was corroborated for those

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Paenibacillus which displayed greater biocontrol of F. graminearum, and the inoculation with one

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of these isolates in combination with the Pantoea isolate resulted in greater chlorophyll content in

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barley seedlings. Our results show a significant ecological potential of some components of the

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wheat seed endophytic community.

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Keywords: barley, bioassays, Fusarium, Paenibacillus, Pantoea, wheat-endophytes

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Introduction

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Wheat is a very important crop for Argentine agriculture and has become a key factor for the

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preservation of soil fertility because many argentine soils are under a continuous soybean-wheat

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rotation scheme, and soybean is recognized as a very soil-exhausting crop (Shurtleff and Aoyagi,

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2014). About 14.5 million tons of total argentine agriculture production corresponded to wheat at

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the 2012/2013 campaign. Argentine was sixth among wheat exporter countries, and wheat

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exportations represented for our economy an income of 2.5 million dollars in that period (Barberis,

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2014).

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During last decades many researchers reported the presence of seed endophytes in several plant

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species including gramineous plants such as rice and maize, where Proteobacteria, Actinobacteria

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and Firmicutes were particularly dominant (Rijavec et al., 2007; Kaga et al., 2009; Ruiz et al.,

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2011).

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Seed endophytes may come from different plant organs, being transferred to seeds via vascular

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connections or through gametes, resulting in colonisation of embryo and endosperm; and

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reproductive meristems may also be the source (Malfanova et al., 2013). Vertical transmition from

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one plant generation to the following may then occur, as suggested by several authors (Ringelberg

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et al., 2012; Liu et al., 2012; Gagne-Bourgue et al., 2013) and depicted recently in Truyens et al.

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(2015).

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After seed germination, these populations are expected to increase in number and to colonise

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different tissues including roots, reaching the endorizosphere and probably also the exorizosphere.

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Mano et al. (2006) observed that although rice seed endophytes mainly colonised shoots, some

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strains were able to spread out into the rhizosphere and soil. Similar observations were made by

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Hardoim et al. (2012). López-López et al. (2010) could recover almost all bacterial genera isolated

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from bean seeds also from the roots of bean seedlings. Under this scenario, introduced

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microorganisms are expected to compete and/or to share their ecological niche with endophytic

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communities established in the rhizosphere, for which it is particularly necessary to gain more

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knowledge about seed endophytic communities in crops which are increasingly being inoculated

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with plant growth promoting microorganisms (PGPM), such as wheat or maize. In this sense and

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regarding particularly corn crop, of note are the findings of Johnston-Monje and Raizada (2011),

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who demonstrated that in maize, a core microbiota consisting of the same bacterial species is

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conserved apparently from teosinte’s times, in spite of evolutive and selective changes, even across

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the huge American continent.

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The role of seed endophytes has not been unravelled yet. It has been demonstrated that some of

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them can increase plant growth due to the production of plant hormones or to their contribution in

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plant nutrients acquisition, specially nitrogen and phosphorus (Gagne-Bourgue et al., 2013;

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Johnston-Monje and Raizada, 2011; Xu et al., 2014). For cacti, the ecological significance of seed

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endophytes was demonstrated by Puente et al. (2009a, 2009b). On another hand, antifungal activity

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of several bacterial seed endophytes has also been recognized, involving lipopeptides like surfactin,

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iturin and mycobacillin (Gagne-Bourgue et al., 2013). Some strains of bacteria frequently

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mentioned as seed endophytes (such as Bacillus and Pseudomonas) were found to have antagonistic

86

effects on F. oxysporum f. sp. lycopersici (Fol), the causative agent of tomato wilt (Sundaramoorthy

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and Balabaska, 2013). Volatile anti-fungal compounds were also found to be involved in the

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biocontrol displayed by endophytic Enterobacter strains obtained from rice (Mukhopadhyay et al.,

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1996).

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Fusarium gramineaurum is the causative agent of wheat head blight, a worldwide fungal plant

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pathogen impacting severely on cereals production and quality, as this microorganism is a source of

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mycotoxins, which affect human and animal health. The purpose of this work was to survey the

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presence of bacterial endophytes in the seeds of a commercial wheat cultivar widely sown in

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Argentina called 75 Aniversario, to identify the most abundant genera and to screen these isolates

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for some features which are involved in direct or indirect mechanisms of plant growth promotion.

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Their abilities to inhibit the growth of the plant pathogen F. graminearum and to promote barley

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growth were also investigated.

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98 99

Materials and methods

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Isolation and identification of bacterial endophytes

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Seeds of wheat (Triticum aestivum L.) cultivar 75 Aniversario (seed supplier: Buck S.A.) were

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surface disinfected using ethanol 70% (30 sec), followed by 1% active Cl2 (2.5 min) and again,

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ethanol 70% (30 sec), with three rinses in sterilised distilled water. One ml of the last rinse water

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was added to 10 ml of liquid LB culture medium and incubated for 48 h to check complete external

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disinfection. Ten intact disinfected seeds were placed on Petri dishes containing LB and incubated

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at 24 °C. Five replicates were prepared. After 7 days of incubation at 28 °C, some representative

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colonies appearing on the majority of the plates were selected and phenotypically characterised;

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following repeated subcultures several isolates were obtained. Standard identification protocols

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based on morphology, Gram staining, spore formation and certain biochemical properties were

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investigated to characterise these isolates. 16S rDNA partial sequencing was performed to get

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identification at genus level; comparisons with deposited sequences in BLAST database were made

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for this purpose. Total DNA was extracted from randomly chosen colonies of each selected isolate

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using the AxyPrep bacterial genomic DNA Miniprep Kit (Axygen Biosciences). A fragment of

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around 1500 bp of the 16S rRNA gene was amplified using the universal primers 27F and 1492R.

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PCR products were purified with the AND-Clean UP (PB-L Productos Bio-Lógicos) and sequenced

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at the Genomic Unit of the Biotechnology Institute of CNIA-INTA (http://www.inta.gov.ar/biotec)

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using a capillary automatic sequencer model ABI3130XL (Applied Biosystems, USA). The Naïve

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Bayesian Classifier utility (Wang et al., 2007) from the RDP Release 10 (http://rdp.cme.msu.edu)

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was used to assign the obtained sequences into the new bacterial taxonomy at genus level with 95%

121

of confidence.

122 123

Indolacetic acid and siderophore production

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In vitro IAA biosynthetic ability of the isolates obtained was estimated by Salkowski colorimetric

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technique in 1 ml-supernatant aliquots of 5-days old cultures grown on LB amended with L-

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tryptophan (100 µg ml-1), as described by Glickman and Dessaux (1995); cultures were run in

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triplicate and three supernatant aliquots of each bacterial culture were processed. Final bacterial

128

counts were calculated by the drop plate technique, as described by Herigstad et al. (2001); IAA

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production was expressed on 107 CFU basis.

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Siderophore production was investigated using the O-CAS assay (Pérez-Miranda et al., 2007), a fast

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and universal method of siderophore detection in which an overlay of the CAS medium of Schwyn

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and Neilands (1987) without nutrients is applied on agar plates containing cultivated

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microorganisms to reveal siderophore production. Halos surrounding colonies demonstrate

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siderophore production.

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Phosphate solubilisation in solid media

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Basal Sperber medium supplemented with 2.5 g l-1 of Ca3(PO4)2 (TCP) was used to test the ability

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of the isolates to solubilise inorganic phosphate, as described by Alikhani et al. (2006). The pH of

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the medium was adjusted to 7.2. The surface of the solidified medium was divided into equal parts

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at the centre of which a 5-µl drop of each bacterial culture (OD=1.00) was applied. Inoculated

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plates were incubated in dark at 25 ºC; at day 10 plates were observed in order to establish the

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formation of clear zones (halo) surrounding colonies capable of solubilise TCP.

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In vitro antagonism against Fusarium graminearum

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These experiments were based on those described by Abdulkarem et al. (2014) with some

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modifications.

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The possibility of antagonistic effects of the isolates obtained against F. graminearum was assessed

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on dual cultures on nutrient agar. Each bacterial strain (overnight cultures; OD=1.00) was drop-

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inoculated (10 µl) at four equidistant points of the plate and incubated at 25 ºC for 3 days. Then, a 1

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150

cm2 plug of F. graminearum obtained from an actively growing culture was placed at the centre of

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the plates, and plates were further incubated for 6 days. Control cultures containing only the fungus

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plugs at the centre served as control units.

153

Involvement of diffusible substances with antifungal activity released by these endophytes into the

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culture medium was also investigated. Four-days grown cultures in LB were centrifuged (23400 g,

155

10 min) twice and filter-sterilized (Millipore-45 µm). The supernatants thus obtained were mixed

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with melted nutrient agar (45-50°C) at a proportion of 1:4 (v/v) before filling the plates. Once

157

solidified, a 1 cm2 plug of F. graminearum obtained from an actively growing culture was placed at

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the centre of the plates, and further incubated for 6 days. Control plates were prepared by mixing

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fresh LB medium with agar nutrient (at the same proportion) before placing the fungal plug.

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The production of volatile metabolites with activity against F. graminearum growth was also

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investigated. Each bacterial strain (overnight cultures; OD=1.00) was drop-inoculated (10 µl) at

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four equidistant points of the plate and incubated at 25 ºC for 3 days. Then, the lids of these plates

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were replaced by the bottom of a plate containing nutrient agar inoculated with a fresh mycelial

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plug of F. graminearum. Plates were sealed together with sticky tape to minimize gas exchange and

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further incubated for 6 days. Controls were prepared in a similar manner but the bottom plate

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contained no bacteria.

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In all these experiments, the radial size of the fungal colony was measured and compared to that of

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control units.

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Biofilm formation

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Biofilm formation was assessed in static conditions using the microtiter dish assay described by

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O'Toole (2011). In this assay, the extent of the biofilm formed by bacterial cultures on the wall

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and/or bottom of a microtiter dish was measured (after the removal of the liquid phase) using a

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0.1% solution of crystal violet in water. To solubilise the dye, 30% acetic acid in water was used.

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Absorbance was read at 550 nm. Finally, data were normalised by total growth estimated by OD at

176

540 nm.

177 178

Antibiotic resistance

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Increasing concentrations (25, 50, and 100 µg ml-1) of ampicillin, kanamycin, chloramphenicol and

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tetracycline (SIGMA Aldrich, USA) were tested in solid LB to characterise the antibiotic resistance

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pattern of the seed endophytes obtained. Four replicated Petri dishes were prepared for each

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antibiotic concentration and for controls.

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Kernel bioassay

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Surface disinfected (as described earlier) seeds of wheat cv. 75 Aniversario and barley cv. Josefina

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INTA were immersed for 2 h at room temperature in a diluted cell suspension of isolates 5a, 5b, 5c

187

or 2. These suspensions were obtained by centrifuging (23400 g 15 min) overnight-grown cultures

188

in LB and resuspending the pellets in sterile saline solution (0.85% NaCl), to reach a cell density of

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about 1 x 105 CFU ml-1. Seeds were then placed (half and half at the same plate) on plates

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containing nutrient agar previously inoculated with 100 µl of a spore suspension (2 x 105 spores ml-

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1

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for two hours. Plates were incubated at room temperature; fungal advance on the seeds was checked

193

15 days later.

) of F. graminearum. Controls were prepared by immersing seeds in distilled sterile saline solution

194 195

Inoculation assays

196

Seeds of barley cv. Josefina INTA were sown on vermiculite. Seedlings were inoculated with the

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endophytes according to the following treatments: 1) isolate 5c inoculated at sowing (5c); 2) isolate

198

2 inoculated at sowing (P); 3) isolate 5c+2, both inoculated at sowing (5c+Pi); 4) isolate 5c+2; the

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second one inoculated 5 days after sowing (5c+P5); 5) uninoculated (C). Inoculation was

200

performed by adding on the crown of each emerging plantlet 10 µl of the corresponding cell

8

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suspension in sterile saline. Bacterial concentrations were in the order of 108 CFU ml-1. Control

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plants received sterile saline instead of bacterial inoculum. Plants developed for 20 days in a growth

203

chamber at 20 °C under a photoperiod of 10/14 (light/dark); light intensity was 5000 lux. During

204

this period, plants were watered with Hoagland solution diluted at half every other day. Three pots

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with five plants each were prepared, pots were distributed in the chamber following a completely

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randomized block design. Fresh and dry biomass and chlorophyll content were evaluated at day 20.

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In vitro compatibility of endophytes 2 and 5c

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Isolate 2 (100 µl; overnight culture) was spread on the surface of Petri dishes with nutrient agar,

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then isolate 5c was drop-inoculated (10 µl; overnight culture) at four equidistant points of the plate;

211

and vice versa. Plates were incubated at 25 ºC for 3 days and bacterial development was assessed.

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Four replicated plates were prepared.

213 214

Statistical analysis

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Data shown in figures 3 and 5 are mean values of three independent experiments. Standard errors of

216

the means (SEM) are presented. Differences among treatments were analyzed by one-way ANOVA

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followed by Tukey’s multiple range test using InStat™ software (Graph Pad Software, San Diego,

218

CA, USA), at different significance levels (* P< 0.05, ** P<0.01).

219 220

Results and discussion

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Much effort has been made to increase crop yields without taking into account the role of

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endophytic communities of seeds, still poorly characterised. In this research, a narrow range of

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endophytic microorganisms were obtained from wheat seeds following a culture-dependent

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protocol. Four distinct isolates (originally designated 1, 2, 5 and 6) were considered representative

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of the bacterial cultivable community of these seeds. Isolate 1, 2 and 6 formed small round colonies

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and isolate 5 developed an extensive, and massive, and irregular growth. As the appearance of this

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material was not uniform, particularly under longer incubation periods (≥ 6 days), three distinct

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parts of this massive culture were subcultured and maintained separately, handled as distinct

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isolates. This rendered a total of 6 isolates designated 1, 2, 5a, 5b, 5c and 6 (Figure 1). Low

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numbers of bacterial genera recovered from seeds were generally related to the challenges imposed

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by the specific habitat they are derived from and to the limitations of culture-dependent techniques

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(Truyens et al., 2015).

233

Taxonomic features and some metabolic properties of the isolated bacteria are summarised in Table

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1. A clear predominance of sporulated bacilli was found. Most of them were assignated to

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Paenibacillus genus according to their 16S rRNA sequencing (isolate 1, 5a, 5b and 5c), while

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isolate 2 and 6 were assigned to genus Pantoea and Fictibacillus/Bacillus, respectively. All

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nucleotide sequences have been submitted to GenBank Database and received the accession

238

numbers shown on Table 1.

239

Most seed endophytic bacteria isolated from plants belong to the phylum γ-Proteobacteria;

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Actinobacteria, Firmicutes and Bacteroidetes were also found inside plant seeds in a lesser extent.

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Bacillus and Pseudomonas were the most frequently genera found in plant seeds, Paenibacillus,

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Micrococcus, Staphylococcus, Pantoea and Acinetobacter were also reported (Truyens et al., 2015

243

and references therein). In a previous work, eight isolates assigned to Pseudomonas, Azospirillum,

244

and Bacillus genera were retrieved from barley seeds under selective pressure for nitrogen-fixing

245

microorganisms (Zawoznik et al., 2014).

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Differences in bacterial genera found in the seeds of different plant species could be related to the

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type of plant exudates that attract specific microorganisms; also to the ability of microorganisms to

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colonise plant tissues, to survive inside the plant and to be transmitted to the seeds (Truyens et al.,

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2015 and references therein).

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In this work, the only gammaproteobacterium isolated, presumably an Enterobactereaceae of

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Pantoea genus designated as isolate 2, was particularly active as IAA (Table 1) and siderophore

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producer, and also solubilised phosphate (Figure 2A and 2B). Only this microorganism could grow

253

on N-free semisolid medium.

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Table 2 shows the antibiotic resistance pattern of the isolates obtained. It may be noted that isolate 1

255

resisted up to 100 ppm of ampicillin, but was sensitive to the other antibiotics. Isolates 5a, 5b and

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5c showed moderate resistance to ampicillin (up to 50 ppm), but high resistance to kanamycin (up

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to 100 ppm). All the isolates were sensitive to tetracycline, while only isolate 2 resisted a low level

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(25 ppm) of chloramphenicol.

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Isolate assigned to Pantoea genus (2) and three of the four isolates assigned to Paenibacillus genus

260

(5a, 5b and 5c) restrained F. graminearum growth on dual culture, as may be observed on Figure

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3. Among these isolates, only those assigned to Paenibacillus seemed to have released to the culture

262

medium antifungal substances capable to restrain F. graminearum growth by themselves (Figure 3).

263

No effect attributable to volatile substances could be proved (data not shown).

264

We considered of interest to validate our in vitro results regarding biocontrol potential of

265

Paenibacillus and Pantoea isolates in a setting in which the kernel, as a mainstay of any beneficial-

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pathogenic interaction during plant life cycle, could be included. Only a few papers investigated

267

bacterial isolates as biocontrol agents on kernels (Shi et al, 2014; Abd El Daim et al, 2015). To

268

address this issue, we designed a simple bioassay suitable to expand our analysis by including at the

269

same experimental units another graminaceous species usually attacked by F. graminearum: barley.

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As depicted on Figure 4, the growth of this phytopathogen on the kernels was markedly restrained

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when wheat and barley seeds were previously immersed in diluted cell suspensions of isolates 5b,

272

5c and 2, while isolate 5a had a minimum effect on fungal advance.

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Isolates 5c and 5b displayed an outstanding ability to form biofilm on an inert surface, of about 7-

274

fold as compared to the well-recognized PGPR Azospirillum brasilense strain Az39, which was

275

used here as reference strain (Figure 5A and 5B). The production of biofilm is generally regarded as

276

a mechanism of plant protection against pathogenic bacteria, since once established on plant roots,

277

these microbial biofilms may act as physical and chemical barriers. Likewise, these biological

11

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matrexes can also limit the release of root exudates avoiding pathogen growth (Haggag, 2010).

279

Haggag and Timmusk (2008) also highlighted the importance of biofilm formation by Paenibacillus

280

polymyxa strains in controlling crown root rot disease, associated to Aspergillus niger.

281

It has been reported that members of Bacillus and Paenibacillus have yielded several potent

282

antimicrobial lipopeptides including iturins, surfactins, fengycins, fusaricidins, polymyxins and

283

others (Cochrane and Vederas, 2014). A Paenibacillus ehimensis strain produced extracellular

284

organic compounds that inhibited F. oxysporum f.sp. lycopersici conidial germination in in vitro

285

assays (Naing et al., 2015), and Paenibacillus polymyxa BRF-1 showed in vitro antifungal activity

286

on the pathogenic fungus Phialophora gregata, the causative agent of the brown stem rot in

287

soybean (Zhou et al., 2008). Very recently, Nguyen et al. (2015) reported for the first time the

288

antagonistic activity of butyl 2,3-dihydroxybenzoate (B2,3DB) isolated from Paenibacillus elgii

289

HOA73 against Fusarium oxysporum f.sp. lycopersici.

290

Regarding biocontrol abilities among Paenibacillus recovered as endophytes from wheat seeds, our

291

results placed isolate 1 apart from isolates 5. Genotypic relationships in line with these findings

292

were already established through a phylogenetic analysis (Grossi et al., 2015).

293

Pantoea genus includes several species that are generally associated with plants, either as epiphytes

294

or as pathogens, and some species can cause disease in humans (Deletoile et al., 2009). At present

295

23 species are included in the genus with valid names (http://www.bacterio.net/-allnamesmr.html).

296

Strains belonging to the most recently proposed species have been isolated from eucalyptus leaves

297

showing symptoms of bacterial blight and die-back in Uganda, Uruguay and Argentina, and from

298

maize suffering from brown stalk rot in South Africa (Brady et al., 2009). Kang et al. (2007)

299

demonstrated that a Pantoea strain colonised pepper stems in high numbers; this bacterial

300

endophyte lacked of plant pathogenicity and stimulated specially root growth. On another hand, it

301

also elicited induced systemic resistance against the leaf spot disease causative agent, Xanthomonas

302

axonopodis pv.vesicatoria. Pantoea vagans strain A-9 (formerly Pantoea agglomerans) is

303

commercially registered for biological control of fire blight, another bacterial disease of pear and

12

304

apple trees caused by Erwinia amylovora. Biological control of fungal phytopathogens was also

305

documented for several Pantoea isolates: a Pantoea agglomerans strain isolated from a rhizospheric

306

Mexican soil showed antifungal activity against the corn phytopathogenic fungi Stenocarpella

307

maydis (Petatán-Sagahón et al., 2011), while a phosphate-solubilizing Pantoea strain isolated from

308

Indian soils was reported to possess multiple plant growth attributes (IAA production, siderophore

309

production, HCN production) and to antagonise with the phytopathogenic fungi Penicillium

310

chrysogenum, Aspergillus niger and Geotrichum candidum in vitro (Dastager et al., 2009).

311

Endophytic Pantoea isolated from different cotton cultivars were reported to show antagonistic

312

potential against some strains of Verticillium dahliae and Fusarium oxysporum f. sp. vasinfectum

313

(Li et al., 2010). Of note is that several Fusarium genes related to fungal defense and/or virulence

314

and cell division (among others) were induced or repressed by Pantoea agglomerans (Pandolfi et

315

al., 2010).

316

Isolate 2, which showed high identity to Pantoea agglomerans at NCBI database, displayed several

317

plant growth promotion features in vitro (Table 1). Several reports documented growth promotion

318

caused by Pantoea agglomerans inoculation in tomato, cucumber, maize, chickpea and rice

319

(Dursun et al., 2010; Taurian et al., 2010; Mishra et al., 2011; Fei et al., 2011). Both Paenibacillus

320

and Pantoea are known to produce auxins and auxins-like compounds, also cytokinins and

321

gibberellins. In conjunction with nitrogen fixation and phosphate solubilisation, these features were

322

related to plant growth promotion (Chauhan et al., 2015).

323

Isolate 2 restrained F. graminearum growth both in dual culture (Figure 3) and in a tripartite

324

bioassay (Figure 4). This isolate also increased root growth and mitigated some adverse effects of

325

saline stress in barley seedlings in a previous pot experiment in which barley plants were grown for

326

10 days (unpublished data). Now we conducted a new pot experiment of greater duration (20 days)

327

and included the combination of two isolates: 5c and 2. Under our experimental conditions,

328

inoculated treatments did not show significant changes in plant biomass; however, an increased

329

chlorophyll content –normally considered an index of improved plant nitrogen status–, was found

13

330

under coinoculation with isolate 5c and isolate 2 (Figure 6A). In an in vitro compatibility assay,

331

these isolates showed no any adverse interaction between them (Figure 6B).

332

Several bacteria are emerging as novel PGPR, with a wide range of positive effects on plant

333

production, including the biocontrol of different fungal and bacterial plant diseases, and

334

Paenibacillus and Pantoea members –found in this study as wheat seed endophytes− repeatedly

335

appear among these novel PGPR in current literature (Chahuan et al. 2015 and references therein).

336

These microorganisms stimulated plant growth and increased plant yields by acting alone or in

337

combination with other well-known rhizospheric microbes; however as far as we know, there are no

338

reports in which the combination of these two microorganisms had been tested.

339 340

Conclusions

341

Our results show a significant ecological potential of some components of the wheat seed

342

endophytic community. Seed endophytic microorganisms probably play important roles during

343

plant development; in fact, they often possess attractive characteristics that could turn into

344

biotechnological applications, including biological control. New inoculants based on spermospheric

345

communities may not only improve cereal yields, they may also contribute to reduce the incidence

346

of fusariosis, and combinations of strains are usually tested. However, more research is needed to

347

shed light on the determinants which define seed colonisation. For instance, it is necessary to

348

understand if seed endophytes are actively selected by their host plants to their own benefits or if

349

seeds are just a biological vehicle to survive and eventually colonise new habitats. Our

350

understanding of microbe interactions at seed stage and their biological relevance may just be

351

starting.

352 353 354 355 14

356

Acknowledgments

357

This work was supported by the University of Buenos Aires (UBACYT 20020130200052BA).

358

María D. Groppa is researcher and Silvana Díaz Herrera is fellowship at the Consejo Nacional de

359

Investigaciones Científicas y Técnicas (CONICET).

360

We thank Ing. Agr. Alejandro Perticari (IMYZA-INTA) for providing the Azospirillum brasilense

361

Az39 strain used in this work.

362

15

363

References

364

Abd El Daim IA, Häggblom P, Karlsson M, Stenström E,Timmusk S. Paenibacillus polymyxa A26

365

Sfp-type PPTase inactivation limits bacterial antagonism against Fusarium graminearum but not of

366

F.culmorum in kernel assay. Front Plant Sci 2015; 6:368, doi: 10.3389/fpls.2015.00368.

367

Abdulkareem M, Aboud HM, Saood HM, Shibly MK. Antagonistic activity of some plant growth

368

rhizobacteria to Fusarium graminearum. Int J Phytopathol 2014; 3:49-54.

369

Alikhani HA, Saleh-Rastin N, Antoun H. Phosphate solubilization activity of rhizobia native to

370

Iranian soils. Plant Soil 2006; 287:35-41.

371

Barberis NA. Evolución y perspectiva mundial y nacional de la producción y el comercio de trigo.

372

Cartilla Digital Manfredi 2014/4, Ediciones Instituto Nacional de Tecnología Agropecuaria, INTA -

373

EEA Manfredi.

374

Brady CL,Venter SN, Cleenwerck I, Engelbeen K, Vancanneyt M, Swings J, Coutinho TA. Pantoea

375

vagans sp. nov., Pantoea eucalypti sp. nov., Pantoea deleyi sp. nov. and Pantoea anthophila sp.

376

nov. Int J Syst Evol Microbiol 2014; 59:2339-2345.

377

Chauhan H, Bagyaraja DJ, Selvakumarb G, Sundaramc SP. Novel plant growth promoting

378

rhizobacteria—Prospects and potential. Appl Soil Ecol 2015; 95:38-53.

379

Cochrane SA, Vederas JC. Lipopeptides from Bacillus and Paenibacillus spp.: A gold mine of

380

antibiotic candidates. Med Res Rev 2014. doi: 10.1002/med.21321.

381

Dastager SG, Deepa CK, Puneet SC, Nautiyal CS, Pandey A. Isolation and characterization of plant

382

growth-promoting strain Pantoea NII-186 from Western Ghat Forest soil. Ind Lett Appl Microbiol

383

2009; 49, 20-25.

384

Deletoile A, Decré D, Courant S, Passet V, Audo J, Grimont P, Arlet G, Brisse1 S. Phylogeny and

385

identification of Pantoea species and typing of Pantoea agglomerans strains by multilocus gene

386

sequencing. J Clin Microbiol 2009; 47:300-310.

16

387

Dursun A, Ekinci M, Donmez MF. Effects of foliar application of plant growth promoting

388

bacterium on chemical contents, yield and growth of tomato (Lycopersicon esculentum L.) and

389

cucumber (Cucumis sativus L.). Pak J Bot 2010; 42: 3349-3356.

390

Fei L, Ya W, Qing Gui Z, Ri Ming Y, Zhi Bin Z, Du Z. Diversity and plant growth promoting

391

activities of the cultivable rhizo-bacteria of Dongxiang wild rice (Oryza rufipogon). Biodiver Sci

392

2011; 19: 476-484.

393

Figueiredo MVB, Martinez CR, Burity HA, Chanway CP. Plant growth promoting rhizobacteria for

394

improving nodulation and nitrogen fixation in the common bean Phaseolus vulgaris L. World J

395

Microbiol Biotechnol 2008; 24:1187-1193.

396

Gagne-Bourgue F, Aliferis KA, Seguin P, Rani M, Samson R, Jabaji S. Isolation and

397

characterization of indigenous endophytic bacteria associated with leaves of switchgrass (Panicum

398

virgatum L.) cultivars. J Appl Microbiol 2013; 114:836-853.

399

Glickmann E, Dessaux Y. A critical examination of the specificity of the Salkowski reagent for

400

indolic compounds produced by phytopathogenic bacteria. Appl Environ Microbiol 1995; 61:793-

401

796.

402

Grossi CEM, Díaz Herrera SM, Zawoznik MS, Groppa MD. Endófitos de semillas de trigo. BAG

403

2015; 26 (Suppl I). On-line version ISSN 1852-6233.

404

Haggag W. The role of biofilm exopolysaccharides on biocontrol of plant diseases. In: Elnashar M,

405

editor. Biopolymers, Sciyo; 2010. http://www.intechopen.com/books/biopolymers/the-role-of-

406

biofilm-exopolysaccharides-on-biocontrol-of-plantdiseases.

407

Haggag WM, Timmusk S. Colonization of peanut roots by biofilm forming Paenibacillus polymyxa

408

initiates biocontrol against crown rot disease. J Appl Microbiol 2008; 104:961-969.

409

Hardoim PR, Hardoim CC, van Overbeek LS, van Elsas JD. Dynamics of seed-borne rice

410

endophytes on early plant growth stages. PLoS One 2012; 7:e30438. doi:

411

10.1371/journal.pone.0030438.

17

412

Herigstad B, Hamilton M, Heersink J. How to optimize the drop plate method for enumerating

413

bacteria. J Microbiol Methods 2001; 44:121-129

414

Johnston-Monje D, Raizada MN. Conservation and diversity of seed associated endophytes in Zea

415

across boundaries of evolution, ethnography and ecology. PLoS ONE 2011; 6: e20396.

416

Kaga H, Mano H, Tanaka F, Watanabe A, Kaneko S, Morisaki H. Rice seeds as sources of

417

endophytic bacteria. Microbes Environ 2009; 24:154-162.

418

Kang SH, Cho HS, Cheong H, Ryu CM, Kim JF, Park SH. Two bacterial entophytes eliciting both

419

plant growth promotion and plant defense on pepper (Capsicum annuum L.). J Microbiol

420

Biotechnol 2007; 17:96-103.

421

Li CH, Zhao MW, Tang CM, Li SP. Population dynamics and identification of endophytic bacteria

422

antagonistic toward plant-pathogenic fungi in cotton root. Microbiol Ecol 2010; 59: 344-356.

423

Liu Y, Zuo S, Xu L, Zou Y, Song W. Study on diversity of endophytic bacterial communities in

424

seeds of hybrid maize and their parental lines. Arch Microbiol 2012; 194:1001-1012.

425

López-López A, Rogel MA, Ormeño-Orillo E, Martínez-Romero J, Martínez-Romero E. Phaseolus

426

vulgaris seed-borne endophytic community with novel bacterial species such as Rhizobium

427

endophyticum sp. nov. Syst Appl Microbiol 2010; 33:322-327.

428

Malfanova N, Lugtenberg BJJ, Berg G. Bacterial endophytes: who and where, and what are they

429

doing there?, In: de Bruijn FJ, editor. Molecular microbial ecology of the rhizosphere. NJ, USA:

430

Wiley-Blackwell, Hoboken; 2013. p. 391-403.

431

Mano H, Tanaka F, Watanabe A, Kaga H, Okunishi S, Morisaki H. Culturable surface and

432

endophytic bacterial flora of the maturing seeds of rice plants (Oryza sativa) cultivated in a paddy

433

field. Microbes Environ 2006; 2:86-100.

434

Mishra A, Chauhan PS, Chaudhry V, Tripathi M, Nautiyal CS. Rhizosphere competent Pantoea

435

agglomerans enhances maize (Zea mays) and chickpea (Cicer arietinum L.) growth, without

436

altering the rhizosphere functional diversity. A van Leeuw 2011; 100:405-413.

18

437

Mukhopadhyay K, Garrison NK, Hinton DM, Bacon CW, Khush GS, Peck HD, Data N.

438

Identification and characterization of bacterial endophytes of rice. Mycopathologia 1996; 134:151-

439

159.

440

Naing KW, Nguyen XH, Anees M, Lee YS, Kim YC, Kim SJ, Kim MH, Kim YH, Kim KY.

441

Biocontrol of Fusarium wilt disease in tomato by Paenibacillus ehimensis KWN38. World J

442

Microbiol Biotechnol 2015; 31:165-174. doi: 10.1007/s11274-014-1771-4.

443

Nguyen XH, Naing KW, Lee YS, Kim KY. Isolation of butyl 2,3-dihydroxybenzoate from

444

Paenibacillus elgii HOA73 against Fusarium oxysporum f.sp. Lycopersici. J. Phytopathol 2015;

445

163:342-352.

446

O’Toole GA. Microtiter dish biofilm formation assay. JoVE 2011; 47. doi: 10.3791/2437.

447

Pandolfi V, Jorge EC, Melo CMR, Albuquerque ACS, Carrer H. Gene expression profile of the

448

plant pathogen Fusarium graminearum under the antagonistic effect of Pantoea agglomerans.

449

Genet Mol Res 2010; 9:1298-1311.

450

Pérez-Miranda S, Cabirol N, George-Téllez R, Zamudio-Rivera LS, Fernández FJ. O-CAS, a fast

451

and universal method for siderophore detection. J Microbiol Meth 2007; 70:127-131.

452

Petatán-Sagahón I, Anducho-Reyes MA, Silva-Rojas HV, Arana-Cuenca A, Tellez-Jurado A,

453

Cárdenas-Álvarez IO, Mercado-Flores Y. Isolation of bacteria with antifungal activity against the

454

phytopathogenic fungi Stenocarpella maydis and Stenocarpella macrospora. Int J Mol Sci 2011;

455

12:5522-5537.

456

Puente ME, Li CY, Bashan Y. Endophytic bacteria in cacti seeds can improve the development of

457

cactus seedlings. Environ Exp Bot 2009a; 66:402-408.

458

Puente ME, Li CY, Bashan Y. Rock-degrading endophytic bacteria in cacti. Environ Exp Bot

459

2009b; 66:389-401.

460

Rijavec T, Lapanje A, Dermastia M, Rupnik M. Isolation of bacterial endophytes from germinated

461

maize kernels. Can J Microbiol 2007; 3:802-808.

19

462

Ringelberg D, Foley K, Reynolds CM. Bacterial endophyte communities of two wheatgrass

463

varieties following propagation in different growing media. Can J Microbiol 2012; 58:67-80.

464

Ruiz D, Agaras B, Werra P, Wall LG, Valverde C. Characterization and screening of plant probiotic

465

traits of bacteria isolated from rice seeds cultivated in Argentina. J Microbiol 2011; 49:902-912.

466

Schwyn B, Neilands JB. Universal chemical assay for the detection and determination of

467

siderophores. Anal Biochem 1987; 160:47-56.

468

Shi C, Yan P, Li J, Wu H, Li Q, Guan S. Biocontrol of Fusarium graminearum growth and

469

deoxynivalenol production in wheat kernels with bacterial antagonists. Int J Environ Res Public

470

Health 2014, 11:1094-1105.

471

Shurtleff W, Aoyagi A. Early history of soybeans and soyfoods worldwide (1024 BCE to 1899):

472

Extensively annotated bibliography and sourcebook, Soyinfo Center, Lafayette, CA, USA; 2014.

473

Sundaramoorthy S, Balabaska P. Evaluation of combined efficacy of Pseudomonas fluorescens and

474

Bacillus subtilis in managing tomato wilt caused by Fusarium oxysporum f. sp. lycopersici (Fol).

475

Plant Pathol J 2013; 12:154-161.

476

Taurian T, Anzuay MS, Angelini JG, Tonelli ML, Ludueña L., Pena D, Ibáñez F, Fabra A.

477

Phosphate-solubilizing peanut associated bacteria: screening for plant growth-promoting activities.

478

Plant Soil 2010; 329: 421-431.

479

Truyens S, Weyens N, Cuypers A, Vangronsveld J. Bacterial seed endophytes: genera, vertical

480

transmission and interaction with plants. Environ. Microbiol Rep 2015; 7:40-50.

481

Wang Q, Garrity GM, Tiedje JM, Cole JR. Naïve Bayesian classifier for rapid assignment of rRNA

482

sequences into the new bacterial taxonomy. Appl Environ Microbiol 2007; 73:5261-5267.

483

Xu M, Sheng J, Chen L, Men Y, Gan L, Guo S, Shen L. Bacterial community compositions of

484

tomato (Lycopersicum esculentum Mill.) seeds and plant growth promoting activity of ACC

485

deaminase producing Bacillus subtilis (HYT-12-1) on tomato seedlings. World J Microbiol

486

Biotechnol 2014; 30:835-845.

20

487

Zawoznik MS, Vázquez SC, Díaz Herrera SM, Groppa MD. Search for endophytic diazotrophs in

488

barley sedes. Braz J Microbiol 2014; 45:621-625.

489

Zhou K, Yamagishi M, Osaki M. Paenibacillus BRF-1 has biocontrol ability against Phialophora

490

gregata disease and promotes soybean growth. Soil Sci Plant Nutr 2008; 54:870-875.

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21

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Legends to figures

493

Figure 1. Image illustrates colony morphotype of the representative endophytes isolated by

494

a culture-dependent protocol from seeds of wheat, cultivar 75 Aniversario, according to the

495

description in Materials and methods. Numbers identify the different isolates.

496

Figure 2. Siderophore production (A) and phosphate solubilisation (B) assays. Images are

497

representative of four replicated experimental units, obtained from three independent

498

experiments.

499

Figure 3. Antagonism against Fusarium graminearum. A) Comparative effects of seed

500

endophytes (dual assay) and of their supernatants (diffusible substances) on the radial size

501

of Fusarium graminearum. Four replicated experimental units obtained from three

502

independent experiments were prepared. Bars represent standard error of the mean (SEM).

503

Asterisks indicate significant differences (P < 0.05). Three independent experiments were

504

carried out. B) Illustrative images showing F. graminearum growth in dual assays with

505

isolates 2 (left) and 5c (right).

506

Figure 4. Bioassays including wheat and barley kernels. Seeds of barley and wheat were

507

pretreated with diluted cell suspensions of isolates 5a, 5b, 5c and 2, and then exposed to F.

508

graminearum, as described in Materials and methods section. Left of each plate: barley

509

kernels; right: wheat kernels.

510

Figure 5. Biofilm formation assay by crystal violet staining (A). Absorbances at 550 nm

511

were normalised by total bacterial growth estimated by OD at 540 nm (B). Bars represent

512

standard error of the mean (SEM). Asterisks indicate significant differences (* P < 0.05;

513

** P < 0.01). Three independent experiments were carried out.

514

Figure 6. Inoculation assays. (A) Chlorophyll content of barley seedlings grown in a

515

growth chamber as described in Materials and methods section during 20 days. Treatments: 22

516

5c: isolate 5c inoculated at sowing, P: isolate 2 inoculated at sowing, 5c+Pi: isolate 5c+2, both

517

inoculated at sowing, 5c+P5: isolate 5c+2; the second one inoculated 5 days after sowing, C:

518

uninoculated. Different letters indicate significant differences. (B) Illustrative images

519

showing the results of in vitro compatibility assay. Left: Isolate 2 inoculated at the surface of the

520

plate and isolate 5c drop-inoculated; right: vice versa.

23

Table 1

Table 1. Taxonomic features and some metabolic properties of isolated bacteria

Isolate

1 2 5a 5b 5c 6

Gram Spore staining formation + + + + +

nd N Y Y Y Y

Growth in semisolid Nfb

Phosphate solubilisation

Siderophore production*

IAA production** (µg/107 cfu)

Most probable affiliation based on partial 16S ARNr sequencing***

NCBI Accession Number

N Y N N N N

N Y N N N N

(+) (+++) (+) (+) (++) (+)

5 52 0.03 2.5 6.5 0.002

Paenibacillus sp. Pantoea sp. Paenibacillus sp. Paenibacillus sp. Paenibacillus sp. Bacillus sp./Fictibacillus sp.

KR263165 KT982206 KR263166 KR263167 KR263168 KR263169

nd: Not determined N: no; Y: yes *Comparison based on dissolution halos diameter. ** Determined in 1 ml supernatant aliquots and expressed on 107 cfu basis. *** All isolates showed 99 % percent identity or more with deposited sequences on BLAST-NCBI database.

Table 2

Table 2. Antibiotic resistance pattern of isolated bacteria.

Isolates 1 2 5a 5b 5c 6

25 + + + + + +

Ampicillin (ppm) 50 100 + + + + + + +

25 + + + -

Kanamycin (ppm) 50 100 + + + + + + -

Chloramphenicol (ppm) 25 50 100 + -

25 -

Tetracycline (ppm) 50 100 -

Figure 1

Figure 2

A

B

1 5a

2 5b

6 5c

1 5a

2 5b

6 5c

Figure 3

90,00

A

Dual

Diffusible

Fungal colony diameter (mm)

80,00

70,00 60,00

*

50,00

*

40,00

*

*

30,00

*

20,00

* *

*

10,00 C

B

1

2

2

2

5a

5b

5c

5c

5c

6

Figure 4

Figure 5

B

A Biofilm formation (550 nm) C Az39 1 2 5a 5b 5c 6

Cellular growth (540 nm)

Figure 6

2200

Chlorophyll content (µg g FW-1)

A

b

2100 b

2000 1900 1800

a

ab

a

1700 1600 C

B

P

5c

5c+Pi

5c+P5