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.
Wheat seeds harbour bacterial endophytes with potential as plant growth promoters and
biocontrol agents of Fusarium graminearum
Universidad de Buenos Aires, Junín 956, Buenos Aires, Argentina.
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,
8 9 10 11 12 13
Myriam S. Zawoznik
Cátedra de Química Biológica Vegetal, Facultad de Farmacia y Bioquímica, Universidad de Buenos
Junín 956, (1113) Ciudad Autónoma de Buenos Aires, Argentina.
E-mail: [email protected]
The role of endophytic communities of seeds is still poorly characterised. The purpose of this work
was to survey the presence of bacterial endophytes in the seeds of a commercial wheat cultivar
widely sown in Argentina and to look for plant growth promotion features and biocontrol abilities
against F. graminearum among them. Six isolates were obtained from wheat seeds following a
culture-dependent protocol. Four isolates were assignated to Paenibacillus genus according to their
16S rRNA sequencing. The only gammaproteobacteria isolated, presumably an Enterobactereaceae
of Pantoea genus, was particularly active as IAA and siderophore producer, and also solubilised
phosphate and was the only one that grew on N-free medium. Several of these isolates demonstrated
ability to restrain F. graminearum growth on dual culture and in a bioassay using barley and wheat
kernels. An outstanding ability to form biofilm on an inert surface was corroborated for those
Paenibacillus which displayed greater biocontrol of F. graminearum, and the inoculation with one
of these isolates in combination with the Pantoea isolate resulted in greater chlorophyll content in
barley seedlings. Our results show a significant ecological potential of some components of the
wheat seed endophytic community.
Keywords: barley, bioassays, Fusarium, Paenibacillus, Pantoea, wheat-endophytes
38 39 40 41 42 43 44 45
Wheat is a very important crop for Argentine agriculture and has become a key factor for the
preservation of soil fertility because many argentine soils are under a continuous soybean-wheat
rotation scheme, and soybean is recognized as a very soil-exhausting crop (Shurtleff and Aoyagi,
2014). About 14.5 million tons of total argentine agriculture production corresponded to wheat at
the 2012/2013 campaign. Argentine was sixth among wheat exporter countries, and wheat
exportations represented for our economy an income of 2.5 million dollars in that period (Barberis,
During last decades many researchers reported the presence of seed endophytes in several plant
species including gramineous plants such as rice and maize, where Proteobacteria, Actinobacteria
and Firmicutes were particularly dominant (Rijavec et al., 2007; Kaga et al., 2009; Ruiz et al.,
Seed endophytes may come from different plant organs, being transferred to seeds via vascular
connections or through gametes, resulting in colonisation of embryo and endosperm; and
reproductive meristems may also be the source (Malfanova et al., 2013). Vertical transmition from
one plant generation to the following may then occur, as suggested by several authors (Ringelberg
et al., 2012; Liu et al., 2012; Gagne-Bourgue et al., 2013) and depicted recently in Truyens et al.
After seed germination, these populations are expected to increase in number and to colonise
different tissues including roots, reaching the endorizosphere and probably also the exorizosphere.
Mano et al. (2006) observed that although rice seed endophytes mainly colonised shoots, some
strains were able to spread out into the rhizosphere and soil. Similar observations were made by
Hardoim et al. (2012). López-López et al. (2010) could recover almost all bacterial genera isolated
from bean seeds also from the roots of bean seedlings. Under this scenario, introduced
microorganisms are expected to compete and/or to share their ecological niche with endophytic
communities established in the rhizosphere, for which it is particularly necessary to gain more
knowledge about seed endophytic communities in crops which are increasingly being inoculated
with plant growth promoting microorganisms (PGPM), such as wheat or maize. In this sense and
regarding particularly corn crop, of note are the findings of Johnston-Monje and Raizada (2011),
who demonstrated that in maize, a core microbiota consisting of the same bacterial species is
conserved apparently from teosinte’s times, in spite of evolutive and selective changes, even across
the huge American continent.
The role of seed endophytes has not been unravelled yet. It has been demonstrated that some of
them can increase plant growth due to the production of plant hormones or to their contribution in
plant nutrients acquisition, specially nitrogen and phosphorus (Gagne-Bourgue et al., 2013;
Johnston-Monje and Raizada, 2011; Xu et al., 2014). For cacti, the ecological significance of seed
endophytes was demonstrated by Puente et al. (2009a, 2009b). On another hand, antifungal activity
of several bacterial seed endophytes has also been recognized, involving lipopeptides like surfactin,
iturin and mycobacillin (Gagne-Bourgue et al., 2013). Some strains of bacteria frequently
mentioned as seed endophytes (such as Bacillus and Pseudomonas) were found to have antagonistic
effects on F. oxysporum f. sp. lycopersici (Fol), the causative agent of tomato wilt (Sundaramoorthy
and Balabaska, 2013). Volatile anti-fungal compounds were also found to be involved in the
biocontrol displayed by endophytic Enterobacter strains obtained from rice (Mukhopadhyay et al.,
Fusarium gramineaurum is the causative agent of wheat head blight, a worldwide fungal plant
pathogen impacting severely on cereals production and quality, as this microorganism is a source of
mycotoxins, which affect human and animal health. The purpose of this work was to survey the
presence of bacterial endophytes in the seeds of a commercial wheat cultivar widely sown in
Argentina called 75 Aniversario, to identify the most abundant genera and to screen these isolates
for some features which are involved in direct or indirect mechanisms of plant growth promotion.
Their abilities to inhibit the growth of the plant pathogen F. graminearum and to promote barley
growth were also investigated.
Materials and methods
Isolation and identification of bacterial endophytes
Seeds of wheat (Triticum aestivum L.) cultivar 75 Aniversario (seed supplier: Buck S.A.) were
surface disinfected using ethanol 70% (30 sec), followed by 1% active Cl2 (2.5 min) and again,
ethanol 70% (30 sec), with three rinses in sterilised distilled water. One ml of the last rinse water
was added to 10 ml of liquid LB culture medium and incubated for 48 h to check complete external
disinfection. Ten intact disinfected seeds were placed on Petri dishes containing LB and incubated
at 24 °C. Five replicates were prepared. After 7 days of incubation at 28 °C, some representative
colonies appearing on the majority of the plates were selected and phenotypically characterised;
following repeated subcultures several isolates were obtained. Standard identification protocols
based on morphology, Gram staining, spore formation and certain biochemical properties were
investigated to characterise these isolates. 16S rDNA partial sequencing was performed to get
identification at genus level; comparisons with deposited sequences in BLAST database were made
for this purpose. Total DNA was extracted from randomly chosen colonies of each selected isolate
using the AxyPrep bacterial genomic DNA Miniprep Kit (Axygen Biosciences). A fragment of
around 1500 bp of the 16S rRNA gene was amplified using the universal primers 27F and 1492R.
PCR products were purified with the AND-Clean UP (PB-L Productos Bio-Lógicos) and sequenced
at the Genomic Unit of the Biotechnology Institute of CNIA-INTA (http://www.inta.gov.ar/biotec)
using a capillary automatic sequencer model ABI3130XL (Applied Biosystems, USA). The Naïve
Bayesian Classifier utility (Wang et al., 2007) from the RDP Release 10 (http://rdp.cme.msu.edu)
was used to assign the obtained sequences into the new bacterial taxonomy at genus level with 95%
Indolacetic acid and siderophore production
In vitro IAA biosynthetic ability of the isolates obtained was estimated by Salkowski colorimetric
technique in 1 ml-supernatant aliquots of 5-days old cultures grown on LB amended with L-
tryptophan (100 µg ml-1), as described by Glickman and Dessaux (1995); cultures were run in
triplicate and three supernatant aliquots of each bacterial culture were processed. Final bacterial
counts were calculated by the drop plate technique, as described by Herigstad et al. (2001); IAA
production was expressed on 107 CFU basis.
Siderophore production was investigated using the O-CAS assay (Pérez-Miranda et al., 2007), a fast
and universal method of siderophore detection in which an overlay of the CAS medium of Schwyn
and Neilands (1987) without nutrients is applied on agar plates containing cultivated
microorganisms to reveal siderophore production. Halos surrounding colonies demonstrate
Phosphate solubilisation in solid media
Basal Sperber medium supplemented with 2.5 g l-1 of Ca3(PO4)2 (TCP) was used to test the ability
of the isolates to solubilise inorganic phosphate, as described by Alikhani et al. (2006). The pH of
the medium was adjusted to 7.2. The surface of the solidified medium was divided into equal parts
at the centre of which a 5-µl drop of each bacterial culture (OD=1.00) was applied. Inoculated
plates were incubated in dark at 25 ºC; at day 10 plates were observed in order to establish the
formation of clear zones (halo) surrounding colonies capable of solubilise TCP.
In vitro antagonism against Fusarium graminearum
These experiments were based on those described by Abdulkarem et al. (2014) with some
The possibility of antagonistic effects of the isolates obtained against F. graminearum was assessed
on dual cultures on nutrient agar. Each bacterial strain (overnight cultures; OD=1.00) was drop-
inoculated (10 µl) at four equidistant points of the plate and incubated at 25 ºC for 3 days. Then, a 1
cm2 plug of F. graminearum obtained from an actively growing culture was placed at the centre of
the plates, and plates were further incubated for 6 days. Control cultures containing only the fungus
plugs at the centre served as control units.
Involvement of diffusible substances with antifungal activity released by these endophytes into the
culture medium was also investigated. Four-days grown cultures in LB were centrifuged (23400 g,
10 min) twice and filter-sterilized (Millipore-45 µm). The supernatants thus obtained were mixed
with melted nutrient agar (45-50°C) at a proportion of 1:4 (v/v) before filling the plates. Once
solidified, a 1 cm2 plug of F. graminearum obtained from an actively growing culture was placed at
the centre of the plates, and further incubated for 6 days. Control plates were prepared by mixing
fresh LB medium with agar nutrient (at the same proportion) before placing the fungal plug.
The production of volatile metabolites with activity against F. graminearum growth was also
investigated. Each bacterial strain (overnight cultures; OD=1.00) was drop-inoculated (10 µl) at
four equidistant points of the plate and incubated at 25 ºC for 3 days. Then, the lids of these plates
were replaced by the bottom of a plate containing nutrient agar inoculated with a fresh mycelial
plug of F. graminearum. Plates were sealed together with sticky tape to minimize gas exchange and
further incubated for 6 days. Controls were prepared in a similar manner but the bottom plate
contained no bacteria.
In all these experiments, the radial size of the fungal colony was measured and compared to that of
Biofilm formation was assessed in static conditions using the microtiter dish assay described by
O'Toole (2011). In this assay, the extent of the biofilm formed by bacterial cultures on the wall
and/or bottom of a microtiter dish was measured (after the removal of the liquid phase) using a
0.1% solution of crystal violet in water. To solubilise the dye, 30% acetic acid in water was used.
Absorbance was read at 550 nm. Finally, data were normalised by total growth estimated by OD at
Increasing concentrations (25, 50, and 100 µg ml-1) of ampicillin, kanamycin, chloramphenicol and
tetracycline (SIGMA Aldrich, USA) were tested in solid LB to characterise the antibiotic resistance
pattern of the seed endophytes obtained. Four replicated Petri dishes were prepared for each
antibiotic concentration and for controls.
Surface disinfected (as described earlier) seeds of wheat cv. 75 Aniversario and barley cv. Josefina
INTA were immersed for 2 h at room temperature in a diluted cell suspension of isolates 5a, 5b, 5c
or 2. These suspensions were obtained by centrifuging (23400 g 15 min) overnight-grown cultures
in LB and resuspending the pellets in sterile saline solution (0.85% NaCl), to reach a cell density of
about 1 x 105 CFU ml-1. Seeds were then placed (half and half at the same plate) on plates
containing nutrient agar previously inoculated with 100 µl of a spore suspension (2 x 105 spores ml-
for two hours. Plates were incubated at room temperature; fungal advance on the seeds was checked
15 days later.
) of F. graminearum. Controls were prepared by immersing seeds in distilled sterile saline solution
Seeds of barley cv. Josefina INTA were sown on vermiculite. Seedlings were inoculated with the
endophytes according to the following treatments: 1) isolate 5c inoculated at sowing (5c); 2) isolate
2 inoculated at sowing (P); 3) isolate 5c+2, both inoculated at sowing (5c+Pi); 4) isolate 5c+2; the
second one inoculated 5 days after sowing (5c+P5); 5) uninoculated (C). Inoculation was
performed by adding on the crown of each emerging plantlet 10 µl of the corresponding cell
suspension in sterile saline. Bacterial concentrations were in the order of 108 CFU ml-1. Control
plants received sterile saline instead of bacterial inoculum. Plants developed for 20 days in a growth
chamber at 20 °C under a photoperiod of 10/14 (light/dark); light intensity was 5000 lux. During
this period, plants were watered with Hoagland solution diluted at half every other day. Three pots
with five plants each were prepared, pots were distributed in the chamber following a completely
randomized block design. Fresh and dry biomass and chlorophyll content were evaluated at day 20.
In vitro compatibility of endophytes 2 and 5c
Isolate 2 (100 µl; overnight culture) was spread on the surface of Petri dishes with nutrient agar,
then isolate 5c was drop-inoculated (10 µl; overnight culture) at four equidistant points of the plate;
and vice versa. Plates were incubated at 25 ºC for 3 days and bacterial development was assessed.
Four replicated plates were prepared.
Data shown in figures 3 and 5 are mean values of three independent experiments. Standard errors of
the means (SEM) are presented. Differences among treatments were analyzed by one-way ANOVA
followed by Tukey’s multiple range test using InStat™ software (Graph Pad Software, San Diego,
CA, USA), at different significance levels (* P< 0.05, ** P<0.01).
Results and discussion
Much effort has been made to increase crop yields without taking into account the role of
endophytic communities of seeds, still poorly characterised. In this research, a narrow range of
endophytic microorganisms were obtained from wheat seeds following a culture-dependent
protocol. Four distinct isolates (originally designated 1, 2, 5 and 6) were considered representative
of the bacterial cultivable community of these seeds. Isolate 1, 2 and 6 formed small round colonies
and isolate 5 developed an extensive, and massive, and irregular growth. As the appearance of this
material was not uniform, particularly under longer incubation periods (≥ 6 days), three distinct
parts of this massive culture were subcultured and maintained separately, handled as distinct
isolates. This rendered a total of 6 isolates designated 1, 2, 5a, 5b, 5c and 6 (Figure 1). Low
numbers of bacterial genera recovered from seeds were generally related to the challenges imposed
by the specific habitat they are derived from and to the limitations of culture-dependent techniques
(Truyens et al., 2015).
Taxonomic features and some metabolic properties of the isolated bacteria are summarised in Table
1. A clear predominance of sporulated bacilli was found. Most of them were assignated to
Paenibacillus genus according to their 16S rRNA sequencing (isolate 1, 5a, 5b and 5c), while
isolate 2 and 6 were assigned to genus Pantoea and Fictibacillus/Bacillus, respectively. All
nucleotide sequences have been submitted to GenBank Database and received the accession
numbers shown on Table 1.
Most seed endophytic bacteria isolated from plants belong to the phylum γ-Proteobacteria;
Actinobacteria, Firmicutes and Bacteroidetes were also found inside plant seeds in a lesser extent.
Bacillus and Pseudomonas were the most frequently genera found in plant seeds, Paenibacillus,
Micrococcus, Staphylococcus, Pantoea and Acinetobacter were also reported (Truyens et al., 2015
and references therein). In a previous work, eight isolates assigned to Pseudomonas, Azospirillum,
and Bacillus genera were retrieved from barley seeds under selective pressure for nitrogen-fixing
microorganisms (Zawoznik et al., 2014).
Differences in bacterial genera found in the seeds of different plant species could be related to the
type of plant exudates that attract specific microorganisms; also to the ability of microorganisms to
colonise plant tissues, to survive inside the plant and to be transmitted to the seeds (Truyens et al.,
2015 and references therein).
In this work, the only gammaproteobacterium isolated, presumably an Enterobactereaceae of
Pantoea genus designated as isolate 2, was particularly active as IAA (Table 1) and siderophore
producer, and also solubilised phosphate (Figure 2A and 2B). Only this microorganism could grow
on N-free semisolid medium.
Table 2 shows the antibiotic resistance pattern of the isolates obtained. It may be noted that isolate 1
resisted up to 100 ppm of ampicillin, but was sensitive to the other antibiotics. Isolates 5a, 5b and
5c showed moderate resistance to ampicillin (up to 50 ppm), but high resistance to kanamycin (up
to 100 ppm). All the isolates were sensitive to tetracycline, while only isolate 2 resisted a low level
(25 ppm) of chloramphenicol.
Isolate assigned to Pantoea genus (2) and three of the four isolates assigned to Paenibacillus genus
(5a, 5b and 5c) restrained F. graminearum growth on dual culture, as may be observed on Figure
3. Among these isolates, only those assigned to Paenibacillus seemed to have released to the culture
medium antifungal substances capable to restrain F. graminearum growth by themselves (Figure 3).
No effect attributable to volatile substances could be proved (data not shown).
We considered of interest to validate our in vitro results regarding biocontrol potential of
Paenibacillus and Pantoea isolates in a setting in which the kernel, as a mainstay of any beneficial-
pathogenic interaction during plant life cycle, could be included. Only a few papers investigated
bacterial isolates as biocontrol agents on kernels (Shi et al, 2014; Abd El Daim et al, 2015). To
address this issue, we designed a simple bioassay suitable to expand our analysis by including at the
same experimental units another graminaceous species usually attacked by F. graminearum: barley.
As depicted on Figure 4, the growth of this phytopathogen on the kernels was markedly restrained
when wheat and barley seeds were previously immersed in diluted cell suspensions of isolates 5b,
5c and 2, while isolate 5a had a minimum effect on fungal advance.
Isolates 5c and 5b displayed an outstanding ability to form biofilm on an inert surface, of about 7-
fold as compared to the well-recognized PGPR Azospirillum brasilense strain Az39, which was
used here as reference strain (Figure 5A and 5B). The production of biofilm is generally regarded as
a mechanism of plant protection against pathogenic bacteria, since once established on plant roots,
these microbial biofilms may act as physical and chemical barriers. Likewise, these biological
matrexes can also limit the release of root exudates avoiding pathogen growth (Haggag, 2010).
Haggag and Timmusk (2008) also highlighted the importance of biofilm formation by Paenibacillus
polymyxa strains in controlling crown root rot disease, associated to Aspergillus niger.
It has been reported that members of Bacillus and Paenibacillus have yielded several potent
antimicrobial lipopeptides including iturins, surfactins, fengycins, fusaricidins, polymyxins and
others (Cochrane and Vederas, 2014). A Paenibacillus ehimensis strain produced extracellular
organic compounds that inhibited F. oxysporum f.sp. lycopersici conidial germination in in vitro
assays (Naing et al., 2015), and Paenibacillus polymyxa BRF-1 showed in vitro antifungal activity
on the pathogenic fungus Phialophora gregata, the causative agent of the brown stem rot in
soybean (Zhou et al., 2008). Very recently, Nguyen et al. (2015) reported for the first time the
antagonistic activity of butyl 2,3-dihydroxybenzoate (B2,3DB) isolated from Paenibacillus elgii
HOA73 against Fusarium oxysporum f.sp. lycopersici.
Regarding biocontrol abilities among Paenibacillus recovered as endophytes from wheat seeds, our
results placed isolate 1 apart from isolates 5. Genotypic relationships in line with these findings
were already established through a phylogenetic analysis (Grossi et al., 2015).
Pantoea genus includes several species that are generally associated with plants, either as epiphytes
or as pathogens, and some species can cause disease in humans (Deletoile et al., 2009). At present
23 species are included in the genus with valid names (http://www.bacterio.net/-allnamesmr.html).
Strains belonging to the most recently proposed species have been isolated from eucalyptus leaves
showing symptoms of bacterial blight and die-back in Uganda, Uruguay and Argentina, and from
maize suffering from brown stalk rot in South Africa (Brady et al., 2009). Kang et al. (2007)
demonstrated that a Pantoea strain colonised pepper stems in high numbers; this bacterial
endophyte lacked of plant pathogenicity and stimulated specially root growth. On another hand, it
also elicited induced systemic resistance against the leaf spot disease causative agent, Xanthomonas
axonopodis pv.vesicatoria. Pantoea vagans strain A-9 (formerly Pantoea agglomerans) is
commercially registered for biological control of fire blight, another bacterial disease of pear and
apple trees caused by Erwinia amylovora. Biological control of fungal phytopathogens was also
documented for several Pantoea isolates: a Pantoea agglomerans strain isolated from a rhizospheric
Mexican soil showed antifungal activity against the corn phytopathogenic fungi Stenocarpella
maydis (Petatán-Sagahón et al., 2011), while a phosphate-solubilizing Pantoea strain isolated from
Indian soils was reported to possess multiple plant growth attributes (IAA production, siderophore
production, HCN production) and to antagonise with the phytopathogenic fungi Penicillium
chrysogenum, Aspergillus niger and Geotrichum candidum in vitro (Dastager et al., 2009).
Endophytic Pantoea isolated from different cotton cultivars were reported to show antagonistic
potential against some strains of Verticillium dahliae and Fusarium oxysporum f. sp. vasinfectum
(Li et al., 2010). Of note is that several Fusarium genes related to fungal defense and/or virulence
and cell division (among others) were induced or repressed by Pantoea agglomerans (Pandolfi et
Isolate 2, which showed high identity to Pantoea agglomerans at NCBI database, displayed several
plant growth promotion features in vitro (Table 1). Several reports documented growth promotion
caused by Pantoea agglomerans inoculation in tomato, cucumber, maize, chickpea and rice
(Dursun et al., 2010; Taurian et al., 2010; Mishra et al., 2011; Fei et al., 2011). Both Paenibacillus
and Pantoea are known to produce auxins and auxins-like compounds, also cytokinins and
gibberellins. In conjunction with nitrogen fixation and phosphate solubilisation, these features were
related to plant growth promotion (Chauhan et al., 2015).
Isolate 2 restrained F. graminearum growth both in dual culture (Figure 3) and in a tripartite
bioassay (Figure 4). This isolate also increased root growth and mitigated some adverse effects of
saline stress in barley seedlings in a previous pot experiment in which barley plants were grown for
10 days (unpublished data). Now we conducted a new pot experiment of greater duration (20 days)
and included the combination of two isolates: 5c and 2. Under our experimental conditions,
inoculated treatments did not show significant changes in plant biomass; however, an increased
chlorophyll content –normally considered an index of improved plant nitrogen status–, was found
under coinoculation with isolate 5c and isolate 2 (Figure 6A). In an in vitro compatibility assay,
these isolates showed no any adverse interaction between them (Figure 6B).
Several bacteria are emerging as novel PGPR, with a wide range of positive effects on plant
production, including the biocontrol of different fungal and bacterial plant diseases, and
Paenibacillus and Pantoea members –found in this study as wheat seed endophytes− repeatedly
appear among these novel PGPR in current literature (Chahuan et al. 2015 and references therein).
These microorganisms stimulated plant growth and increased plant yields by acting alone or in
combination with other well-known rhizospheric microbes; however as far as we know, there are no
reports in which the combination of these two microorganisms had been tested.
Our results show a significant ecological potential of some components of the wheat seed
endophytic community. Seed endophytic microorganisms probably play important roles during
plant development; in fact, they often possess attractive characteristics that could turn into
biotechnological applications, including biological control. New inoculants based on spermospheric
communities may not only improve cereal yields, they may also contribute to reduce the incidence
of fusariosis, and combinations of strains are usually tested. However, more research is needed to
shed light on the determinants which define seed colonisation. For instance, it is necessary to
understand if seed endophytes are actively selected by their host plants to their own benefits or if
seeds are just a biological vehicle to survive and eventually colonise new habitats. Our
understanding of microbe interactions at seed stage and their biological relevance may just be
352 353 354 355 14
This work was supported by the University of Buenos Aires (UBACYT 20020130200052BA).
María D. Groppa is researcher and Silvana Díaz Herrera is fellowship at the Consejo Nacional de
Investigaciones Científicas y Técnicas (CONICET).
We thank Ing. Agr. Alejandro Perticari (IMYZA-INTA) for providing the Azospirillum brasilense
Az39 strain used in this work.
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Legends to figures
Figure 1. Image illustrates colony morphotype of the representative endophytes isolated by
a culture-dependent protocol from seeds of wheat, cultivar 75 Aniversario, according to the
description in Materials and methods. Numbers identify the different isolates.
Figure 2. Siderophore production (A) and phosphate solubilisation (B) assays. Images are
representative of four replicated experimental units, obtained from three independent
Figure 3. Antagonism against Fusarium graminearum. A) Comparative effects of seed
endophytes (dual assay) and of their supernatants (diffusible substances) on the radial size
of Fusarium graminearum. Four replicated experimental units obtained from three
independent experiments were prepared. Bars represent standard error of the mean (SEM).
Asterisks indicate significant differences (P < 0.05). Three independent experiments were
carried out. B) Illustrative images showing F. graminearum growth in dual assays with
isolates 2 (left) and 5c (right).
Figure 4. Bioassays including wheat and barley kernels. Seeds of barley and wheat were
pretreated with diluted cell suspensions of isolates 5a, 5b, 5c and 2, and then exposed to F.
graminearum, as described in Materials and methods section. Left of each plate: barley
kernels; right: wheat kernels.
Figure 5. Biofilm formation assay by crystal violet staining (A). Absorbances at 550 nm
were normalised by total bacterial growth estimated by OD at 540 nm (B). Bars represent
standard error of the mean (SEM). Asterisks indicate significant differences (* P < 0.05;
** P < 0.01). Three independent experiments were carried out.
Figure 6. Inoculation assays. (A) Chlorophyll content of barley seedlings grown in a
growth chamber as described in Materials and methods section during 20 days. Treatments: 22
5c: isolate 5c inoculated at sowing, P: isolate 2 inoculated at sowing, 5c+Pi: isolate 5c+2, both
inoculated at sowing, 5c+P5: isolate 5c+2; the second one inoculated 5 days after sowing, C:
uninoculated. Different letters indicate significant differences. (B) Illustrative images
showing the results of in vitro compatibility assay. Left: Isolate 2 inoculated at the surface of the
plate and isolate 5c drop-inoculated; right: vice versa.
Table 1. Taxonomic features and some metabolic properties of isolated bacteria
1 2 5a 5b 5c 6
Gram Spore staining formation + + + + +
nd N Y Y Y Y
Growth in semisolid Nfb
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. 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 + -
Tetracycline (ppm) 50 100 -
Fungal colony diameter (mm)
A Biofilm formation (550 nm) C Az39 1 2 5a 5b 5c 6
Cellular growth (540 nm)
Chlorophyll content (µg g FW-1)
2000 1900 1800
1700 1600 C