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A combination of humic substances and

Herbaspirillum seropedicae inoculation
enhances the growth of maize (Zea mays
L.)
Dataset in Plant and Soil March 2013
Impact Factor: 2.95 DOI: 10.1007/s11104-012-1382-5

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Plant Soil
DOI 10.1007/s11104-012-1382-5

REGULAR ARTICLE

A combination of humic substances and Herbaspirillum seropedicae

inoculation enhances the growth of maize (Zea mays L.)
Luciano Pasqualoto Canellas & Dariellys Martnez Balmori &
Leonardo Oliveira Mdici & Natlia Oliveira Aguiar &
Eliemar Campostrini & Raul C. C. Rosa &
Arnoldo R. Faanha & Fbio Lopes Olivares

Received: 13 March 2012 / Accepted: 12 July 2012

# Springer Science+Business Media B.V. 2012

Abstract
Background Endophytic diazotrophic bacteria colonize several non-leguminous plants and promote plant
growth. Different mechanisms are involved in
bacteria-induced plant growth promotion, including
biological nitrogen fixation (BNF), mineral solubilization, production of phytohormones, and pathogen
Responsible Editor: Euan K. James.
L. P. Canellas : D. M. Balmori : N. O. Aguiar :
A. R. Faanha : F. L. Olivares (*)
Ncleo de Desenvolvimento de Insumos Biolgicos
para a Agricultura (NUDIBA), Universidade Estadual
do Norte Fluminense Darcy Ribeiro (UENF),
Av. Alberto Lamego, 2000,
Campos dos Goytacazes 28013-602 Rio de Janeiro, Brazil
e-mail: fabioliv@uenf.br
L. O. Mdici
Departamento de Cincias Fisiolgicas,
Universidade Federal Rural do Rio de Janeiro,
km7 BR 467,
Seropdica, Rio de Janeiro, Brazil
E. Campostrini
Laboratrio de Melhoramento Gentico Vegetal,
Universidade Estadual do Norte
Fluminense Darcy Ribeiro (UENF),
Av. Alberto Lamego, 2000,
Campos dos Goytacazes 28013-602 Rio de Janeiro, Brazil
R. C. C. Rosa
Embrapa Mandioca e Fruticultura,
Rua Embrapa, s/n.,
CEP 44380-000 Cruz das Almas, BA, Brasil

biocontrol. Herbaspirillum seropedicae is a broadhost-range endophyte that colonizes sugarcane, rice,

wheat, sorghum, and maize, and has been used as a
biofertilizer. Contrasting results between greenhouse
and field experiments have prompted efforts to
improve the consistency of the plant response to
microbial stimulation.
Aims The aim of this study was to evaluate the effect
of the presence of humic substances on inoculation of
maize (Zea mays L.) with H. seropedicae.
Methods Two experiments were conducted: one in the
greenhouse using sand and nutrient solution and the
other a field trial in soil with low natural fertility and
to which was applied N in the form of urea (50 kg ha1).
In the greenhouse, pre-emerging seeds were inoculated
with a solution of H. seropedicae (109 cells mL1) in the
presence of humic substances isolated from vermicompost (10, 20, or 30 mg CL1); in the field trial, bacteria
combined with humate were added as a foliar spray
(450 Lha1).
Results At early stages (7 and 45 days old) in the
greenhouse, the treatment activated plant metabolism including enhancement of plasma membrane
H+-ATPase activity, alteration of sugar and N metabolism, and greater net photosynthesis. The number of viable bacterial cells was higher in root
tissues when inoculation was in the presence of
soluble humic substances. Foliar application of
endophytic diazotrophic bacteria and humic substances increased maize grain production 65 %
under field conditions. These results show a promising

Plant Soil

use of humic substances to improve the benefit of

endophytic diazotrophic inoculation.
Keywords Zea mays L. . Endophytes . Diazotrophs .
Humates

Introduction
Current trends in agriculture are focused on enhancing
the efficiency of fertilizer use, since approximately
65 % of applied mineral nitrogen is lost from the
plantsoil system through gaseous emissions, runoff,
erosion, and leaching (Bhattacharjee et al. 2008;
Adesemoye and Kloepper 2009). The major part of
world mineral nitrogen use is for sustaining cereal
production. Enhancement of biological nitrogen
fixation (BNF) is particularly important for these crops
(Cocking 2003).
Biofertilizers using microbes can increase crop
growth through a combination of BNF, growth promoting by hormonal substances, increased availability
of soil nutrients, and disease control (Cocking 2003).
Endophytic diazotrophic bacteria (EDB) are one of the
most efficient plant growth-promoting bacteria and are
used as inoculants for non-leguminous plants; they
have proved to be an efficient source of N that can
partly substitute for urea in cultivation (Baldani et al.
2000). Boddey et al. (1995) suggested that 3060 N
ha 1 crop 1 may be obtained from sugarcaneassociated BNF. Recently, Taul et al. (2011) using
15
N-dilution techniques and Urquiaga et al. (2011) by
natural 15N abundance also verified a significant contribution of BNF (3560 %) to sugarcane. Roesch et
al. (2005, 2008) using microbiological, molecular
tools, and statistical inference found larger diazotrophic bacteria diversity on maize and verified a variable
yield response to inoculation. A number of greenhouse
and field experiments with inoculation of maize suggest the potential to stimulate maize growth and production by EDB (Estrada et al. 2005; Suman et al.
2005). However, one aspect required to realize the
potential of endophytes in agribusiness is increasing
the EDB delivery to host plants, since a general decrease in performance is observed when plants inoculated in pots are shifted to the field (Bhattacharjee et
al. 2008).
According to Kirchhof et al. (1997), rhizosphere
associated N2-fixing bacteria are distant from the main

source of plant assimilates and are in heavy competition with other microorganisms for root exudates.
Otherwise, the endophytic niche represents a suitable
environment for enhanced activity of beneficial bacteria with less biotic and abiotic restrictions. Ecological
studies involving endophytes point to the root systems
as a main site for colonization and establishment of the
interaction. Successful entry into the host plant by
endophytes is made through root tips, root cracks at
the point of emergence of lateral roots, injured sites on
the root epidermis, stomata apertures, and damaged
thichomes (James et al. 2002). James and Olivares
(1998) considered both the intercellular space and
the lumen of the xylem, collectively called the apoplast compartment, a suitable place for endophyte
location since it includes a constant supply of nutrients
and circulation systems for beneficial products from
the associated bacteria. The localization in these sites
favors the endophytic spreading into the whole plant
body, including further establishment in the aerial
parts of the host plant such as intercellular spaces
through the vegetative plant axis (James and Olivares
1998).
Seghers et al. (2004), studying the impact of agricultural practices on the maize endophytic community,
found that organic fertilization enhances the diversity
and the enrichment of endophytes with respect to other
communities.
Humic substances comprise a major part of organic
matter, and their influence on soil properties is well
known and could be used to improve microbial activity. In addition, humic substances can directly affect
root growth (Nardi et al. 2009), especially lateral root
emergence and proliferation of root mitotic sites
(Canellas et al. 2002). A mechanism for humic substance stimulation of root growth was proposed based
on the classical acid growth theory, describing an
auxin-like induction of protein synthesis and activation of the plasma membrane (PM) H+-ATPase in
maize roots (Canellas et al. 2002; Zandonadi et al.
2007). In fact, Quaggiotti et al. (2004) demonstrated
the induction by humic substances of the Mha2 gene,
which encodes one of the main P-type H+-ATPase
isoforms expressed in maize cell roots. This resembles
auxin-dependent activation (observed by DR5::GUS
gene reporter expression; Canellas et al. 2011) and
induction of de novo synthesis of the PM H+-ATPase,
enhancing apoplast acidification, which in turn is essential for activation of enzymatic cell wall plasticity

Plant Soil

(classical acid growth theory; Hager et al. 1991). Remarkable changes in root architecture, such as root
hair and lateral root emergence induction in nonleguminous plants by humic substances (Nardi et al.
1996; Canellas et al. 2010), may favor the fitness of
the bacteria plant interaction due the enhancement of
attachment and infection sites of the root. Additionally, a previous report had shown that, in some circumstances, when bacteria gain entrance into the plant
tissue, it could occur by cell wall-hydrolyzing
enzymes that support the process of bacteria invasion
and dissemination in the host (James et al. 2002).
These enzymes had increased activity under low pH,
which is compatible with H+-ATPase induction by
humic substances, which reinforce our hypothesis that
humic substances can make the delivery process of the
EDB more efficient to the host plant.
In this work, two studies were conducted. The
objective of the first was to verify the effects of humic
substances and EDB on some biometric and physiological parameters including root area, H+-ATPase
activity, plant metabolism, and leaf colonization using
a pot-based growth medium with maize seedlings
evaluated in early stages of cultivation. The second
study was a trial experiment with the objective of
evaluating foliar application of EDB and humic substances in a commercial maize crop using a low N
level (50 kg urea-N ha1).

was determined by the most probable number technique (MPN) by pellicle formation using three replications and expressed as the log of the cell number g1
root fresh mass after growth on JNFb N-free semisolid medium according to Dbereiner et al. (1995).
The bacteria suspension of H. seropedicae strain Z67
was grown on JNFb liquid medium with added NH4Cl
1 g L1 at 34 C for 16 h with shaking (150 rpm). Cells
were pelleted by centrifugation (4,000g for 15 min)
and resuspended in sterilized water at cell densities of
109 colony-forming units (cfu) mL1. The inoculant
was prepared diluting 200 mL of bacterial in 800 mL
of humic substances at pH 7.0 to produce a final
concentration of 10, 20, or 30 mg C per litre and final
bacteria concentration 5108 cells/mL.
Soluble humic substances were extracted from vermicomposted cattle manure with 0.1 M KOH at a 1:20
solidliquid ratio by mechanical shaking for 6 h. The
suspension was centrifuged at 5,000 g and filtered
through a Whatman #42 filter to give the humate.
The humate was dialyzed against water using a
1,000-Da cutoff membrane. Some humate characteristics were: organic matter on a dry weight basis,
36.2 gkg1; total humic substances, 25.8 g1; humic
acids, 12.8 gkg1; fulvic acids, 13.0 gkg1; pH 8.67;
electrical conductivity, 11.7 mS cm1; total nitrogen
content, 1.4 gkg1; total P content (as P2O5), 12.6 g
kg1; ash, 3 %. Humates were diluted to 10, 20, and
30 mg CL1 in ultrapure water to use in plant
bioassays.

Materials and methods

Pot-assay experiments
Microorganisms and humic substances
Herbaspirillum seropedicae strain Z67 was grown in
vials with complete JNFb semisolid medium. The
composition of JNFb-medium per liter is: malic acid
( 5 . 0 g ) ; K 2 HPO 4 (0.6 g) ; K H 2 P O 4 (1.8 g)
MgSO47H2O (0.2 g); NaCl (0.1 g); CaCl2 (0.02 g);
0.5 % bromothymol blue in 0.2N KOH (2 mL); vitamin solution (1 mL); micronutrient solution (2 mL);
1.64 % FeEDTA solution (4 mL); KOH (4.5 g). In
100 mL, the vitamin solution contained: biotin
(10 mg) and pyridoxol-HCl (20 mg); 1 L of the micronutrient solution consisted of: CuSO4 (0.4 g);
ZnSO47H2O (0.12 g); H3BO3 (1.4 g);
Na2MoO42H2O (1.0 g) MnSO4H2O (1.5 g). The
pH was adjusted to 5.8 and 1.9 gL1 of agar was
added (Olivares et al. 1996). The bacterial population

Maize seeds (Zea mays L., var. UENF 506-8), kindly

provided by Dr. Messias Gonzaga from Laboratrio de
Melhoramento Gentico Vegetal (LMGV), were
surface-sterilized by soaking in 0.5 % NaClO for
30 min, followed by rinsing and then soaking in water
for 6 h. Afterward, the seeds were sown in 2.0-L
Leonards pots filled with washed and sterilized sand
wetted with 1/3 strength Furlani nutrient solution
(mol L 1 : 3.527 Ca; 2.310 K; 855 Mg; 45 P;
587 S; 25 B; 77 Fe; 9.1 Mn; 0.63 Cu; 0.83 Mo; 2.29
Zn; 1.74 Na; and 75 EDTA) with the N content adjusted to a low concentration (100 mol L1 NO3 +
NH4). Six replicates were used in an randomized statistical design. After 1 week, the solution was changed
for one-half of the ionic force and after the subsequent
week replaced daily for total ionic force. At 5, 15, and

Plant Soil

25 days after planting (DAP), the maize seedlings

were treated with humates diluted with low N Furlani
nutrient solution at 10, 20,and 30 mg CL1 with and
without a suspension of the endophytic diazotrophic
bacteria Herbaspirillum seropedicae Z67 strain (B) at
109 cells number mL1. Six maize plants were collected at 7 and 45 days after treatment for growth analysis.
The fresh weight of roots and shoots were measured
and the superficial root area was estimated in three
replications by Delta-T scan software (Bouma et al.
2000). For the others, three replications were used for
further analysis.
Plasma membrane (PM) H+-ATPase activity was
measured in root vesicles preparation according to
the procedure of Canellas et al. (2002). Nitrate
reductase (NR), glutamine synthetase (GS), and
the total sugar, reducing sugar, and amino acid
contents were analyzed in 45-day-old plants. The
activity of NR was determined using 0.5 g of
intact tissues cut into 22 mm pieces and incubated for 30 min after vacuum infiltration in 5 mL
of a reaction mixture containing 50 mM phosphate
buffer (pH 7.5), 50 mM KNO3, and 1 % (v/v)
propanol. The NR reaction was terminated by taking 1 mL of the reaction mixture and adding 1 mL
of a solution containing 3 M HCl and 1 % (w/v)
sulfanilamide and 1 mL of a solution of 0.02 %
(w/v) N-(1-naphthyl)ethylenediamine. The NR activity was determined by measuring the absorbance
at 540 nm (Jaworski 1971). Enzyme extraction for
GS assay was carried out as described in Medici et
al. (2003). The plant tissue was reduced to powder
in a mortar and pestle with liquid nitrogen and
then homogenized (3:1 buffer volume:fresh weight
for leaves and 2:1 for roots) with 100 mM Tris
buffer (pH 7.8) containing 1 mM dithiothreitol,
10 % (v/v) glycerol and 0.02 % (w/v) insoluble
polyvinylpyrrolidone. The crude extract was centrifuged at 15,000g for 30 min at 4 C. The GS
assay was started by adding 0.1 mL of supernatant
enzyme extract to 0.4 mL of a reaction mixture
containing 5 mol Tris buffer (pH 7.8), 25 mol
MgSO4, 2.5 mol hydroxylamine, 25 mol Lglutamate, 10 mol ATP. The reaction was incubated for 30 min at 30 C and terminated by
adding 0.7 mL ferric chloride reagent. After centrifugation at 1,000g, GS activity was determined
by measuring the absorbance at 540 nm. The carbohydrates were analyzed by enzymatic methods

using an ELISA procedure (Stitt et al. 1989). Total

amino acids were determined according to Cocking
and Yemm (1954).
Net photosynthesis (A, mol m2 s1), instantaneous transpiration rate (E, mmol m2 s1), and
stomatal conductance (gs, mol m2 s1) were determined by an LI-6200 automatic photosynthesis
analyzer (LI-COR, Lincoln, NE, USA). The photosynthesis data were obtained in total expanded
leaves at nine oclock. The chlorophyll content
was estimated by SPAD analysis (SPAD-502; Minolta,
Japan), with 10 data points for each plant. All
photosynthesis analysis used artificial light with
1,000 mol m2 s1.
Field experiment
A field experiment was carried out on an Ultisol
located at Maca, Rio de Janeiro, Brazil (4155.47
W, 2219S; altitude 14 m). The soil chemical properties of the 020 cm layer were analyzed according to
Embrapa (1997): pH (H2O)05.6; Al00.0 mmolc dm3
(titration against NaOH); Ca030.0 mmolc dm3 and
Mg014.0 mmolc dm3 (titration against EDTA);
P02.0 mg dm3 (Mehlich 1 extraction and colorimetric determination); K072.0 mg dm3 (Mehlich
1 extraction and flame photometric determination).
Total organic carbon and nitrogen contents were,
respectively, 0.6 and 0.04 %. The trial was set up
in a completely randomized design with four repetitions, using one application of humic substances isolated from vermicompost at 20 mg CL1, H. seropedicae
(109cells mL1), a combination of humic substances,
and H. seropedicae, and plants without bacteria
inoculation or humic acid application (control
treatment). The hybrid P6875 from DEKALB was
seeded at 120,000 ha1 at a low N supply (50 kg ha1 as
urea). The treatments consisted of bacterial suspension, humic substances at 20 mg CL1 or its combination which were applied at the same time as
foliar spray at 45 days after germination with a rate
of 450 Lha1 prepared as described for pot assay.
The plots consisted of four rows with 12.5 m long,
with plants spaced 0.2 m apart within rows and only
the two central rows were considered. The grain
production was expressed in kg ha1. The control
treatment consisted of plants without application of
bacteria suspension, humic substances, or a combination
of both.

Plant Soil

Results
In the pot assay, inoculation with H. seropedicae or
addition of soluble humates and the combination of
both (bacteria and humates) increased maize root area
related to the control with exception for sole bacteria
inoculation (B) and its combination with humate at
10 mg CL1 (B+10) evaluated at plantlets stage after
7 days germination (Fig. 1). The results were normalized with respect to control plants (defined as 100 %);
at 7 days, seedlings treated only with humate at
30 mg C L1 (30) or with 20 or 30 mg C L1 of
humates together with H. seropedicae (B+20 and B
+30) showed values above 200 % stimulation of root
surface area. At 45 days, all treatments, even at
10 mg CL1 of humates, showed highest root area
compared with untreated control plants (Fig. 1).
The estimation of the diazotrophic bacteria population associated to roots in the pot assay was performed
in two maize developmental stages (Fig. 2). For all
treatments, except 30 mg CL1 of humates at 45 days,
the number of bacteria was higher in fresh root tissues
compared with uninoculated plants at 7 and 45 days.
The comparison between sole bacteria (B) and its
combination with increased rates of humates (B+10,
B+20, and B+30) reveal that humic substances enhanced the number of viable H. seropedicae cells that
were plant-associated specially at 20 mg C L1 at
45 days, whereas a sharp reduction of the size population at 30 mg CL1 of humates was noted (Fig. 2).
The effects of these treatments on the PM H+ATPase activity of isolated root vesicles at 7 and 45
days are shown in Table 1. The stimulation over
Fig. 1 Root superficial area
stimulation of maize seedlings at 7 () and 45 ()
days after treatments in the
pot assay. Treatments: control plants (C), humic substances at 10, 20, and
30 mg CL1 (10, 20 and 30),
109 cells mL1 of Herbaspirillum seropedicae (B) and
bacteria plus 10, 20, and 30
mg C humic substances L1
(B+10, B+20, and B+30,
respectively). The values
represent the mean standard
deviation

control plants was 55109 % at 7 days and 3896 %

at 45 days. As expected, induction of PM H + ATPase activity by treatments was higher for 7- than
45-day seedlings, which showed average values of
2.55 and 1.28 mol Pi mg prot1 min1, respectively.
A non-significant effect between different concentrations of humate was observed for both harvest times,
but when humate was combined with bacteria inoculation, B+20 treatment was superior compared with B
+10 treatment at 45 days (22 % stimulation). It is
interesting to note that all combined treatments (humate + bacteria) induced a significant stimulation of
the PM H+-ATPase activity in relation to the sole
bacteria inoculation, showing an average effect, respectively, ranging from 42 and 36 % for 7 and 45 days
(Table 1).
Chlorophyll content of maize leaves was indirectly
evaluated by SPAD number at 45 days after germination, being higher than controls only for treatments
combining inoculation with H. seropedicae at 10 or
20 mg CL1 humates (Table 2). Free carbohydrate
content in leaf extracts was lower for humic substances at 20 and 30 mg L1, sole bacteria, and combined
treatments (B+10 and B+20), with the latter showing a
reduction of 62.5 and 50.5 % of free carbohydrate
content related to untreated control plants and plants
treated with 10 mg CL1 humates (Table 2). The total
amino acids content increased 70 % in fresh leaf
tissues of plants treated with H. seropedicae and
humates at 30 mg L1 compared to control, which
had shown no significant differences in relation to
other treatments in total amino acid content. Total N
content (mg N plant1) increased in all treatments

Plant Soil
Fig. 2 Number of bacterial
cells (log cells g1 fresh tissue) on leaves of maize
seedlings at 7 () and 45 ()
days. Treatments: control
plants (C), log 109 cells
mL1 of Herbaspirillum
seropedicae Z 67 (B) and
bacteria plus 10, 20, and
30 mg C humic substances
L1 (B+10, B+20, and B
+30, respectively). The values represent the mean
standard deviation

(except 30 mg L1 of humate) in relation to control

plants with a range of 28 to 84 % (Table 2).
The effect of humate application or H.seropedicae
inoculation and its combination over activity of nitrate
reductase (NR) and glutamine synthetase (GS) in
leaves and roots of maize plants under greenhouse
condition at 45 days after germination can be noted
at Figs. 3 and 4. The treatment combining H. seropedicae and humic substances (B+10, B+20, and B+30)
induced NR activity in leaves (Fig. 3a) ranging between 120 and 230 % in relation to control (C) as well

as sole bacteria inoculation (B). Also, plants treated

with 10 mg L1 of humic substances (without bacteria)
showed a higher level of NR activity than control, and
no difference between bacterial inoculation and control was observed while humic substances at 30 mg C
L1 decreased NR activity in leaves (Fig. 3a). NR
activity in roots was lower and showed more variability than NR activity in leaves, but there was a noticeable effect from bacteria inoculation alone (B) and in
the presence of 10 and 20 mg CL1 (B+10 and B+20)
related to the control (Fig. 3b). At high concentration

Table 1 ATP hydrolysis by plasma membrane H+-ATPase

isolated from root vesicles of maize inoculated with Herbaspirillum seropedicae and humic substances isolated from
vermicompost in low N nutrient solution at concentrations

of 10, 20, and 30 mg CL1 and with 109 log cell mL1 H.
seropedicae Z 67 (Least Significant Difference at 5 % over
20 % stimulation)

Treatment

7-day-old
(mol Pi/mg protein min1)

Stimulation (%)

45-day-old
(mol Pi/mg protein min1)

Stimulation (%)

1.38

0.74

10

2.49

80

1.32

78

20

2.55

84

1.45

96

30

2.52

83

1.34

81

2.14

55

1.02

38

B+10

2.89

109

1.23

66

B+20

2.67

93

1.39

88

B+30

2.61

89

1.24

68

Treatments: C nutrient solution with low N content; 10, 20, and 30 nutrient solution with low N content supplemented with 10, 20 and
30 mg CL1 of humic substances, respectively; B nutrient solution with low N content supplemented with H. seropedicae Z 67 using
109 log cells mL1 ; B+10, B+20, and B+30 nutrient solution with low N content supplemented with H. seropedicae Z 67 using 109
log cells mL1 and 10, 20 and 30 mg CL1 of humic substances, respectively

Plant Soil
Table 2 Chlorophyll content (SPDA units), total carbohydrate and total amino acids of leaves and nitrogen content per plant
Treatment

SPAD

Carbohydrates
(mg g1 fresh tissue)

Total amino acids

(mol g1fresh tissue)

mg N plant1

33.90 b

18.20 a

0.60 bc

66 c

10

37.06 ab

19.79 a

0.33 c

119 a

20

36.90 ab

8.39 bc

0.59b

90 ba

30

34.10 b

10.08 bc

0.74ab

75 c

35.00 ab

8.19 bc

0.63b

110 ab

B+10

42.06 a

10.70 bc

0.70 b

85 b

B+20

41.63 a

6.82 c

0.67 b

88 b

B+30

39.43 ab

14.34 ab

1.02 a

122 a

CV
Significance.

6.77

15.09

11.49

P<0.05

P<0.01

P<0.01

11.53
P<0.01

C nutrient solution with low N content; 10, 20, and 30 nutrient solution with low N content supplemented with 10, 20 and 30 mg CL1
of humic substances, respectively; B nutrient solution with low N content supplemented with H. seropedicae Z 67 using 109 log cells
mL1 ; B+10, B+20, and B+30 nutrient solution with low N content supplemented with H. seropedicae Z 67 using 109 log cells mL1
and 10, 20 and 30 mg CL1 of humic substances, respectively
Means followed by different letters are different by Tukey test

of humic substances in combination with bacteria

(B+30), the NR activity was similar to the control
(Fig. 3b). In addition, a significant effect was observed
for sole humate application at 20 and 30 mg CL1
Fig. 3 In vivo nitrate reductase activity of leaves (a)
and roots (b) of 45-day-old
maize plants treated as controls (c) or with humic substances at 10, 20, and
30 mg CL1 (10, 20, and
30), log 109 cells mL1 of H.
seropedicae (b) and bacteria
plus 10, 20, and 30 mg C
humic substances L1
(B+10, B+20, and B+30,
respectively). The values
represent the mean standard
deviation

related to control plants, which do not differ from the

lower humate dose (10 mg CL1). GS activity was
stimulated only in treatments with a larger humate
concentration (30 mg CL1) in the presence of the

Plant Soil
Fig. 4 In vivo glutamine
synthetase activity of leaves
(a) and roots (b) from 45day-old maize plants treated
as controls or with humic
substances at 10, 20, and
30 mg CL1 (10, 20, and
30), log 109 cells mL1 of H.
seropedicae (b) and bacteria
plus 10, 20, and 30 mg C
humic substances L1
(B+10, B+20 and B+30,
respectively). The values
represent the mean standard
deviation

bacteria in leaves (Fig. 4a). For roots, it was observed

that an average GS-activity was eightfold more than
that for leaves, and for all humate concentrations used
a stimulation of GS activity related to control plants
was noted (Fig. 4b). For all treatments inoculated with
H. seropedicae, there was no significant GS activity
stimulation related to control (Fig. 4b).
The rate of net photosynthesis was significantly
enhanced for all treatments (H. seropedicae, humates,
and their combinations) with respect to control, except
B+10 treatment (Fig. 5a). The combination of bacteria
and humic substances increased the net photosynthetic
rate with the increase of humate concentration, while it
was not observed when comparing different rates of
sole humate application. For stomata conductance and
transpiration rate parameters, the same trend was
obtained with all treatments with significantly increased values related to control plants (except B
+10). In addition, a remarkable influence of the
humates at 10 mg CL1 was observed compared to
other humate doses and bacteria inoculation treatments
(Fig. 5b and c).
The effect of humate application or H.seropedicae inoculation and its combination over different

carbohydrate contents in maize leaves under greenhouse

conditions at 45 days can be found at Fig. 6. Glucose and
fructose contents were significantly higher for control
plants (Fig. 6a, b) with values ranging, respectively, from
3 to 12 and 2 to 10 times, compared to other treatments.
The sucrose content was similar for all treatments
(Fig. 6c). In contrast, the starch content of maize leaves
of the control plants decreased in comparison with other
treatments (Fig. 6d). Humate application at low dose
accumulated significantly more starch in leaves than
other treatments and no differences between bacteria
inoculation treatments were observed (Fig. 6d).
All the biometric and physiological parameters
evaluated above came from pot assay with maize
plants evaluated under greenhouse conditions at 7
and 45 days after inoculation. We designed a field
experiment with the aim of initiating the validation
of the proposed biofertilizer technology using the selected 20 mg CL1 of humate based on the overall
results obtained. The results obtained for grain production had shown a significant and positive effect of
the technology which combined both H. seropedicae
strain Z67 and humic substances over control plants
(65 % increased grain yields). Furthermore, the

Plant Soil
Fig. 5 Rate of net photosynthesis (a), stomatal
conductance (b) and transpiration (c) of 45-day-old
maize plants treated as controls (c) or with humic substances at 10, 20, and
30 mg CL1 (10, 20, and
30), log 109 cells mL1 of H.
seropedicae (b) and bacteria
plus 10, 20, and 30 mg C
humic substances L1
(B+10, B+20, and B+30,
respectively). The values
represent the mean standard
deviation

combined application was superior to the sole application of bacteria or humate with respective increase
of 45 and 48 % in grain production. In addition,
around 20 % positive increases over control plants
were observed for sole bacteria inoculation or
20 mg CL1 of humate application (Fig. 7).

Discussion
Gradual replacement of non-renewed energy source to
produce food, fibers, and energy is part of an underconstruction new paradigm for twenty-first century
agriculture. The increase of microbiological processes,

such as biological nitrogen fixation, phosphate solubilization,and the biostimulation process is particularly
important for key species of the family Poaceae (rice,
sugarcane, wheat, and corn), in view of the magnitude
of the planted area and its strategic importance as a
global food resource.
In Brazil, promising results have recently been
obtained with Azospirillum brasilensis inoculants for a
maize crop (Hungria et al. 2010). Besides the plant
bacteria genotypic screening program, innovative inoculation technologies are also needed to increase the
adoption of inoculants containing microorganisms that
promote plant growth. In the present work, we proposed
to evaluate a new biofertilizer concept based on the

Plant Soil
Fig. 6 Total content of glucose (a), fructose (b), sucrose (c) and starch (d) from
45-day-old maize plants
treated with liquid humus
diluted in nutrient solution
to a concentration and bacteria plus 10, 20, and
30 mg C humic substances
L1 (B+10, B+20,and B+30,
respectively). The values
represent the mean standard
deviation

combination of humic substances and an endophytic

diazotrophic bacterium Herbaspirillum seropedicae
and its effect on maize plant growth and physiology.
It is widely reported that plant growth responds to
vermicomposts and humic substances extracted from it
in several plant species (Arancon et al. 2004; Nardi et al.
2009). Canellas et al. (2002) treated maize seedlings for
7 days with different concentrations of humic acid isolated from vermicompost and showed stimulation of the
root elongation with consequent enhancement of the
root surface area. Similar results were obtained in the

present study for humate application and its combination with bacteria. Such promotion of root superficial
area (Fig. 1) by humates favored H. seropedicae colonization of plants, as shown by the large number of
bacterial cells on the fresh root tissues (Fig. 2). Since
the morphological changes of the root system triggered
by humic substances comprise increases of lateral root
formation sites, root hair density and length (Nardi et al.
2009), as well as overall surface area available for
bacteria attachment, we could expect an increase in the
population size plant-associated coupled with humate

Plant Soil
Fig. 7 Effect on maize
grain production (kg ha1)
in field experiments. Control
plants received 50 kg Nha1
as urea. Treatments consisted of one foliar application (300 Lha1) of humic
substances (20 20 mg CL1),
Herbaspirillum seropedicae
(B log 109 cells mL1) and
humic substances + H.
seropedicae (B+20)

application as shown in Fig. 2. In addition, Canellas et

al. (2008) and Puglisi et al. (2008) observed increased
efflux of organic acids exudates into the rhizosphere of
maize plants treated with humic acid, which could be
used as carbon substrate to support the bacteria population growth associated with the root system.
Herbaspirillum seropedicae is a diazotrophic endophyte bacterium that colonizes mainly graminaceous
plants such as sugarcane, rice, wheat, sorghum, and
maize (Olivares et al. 1996). James and Olivares
(1998) and more recently (Monteiro et al. 2012)
reviewed the infection process of H. seropedicae in
Poaceae root plant and points out that the association
starts with their attraction to the roots, as they provide
carbon sources for the bacteria, followed by attachment of the bacteria to root surfaces and the subsequent colonization of the emergence points of lateral
roots. Presumably, structural changes observed in
roots treated with humic substances could increase
the area available for attachment and the number of
entry sites contributing to an enhanced endophytic
establishment of the bacteria.
The observed increase in the root surface can be
linked with stimulation of PM H+-ATPase (Hager et
al. 1991). Such a stimulatory effect was noted for all
treatments related to untreated control plants (Table 1).
The central role of the PM H+-ATPase in ion uptake
and plant growth is well known, since these enzymes
provide an electrochemical gradient necessary to energize ion transport for cell uptake and induce cell
growth by a mechanism known as acid growth, in

which H+ acts as the intermediate between auxin and

cell wall loosening (Morsomme and Boutry 2000).
The H+-ATPase activity has been widely used as a
biomarker for bioactivity properties of humic substances (Zandonadi et al. 2007; Nardi et al. 2009; Canellas
et al. 2010). Although less studied, inoculation with
plant growth promoting bacteria could also lead to the
plasma membrane proton pumps stimulation (Bashan
and Levanony 1991) enhancing nutrient uptake, which
is the most commonly proposed explanation for the
beneficial effects of plant growth-promoting bacteria
(PGPB) on plant growth.
Although there are no experimental data showing
whether humic substances and PGPB effects on root
morphogenesis share common steps, apparently the
mode of action of both could be partially attributed
to the release of auxin and its relationship with PM
H+-ATPase activity (Muscolo et al. 1998; Zandonadi
et al. 2007). Canellas et al. (2002) reported that humic
acids isolated from vermicompost possess auxin-like
activities that induce H +-ATPase synthesis and
growth of maize roots, and Herbaspirillum seropedicae can produce in vitro the IAA (indole acetic
acid) phytohormone (Radwan et al. 2002).
It is interesting to note that the humic substance
suspension used in the present work at 10, 20, or
30 mg CL1 concentration combines a dual property,
acting per se as a plant growth promoter and as a
source of stabilized organic matter exhibiting hydrophilic and hydrophobic domains under supramolecular
arrangement (Picollo 2002), which is available for

Plant Soil

physical interaction and compartmentalization of bacteria suspension cells (Canellas et al. 2011). Humic substances in solution are a set of relatively small
molecules, which are loosely bound by intermolecular
hydrophobic interactions (Piccolo 2002). Humic substances have a high apparent molecular weight and their
hydrophobic interior may protect labile compounds,
biomolecules, and microorganisms from biodegradation, enhancing their persistence (Spaccini et al. 2002).
Furthermore, organic acids can dissociate humic substances into low and high molecular weight fractions
(Canellas et al. 2008), and bioactive molecules such as
indoleacetic acid may be released from humic substances upon dissociation of the parent material (Nardi et al.
2009). We speculate that humic solutions provide a
more favorable environment for EDB, perhaps due to
an irregular surface that can anchor cells and promote
biofilm development at the plant interface.
We also assessed the influence of the humate application and bacteria inoculation on the complex relationship between carbon and nitrogen metabolism in
maize plants grown under greenhouse. The observed
changes in leaf contents of chlorophyll, total carbohydrate, free amino acids, and nitrogen (Table 2) related
to control plants evoke physiological responses related
to photosynthetic capacity and N-uptake/assimilation.
Humic substances may promote plant growth
through the induction of carbon and nitrogen metabolism (Nardi et al. 2009). The roles of humic substances
in basic plant physiology have been extensively studied, but little is known about the beneficial bacteria. In
one remarkable study performed by Chi et al. (2005),
rice plants inoculated with different endophytic rhizobial species showed increased photosynthetic rate,
stomatal conductance, transpiration velocity, water utilization efficiency, and flag leaf area, and accumulated
higher levels of indoleacetic acid and gibberellins
growth-regulating phytohormones.
Leaf total carbohydrate content decreases around
5354 % compared to those of control plants following inoculation with bacteria or a 20-mg CL1 humate
solution, and except at a higher humate concentration,
a significant reduction in total carbohydrate content
was found in co-inoculation treatments (Table 2).
While glucose and fructose content decreased
(Fig. 6a, b) following treatments, an increase was
observed in starch content (Fig. 6d). Humic substances
induce carbon and nitrogen metabolism by increasing
the activity of enzymes involved in glycolysis, the

Krebs cycle, and N assimilation (Quaggiotti et al.

2004). Nardi et al. (2009) reported that humic substances can inhibit the activity of glucokinase, phosphoglucose isomerase, aldolase, and pyruvate kinase,
enzymes involved in glucose metabolism. Nardi et al.
(2007) showed that the effect of humic substances on
activities of enzymes related to the glycolysis pathway
in maize seedlings can be a stimulation or inhibition,
depending on the concentration and chemical features
of the humic materials.
Because total carbohydrate content as well as reducing sugar (with the exception of starch) decreased
following combined use of humate and bacteria, it is
possible that these metabolites can be used to sustain
growth and enhance N metabolism, since enzymes
linked to N assimilation were stimulated (Figs. 3, 4).
A previous report showed the promotion of different
humic substances on nitrate uptake and enzyme activity induction (Albuzio et al. 1986). In addition, Pinton
et al. (1999) showed that water-soluble humic matter
affected nitrate uptake and PM H+-ATPase activity in
maize roots in a similar way to that observed here
(Table 1; Fig. 3). As a consequence, the total N accumulated in plants was higher (Table 2). Conflicting
results have been observed related to N-assimilation
enzymes in maize plants inoculated with plant growthpromoting bacteria. Machado et al. (1998) showed a
stimulation of the glutamine synthetase activity of the
root and Reis et al. (2008) noted no effect of the
inoculation for GS and NR enzymes. However, the
increase of the nitrate reductase activity in leaves
treated with the combination humates and bacteria
suggest a much more effective uptake coupled with
translocation of nitrate to aerial part, which is close to
the sites of energy generation required for the extensive reduction of nitrate.
An enhancement of net photosynthesis in humate
application and bacteria-inoculated maize plants
(Fig. 5a) was also observed, despite no significant
changes in leaf chlorophyll content (Table 2), but we
found higher stomatal conductance in plants treated with
humates and inoculated with Herbaspirillum (Fig. 4b).
The first evidence of humic effects on stomatal opening
was provided by Russell et al. (2006). According to
these authors, activation of the PM H+-ATPase is a key
step leading to stomatal opening in response to a variety
of stimuli, including auxin. Since this enzyme has been
shown to be activated by humates (Table 1), it is possible
that co-inoculation would stimulate stomatal opening

Plant Soil

and transpiration (Fig. 5c). Finally, given the root growth

promotion, H+-ATPase induction, modification of carbohydrate and nitrogen metabolism, and enhancement
of net photosynthesis in maize seedlings co-inoculated
with H. seropedicae and humates, we expected an enhancement of maize grain production. We carried out
just one experiment in very low natural fertility soil
using 50 kg urea-N ha1. Despite a low level of grain
production (2.800 kg ha1) in control treatments, coinoculation enhanced maize grain production by around
65 %, in the high range of that expected for H. seropedicae crop stimulation (around 1520 % according to
Bhattacharjee et al. 2008). Despite just 1 year of trials,
these results indicate that co-inoculation of EDB with
humic substances may turn out to be an important biotool for low-input agriculture or increased efficiency of
nutrient use for sustainable crop production.

Concluding remarks
Inoculation of maize with H. seropedicae in the presence of humic substances isolated from vermicompost
induced lateral root emergence in the early stages of
plant growth and a clear stimulation of plasma membrane H+-ATPase. Lateral root proliferation favored colonization by H. seropedicae and changes in sugar and N
metabolism, resulting in a more efficient photosynthetic
process under low-N conditions. In a field experiment,
plants co-inoculated with H. seropedicae in the presence
of 20 mg CL1 of humic substances showed an increase
in grain production, suggesting a synergistic effect and a
promising way to expand the benefits of utilizing endophytic diazotrophic microorganisms.
Acknowledgements This work was part of D.B.M thesis and
was supported by the Conselho Nacional de Desenvolvimento
Cientfico e Tecnolgico (CNPq), Fundao de Amparo Pesquisa do Estado do Rio de Janeiro (FAPERJ) and International
Foundation of Science (IFS). Nudiba-UENF is a member of the
National Institute of Science and Technology for Nitrogen
Fixation (INCT Fixao Biolgica de Nitrognio). We also
grateful to CAPES/MES PEC-PG for the D.B.M doctoral
fellowship at Nudiba.

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