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Foliar application of manganese increases sugarcane resistance to orange rust

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Orange rust (Puccinia kuehnii) is a serious disease compromising the sustainability of sugarcane (Saccharum spp.) production. The objective of this research was to study whether supplemental manganese (Mn) supply through foliar spray ameliorates negative effects of orange rust on sugarcane and, if so, to reveal the underlying mechanisms. The experiment was conducted using a sugarcane variety susceptible to the disease; a single spray of Mn at 5.0 g L⁻¹ (Mn0.5%) and 10 g L⁻¹ (Mn1%), plus a control (Mn0%) was performed before pathogen inoculation. Symptom severity, antioxidant metabolism, lignin deposition and anatomical organization were evaluated. Photosynthesis was also measured in newly expanded leaves, and plants were harvested to estimate growth responses. The intensity of disease symptoms was reduced from 15.0% under Mn0% to 2.2% and 0.9% under Mn0.5% and Mn1.0%, respectively. This decrease was accompanied by increases in biomass production in the plants. Scanning and light microscopy images revealed that Mn treatment caused direct damage to the fungi spores and improved lignin deposition in the mesophyll. In the presence of the disease, Mn‐sprayed leaves exhibited lower levels of oxidative stress, in addition to improved structural organization of xylem and phloem vessels compared to the untreated control. The negative effects of orange rust on gas exchange and photochemistry were also ameliorated by Mn application. Our results add insights into the mechanisms underlying augmented sugarcane resistance to orange rust under supplementary foliar Mn spray and will contribute to the development of sustainable crop production systems by offering alternatives to reduce disease damage. This article is protected by copyright. All rights reserved.
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Foliar application of manganese increases sugarcane
resistance to orange rust
G. L. Mesquita
a
, F. A. O. Tanaka
a
*, F. C. B. Zambrosi
b
, R. Chapola
c
, D. Cursi
c
,
G. Habermann
d
, N. S. Massola Jr
a
, V. P. Ferreira
a
, S. A. Gaziola
e
and R. A. Azevedo
e
a
Departamento de Fitopatologia, Universidade de S~
ao Paulo (USP), PO Box 09, Piracicaba, SP, 13418-900;
b
Centro de Solos e Recursos
Ambientais, Instituto Agron^
omico (IAC), PO Box 28, Campinas, SP, 13012-970;
c
Rede Interuniversit
aria para Desenvolvimento do Setor
Sucroenerg
etico (RIDESA), Araras, SP, 13600-970;
d
Universidade Estadual Paulista (UNESP), PO Box 199, Rio Claro, SP, 13506-900; and
e
Departamento de Gen
etica, Universidade de S ~
ao Paulo (USP), PO Box 09, Piracicaba, SP,13418-900, Brazil
Orange rust (Puccinia kuehnii) is a serious disease compromising the sustainability of sugarcane (Saccharum spp.) pro-
duction. The objective of this research was to study whether supplemental manganese (Mn) supplied through foliar
sprays ameliorates negative effects of orange rust on sugarcane and, if so, to reveal the underlying mechanisms. The
experiment was conducted using a sugarcane variety susceptible to the disease; a single spray of Mn at 5 g L
1
(Mn
0.5%
)or10gL
1
(Mn
1%
), plus a control (Mn
0%
) was performed before pathogen inoculation. Symptom severity,
antioxidant metabolism, lignin deposition and anatomical organization were evaluated. Photosynthesis was also mea-
sured in newly expanded leaves and plants were harvested to estimate growth responses. The percentage diseased leaf
area was reduced from 15% under Mn
0%
to 2.2% and 0.9% under Mn
0.5%
and Mn
1%
, respectively. This decrease
was accompanied by increases in biomass production in the plants. Scanning and light microscopy images revealed that
Mn treatment caused direct damage to the fungal spores and improved lignin deposition in the mesophyll. In the pres-
ence of the disease, Mn-sprayed leaves exhibited lower levels of oxidative stress, in addition to improved structural
organization of xylem and phloem vessels compared to the untreated control. The negative effects of orange rust on
gas exchange and photochemistry were also ameliorated by Mn application. The results give insight into the mecha-
nisms underlying augmented sugarcane resistance to orange rust under supplementary foliar Mn spray and contribute
to the development of sustainable crop production systems by offering alternatives for reduction of disease damage.
Keywords: fungal disease, mineral nutrition, Puccinia kuehnii,Saccharum spp.
Introduction
Orange rust, caused by Puccinia kuehnii, is a widespread
fungal disease (Chavarr
ıa et al., 2009; Perez-Vicente et al.,
2010) that leads to significant reductions in sugarcane
(Saccharum spp.) yields and profits. Integrated control
measures are required to minimize such losses. A common
solution recommended for growers to cope with this biotic
stress has been the replacement of susceptible varieties by
resistant ones (Araujo et al., 2013); however, there appear
to be practical constraints that limit the successes of this
strategy when solely adopted under field conditions. For
instance, resistant varieties can become vulnerable to
orange rust over time because of the occurrence of differ-
ent races of the fungus (Moreira et al., 2018b). Moreover,
desirable characteristics that exist in susceptible varieties
and are necessary to promote the viability of sugarcane
production in diverse environments might be lost once
these genotypes are no longer planted. In this context,
alternative and complementary options for both reducing
orange rust severity in susceptible varieties and lowering
disease development in resistant ones could offer a greater
diversity of genotypes when planning varietal distribution
in farming systems.
Other than planting varieties resistant to orange rust, a
common option that might be adopted by growers is
chemical control with synthetic pesticides and there are
commercial products recommended for this purpose.
However, the excessive use of synthetic pesticides signifi-
cantly contributes to the presence of chemical residues in
the environment, reduces the profits of growers and cre-
ates more favourable conditions for the appearance of
fungicide-resistant races of pathogens (Ma & Michai-
lides, 2005). Moreover, the efficacy of chemical control
of orange rust in the field remains to be fully established
because there are uncertainties regarding the best timing
and frequency of fungicide application as well the level
of economic damage that justifies intervention in a given
area. Therefore, implementation of alternative strategies
favouring rational use of fungicides in integrated disease
*E-mail: fatanaka@usp.br
ª2019 British Society for Plant Pathology 1
Plant Pathology (2019) Doi: 10.1111/ppa.13041
management is necessary to sustain sugarcane production
profitability and to decrease environmental pollution.
One suitable option for reducing dependence on indus-
trial fungicides could be the spray of mineral elements
that are essential to plants and simultaneously assist in
reducing pathogen attacks. For example, the beneficial
effect of spray solutions with manganese (Mn)-based
products on reducing disease damage to cultivated plants
has been demonstrated (Hallen et al., 2007; Eskandari
et al., 2018). However, there is a lack of research evalu-
ating the effect of supplementary foliar Mn on resistance
of sugarcane to orange rust.
Research has shown that Mn may directly damage
pathogen structures (antimicrobial activity) and augment
the host defence capability through enhanced lignification
of leaf cells (Rengel et al., 1994; Fones & Preston, 2013).
However, it is unclear how the protective role of Mn fur-
ther influences leaf function or how it affects the overall
performance of infected plants. For instance, diseased
leaves exhibit increased oxidative stress and an impaired
anatomical organization (Apel & Hirt, 2004; Nogueira
et al., 2017) and so stimulation of resistance to orange rust
through Mn spraying might also ameliorate such damaging
effects on treated leaves. Additionally, sourcesink interac-
tions can be disturbed by pathogen infection (Biemelt &
Sonnewald, 2006) and so orange rust may have an impact
on potential sugarcane biomass production. Measurement
of the photosynthetic efficiency of symptomless top visible
dewlap leaves could provide an insight into this impact and
any effects of supplementary Mn sprays because the upper
leaves provide an important contribution to the supply of
carbohydrates (Marchiori et al., 2014).
The aim of this study was to investigate the effect of Mn
foliar spraying on the resistance of sugarcane to orange
rust fungus and to determine the mechanisms of any resis-
tance (such as fungistatic effect and stimulation of lignifi-
cation) and subsequent protection of the mesophyll
against oxidative stress and anatomical disorganization.
This was achieved by spraying plants of a susceptible sug-
arcane variety with Mn, inoculating them with orange rust
spores and evaluating disease severity, plant biomass, leaf
Mn concentration, lignification, antioxidant metabolism,
photosynthetic efficiency and mesophyll structure.
Materials and methods
Experimental conditions and treatments
The experiment was carried out in an unshaded glasshouse from
August to December 2016. The relative humidity of the air ran-
ged from 39% to 98% and the average minimum and maximum
air temperatures were 19 and 34 °C, respectively (registered
daily with a thermohygrometer). The experimental design was a
392 factorial conducted in randomized blocks with three
replications. Factors consisted of fungal disease at two levels
(inoculation and noninoculation with orange rust fungus spores)
and Mn application at three levels (0, 5 and 10 g L
1
spray).
Plants of a sugarcane variety (RB72-454) susceptible to orange
rust were obtained by planting culm segments in plastic trays con-
taining moistened vermiculite with calcium nitrate (1.5 mM).
Seven days after shoot appearance, the plants were fertigated with
a diluted nutrient solution at 25% of the final ionic strength twice
a week for 19 days. Homogenous and more vigorous plants were
selected and transplanted to plastic pots (one plant per pot) filled
with 5.1 kg of washed sand. During the first 10 days after trans-
planting, the plants were fertigated three times per week with a
complete nutrient solution diluted to 50% of the final ionic
strength. The full nutrient solution used in the experiment pre-
sented the following composition: (in mM) 12 N, 0.5 P, 3 K, 5 Ca,
1.3 Mg and 1.3 S; and (in lM) 41.6 B, 1 Cu, 46.7 Fe, 10 Mn, 1.25
Mo and 3.5 Zn (Zambrosi & Mesquita, 2016). The nutrient solu-
tion was prepared using the following salts: Ca(NO
3
)
2
.4H
2
O,
NH
4
NO
3
,KH
2
PO
4
, KCl, MgSO
4
.7H
2
O, H
3
BO
3
, CuSO
4
.5H
2
O,
C
10
H
12
N
2
O
8
FeNa.3H
2
O, MnSO
4
.H
2
O, (NH
4
)
6
Mo
7
O
24
.4H
2
O
and ZnSO
4
.7H
2
O. The plants were fertigated in excess every other
day during the entire experimental period.
The Mn treatments consisted of applying foliar spray solu-
tions containing 5 g L
1
Mn (Mn
0.5%
)or10gL
1
Mn (Mn
1%
)
prepared with MnSO
4
.H
2
O, plus a control exclusively receiving
a spray of deionized water (Mn
0%
). All spray solutions also con-
tained 0.5% mineral oil as a surfactant. The treatments were
applied once until the drip point during the morning at 50 days
after transplanting to the pots when the plants exhibited five or
six fully expanded leaves.
Sugarcane plants were inoculated with the fungus 1 week
after foliar Mn application. For this purpose, spores of the fun-
gus were collected under field conditions from plants with symp-
toms. Sugarcane leaves presenting pustules on the abaxial
portion were sampled 1 day prior to inoculation. In order to
accelerate sporulation, the leaves were maintained in plastic
bags containing moistened paper at room temperature in the
laboratory overnight. The spores of leaves were then removed
with a soft brush and mixed with deionized water. The spore
concentration was quantified under a light microscope, and
sequential dilutions were performed to obtain a concentration of
spores equivalent to 5 910
4
spores mL
1
with 85% viability
(Moreira et al., 2018a). Approximately 30 mL of the spore sus-
pension was sprayed onto the abaxial portion of the leaves of
each plant using a manual sprayer for regular distribution of
small droplets. In the first 24 h after inoculation, the plants
were maintained in a chamber equipped with air humidifiers
that were switched on for 15 min every 2 h to ensure optimal
conditions of temperature and humidity (Chapola et al., 2016).
After inoculation, the plants were returned to the glasshouse
and the two groups of plants (inoculated and noninoculated)
were separated by a barrier of transparent plastic to avoid con-
tamination of the noninoculated plants with the fungal spores.
The plants were allowed to grow for an additional 45 days until
harvesting and, during this period, they were not reinoculated.
The presence of sporulation was confirmed using a 910 hand
lens and the development of symptoms was monitored weekly
from the time at which the first symptoms appeared until the
termination of the experiment. The visual evaluation of the inoc-
ulated plants was based on a foliar chart scale for estimating the
percentage of leaf area with symptoms (Araujo et al., 2013;
Chapola et al., 2016). At the end of the experiment, disease
severity of the whole canopy (percentage of leaf area with symp-
toms per plant) was also estimated by calculating the ratio of
the leaf area with symptoms to the symptomless leaf area.
Plant growth and leaf tissue analysis
At the end of the experiment (i.e. 45 days after inoculation (dai)
with the fungus), the plants were harvested to estimate biomass
accumulation. The leaves were detached from the stalks and the
Plant Pathology (2019)
2G. L. Mesquita et al.
root system was separated from the sand by sieving and washing
with tap water. These plant parts were then rinsed in deionized
water, dried in an oven with forced air circulation (65 °C) for
3 days and weighed to quantify the dry mass (DM) production
of the shoots (leaves plus stalks), roots and whole plants.
The lignin concentration was quantified in leaves by the gravi-
metric method after extraction with acid detergent and cellulose
solubilization by sulphuric acid (Soest & Wine, 1968). The con-
centration of Mn was determined via inductively coupled
plasma optical emission spectrometry following digestion with
nitric-perchloric acid (Bataglia et al., 1983).
Scanning electron microscopy (SEM)
The characteristics of the leaf surfaces were evaluated at 43 dai.
Samples were obtained from the youngest fully expanded leaves
exhibiting symptoms of orange rust in infected plants and from
leaves of the same age from healthy plants. Tissue samples (ap-
proximately 20 mm
2
) from the middle third of the leaves were
cut during the morning from three different plants per treat-
ment, fixed in Karnovsky solution and postfixed for 1 h with
1% osmium tetroxide in 0.05 Mcacodylate. To investigate the
structure of the mesophyll, a subgroup of these fixed samples
was immersed in liquid nitrogen and fragmented with a blade as
described by Mesquita et al. (2016). Subsequently, all samples
were dehydrated in an acetone series of increasing concentration
(30%, 50%, 70%, 90% and 100% (three times)) and dried
until the critical point. These samples were subsequently
attached to stubs and both sides of the leaf surface were gold
coated (MC 50; Balzers). A scanning electron microscope (IT
300; Jeol) was then used for image analysis.
Light microscopy
The anatomical characteristics of the mesophyll were evaluated
in the same leaves sampled for scanning electron microscopy.
Tissue samples (approximately 20 mm
2
) were fixed in Kar-
novsky solution and dehydrated in an increasing ethanol series
(30%, 50%, 70%, 90%, and 100% (three times)). The dehy-
drated samples were infiltrated with acrylic resin glycol-
methacrylate (Leica 1) and 100% ethanol at a ratio of 1:1 for
polymerization and then immersed in pure resin until solidifica-
tion. The obtained blocks were cut on a manual rotatory micro-
tome (RM 2235; Leica) with a C-type stainless steel blade
(Wetzlar; Leica). The sections were stained with 0.05% tolu-
idine blue at pH 2.6 after drying and then mounted on glass
slides. Analyses were performed using a light microscope (Axios-
kop 2; Zeiss), and the images obtained with a camera (MRc;
Zeiss) were recorded as digital files.
For microscopic evaluation of the pattern of lignin deposition
in the mesophyll, resin-embedded samples were sectioned with the
microtome, as described above. These sections were placed in a
phloroglucinol solution for 10 min. Subsequently, drops of con-
centrated hydrochloric acid were added to the sections and they
were immediately examined under a light microscope (Axioskop
2; Zeiss). The degree and location of lignin deposition in the mes-
ophyll were estimated based on the intensity of the red colour
formed from the reaction of lignin with phloroglucinol.
Leaf protein and antioxidant metabolism
On the same day of sampling for the microscopy analyses, the
remaining tissue of the same leaf was immediately frozen in
liquid nitrogen. All leaf tissue was ground in liquid nitrogen and
stored in plastic tubes at 80 °C until further analyses. For pro-
tein quantification, 1 g fresh ground leaf tissue was extracted
with 100 mMpotassium phosphate buffer at pH 7.5, containing
1mMethylenediaminetetraacetic acid, 3 mMDL-dithiothreitol
and 4% insoluble polyvinylpolypyrrolidone (Azevedo et al.,
1998). Lipid peroxidation in the leaves was evaluated by deter-
mining the presence of malondialdehyde (MDA; Cakmak &
Horst, 1991), and the concentration of H
2
O
2
was assessed
according to Alexieva et al. (2001) using a standard curve with
known concentrations of H
2
O
2
. The activities of the enzymes
superoxide dismutase (SOD), catalase (CAT) and ascorbate per-
oxidase (APX) were quantified using the methods described by
Azevedo et al. (1998).
Leaf gas exchange and chlorophyll afluorescence
Measurements of leaf gas exchange and chlorophyll afluores-
cence were taken in the newest fully expanded symptomless
leaves from all treatments immediately before harvesting the
plants (at 45 dai). Leaf CO
2
assimilation (A;lmol m
2
s
1
),
transpiration (E; mmol m
2
s
1
), stomatal conductance (g
s
;
mol m
2
s
1
) and intercellular CO
2
concentration (C
i
;
lmol mol
1
) were measured between 09:00 and 12:00 with an
infrared gas analyser (LI-6400xt; Li-Cor). The instantaneous
carboxylation efficiency (k) was estimated as A/C
i
, and the pho-
tosynthetic water use efficiency was calculated as A/E. These
measurements were conducted under PAR conditions of
2000 lmol m
2
s
1
with an air CO
2
concentration of
400 lmol mol
1
and natural variations in air temperature and
humidity. Chlorophyll afluorescence was measured simultane-
ously with gas exchange in light-exposed leaves, with a model
LCF-Li-Cor 6400-40 fluorometer (Li-Cor) attached to the LI-
6400xt system. The effective quantum efficiency of photosystem
II (ΦPSII) was calculated as ΔF/Fm0=(Fm0F0)/Fm0, where
Fm0and F0are the maximum and steady state fluorescence sig-
nals in light-adapted leaves, respectively. The apparent electron
transport rate (ETR) and photochemical quenching (qP) were
estimated according to Roh
acek (2002).
Statistical analysis
The results were analysed through factorial analysis of variance
(ANOVA; SAS v. 9.1; SAS Institute). When a significant
(P<0.05) two-way interaction between the study factors (Mn
concentration in the spray solution versus inoculation or not
with orange rust) was found, foliar Mn treatments were com-
pared via Tukey’s multiple range test (P<0.05) for a given
inoculation condition, and disease treatments were compared
using the Ftest (P<0.05) within each Mn concentration.
Results
Sugarcane performance
The effect of orange rust on the biomass production of
sugarcane depended on the foliar Mn treatments, as sug-
gested by a significant interaction between the studied
factors (Table 1; Fig. 1AC). For instance, in the pres-
ence of the fungus, the shoot, root and total DM produc-
tion of the plants responded positively to Mn spray; the
difference in these growth parameters between Mn
0%
Plant Pathology (2019)
Manganese and resistance to orange rust 3
and the plants receiving foliar application of the metal ran-
ged from 20% to 75%. In contrast, in healthy plants there
were reductions in root DM under Mn
0.5%
and Mn
1%
and
in both shoot and total DM production under Mn
1%
com-
pared with Mn
0%
. DM partitioning was also influenced by
the tested treatments: the root:shoot DM ratio was lower
in inoculated than noninoculated plants under Mn
0%
, but
an opposite result was found with Mn spray; moreover, the
values of this parameter increased under Mn applications
compared with the control in inoculated plants (Fig. 1D).
Although Mn treatment improved the relative growth of
diseased sugarcane, the presence of orange rust fungus
caused a pronounced negative effect on the overall perfor-
mance of the plants, i.e. the average relative total DM pro-
duction of infected plants compared to healthy plants
across Mn treatments was equivalent to 54%.
Plant nutritional status and leaf lignin concentration
Leaf Mn concentration exhibited a significant interaction
for Mn spray and disease inoculation treatments
(Table 1). There was an increase in the concentration of
Mn in the sprayed leaves in both noninoculated and
inoculated plants compared with Mn
0%
(Fig. 2A); these
increments corresponded to 6.4- and 14.1-fold increases
for Mn
0.5%
and Mn
1%
, respectively. While orange rust
did not affect leaf Mn concentration in both Mn
0%
and
Mn
0.5%
plants, leaf Mn concentration was 14% higher
in infected plants under Mn
1%
.
Table 1 Analysis of variance (mean squares) for responses of
sugarcane plants treated with foliar manganese (Mn) spray and
inoculated or not with orange rust fungus spores (Puccinia kuehnii)
Parameter
Factor
Mn Inoculation Mn 9Inoculation
SDM 1.7 n.s. 24126.7** 2590.8**
RDM 52.8** 3443.2** 3564.7**
TDM 373.6* 66126.7** 7380.9**
Root:shoot ratio 267.5** 23.2* 76.4*
Leaf Mn 5582.7** 941516.2** 5705.5**
Leaf lignin 1358.1** 47.7* 17.7*
SOD 9849.0** 979.3** 777.2**
CAT 49925.9** 21622.1** 9119.2**
APX 87.9** 56.6** 9.26*
H
2
O
2
217.0** 1243.9** 901.9**
A95.7** 604.9** 18.5*
g
s
51.4** 423.2** 28.1**
k1064.3** 616.8** 119.6**
qP 21.7** 95.1** 12.2*
ETR 1185.4** 915.3** 152.9**
PSII 46.1** 22.4** 94.6**
SDM, shoot dry mass; RDM, root dry mass; TDM, total plant dry mass;
SOD, superoxide dismutase activity; CAT, catalase activity; APX,
ascorbate peroxidase activity; H
2
O
2
, leaf peroxide concentration; A,
CO
2
assimilation; g
s
,stomatal conductance; k, instantaneous carboxy-
lation efficiency; qP, photochemical quenching; ETR, electron transport
rate; PSII, effective quantum efficiency of photosystem II.
*
,
**and n.s. indicate P<0.05, P<0.01 and P>0.05, respectively.
Figure 1 Effects of foliar application of
supplementary manganese (Mn) to
sugarcane plants, inoculated or
noninoculated with orange rust spores, on
shoot dry mass (A), root dry mass (B), total
dry mass (C) and root:shoot dry mass ratio
(D), assessed 45 days after inoculation. For
comparison of Mn spray concentrations,
columns with different letters within the same
inoculation condition are significantly
(P<0.05) different based on Tukey’s
multiple range test. For comparison of
disease treatments, the presence of an
asterisk for a given Mn concentration
indicates that noninoculation and inoculation
conditions are significantly (P<0.05)
different based on the Ftest.
Plant Pathology (2019)
4G. L. Mesquita et al.
The effect of orange rust on leaf lignin concentration
was found to be dependent on the Mn rate (Table 1).
In the absence of inoculation, there was no difference
in the concentration of lignin with increasing rates of
Mn (Fig. 2B). However, in the presence of the fungus,
the highest Mn rate was associated with a lignin con-
centration that was 47% higher than that with Mn
0%
and did not differ significantly from the concentration
under Mn
0.5%
. In addition, the lignin concentration in
plants without Mn spray but inoculated with fungal
spores was 21% lower than that in healthy plants
(Fig. 2B).
Disease symptoms, the ratio of the symptomatic to
symptomless leaf area, and SEM observations
The leaves of the noninoculated plants remained green,
without showing any clear evidence of lesions on the
leaf surface, regardless of the Mn rate. In contrast, the
infected sugarcane exhibited the typical symptoms of
orange rust, with uredinial lesions that became chloro-
tic and necrotic spots across the foliar lamina (Fig. 3).
The first symptoms appeared approximately 18 dai, but
at this stage the presence of spores and symptoms was
not observed on the new leaves formed after inocula-
tion. By the end of the experiment (45 days), the
mother shoot of the inoculated plants exhibited four
or five fully expanded leaves showing symptoms of
orange rust. The visual evaluation of these leaves
revealed an estimated severity of 15.0%, 2.2% and
0.9% in the Mn
0%
,Mn
0.5%
and Mn
1%
treatments,
respectively (Fig. 3). Moreover, based on the ratio of
leaf area with symptoms to leaf area without symp-
toms, orange rust intensity at the canopy level corre-
sponded to 6.9%, 0.8% and 0.3% of the whole leaf
area for Mn
0%
,Mn
0.5%
and Mn
1%
treatments, respec-
tively (Fig. 4).
The SEM images confirmed the presence of fungal
spores on the leaf surface of the inoculated plants at
43 dai (Fig. 5ac). However, with visual estimation, a
greater abundance of spores was detected on plants trea-
ted with Mn
0%
than on those treated with foliar Mn. In
addition, some spores exhibited an irregular shape on
plants sprayed with Mn (Fig. 5b,c); these responses were
more pronounced under Mn
1%
than under Mn
0.5%
.
Anatomical study of the leaves
Sugarcane plants that had not been inoculated or trea-
ted with Mn exhibited a well-structured mesophyll
with regularly shaped parenchyma and vascular bundle
cells (Fig. 6a,g). In contrast, the noninoculated plants
subjected to Mn
1%
showed degeneration of scle-
renchyma fibres and deformation of the xylem vessels
(Fig. 6c,i). In the inoculated plants, significant alter-
ations of the leaf structure were observed, and these
responses varied with the Mn rate. For example, the
Mn
0%
plants showed degeneration of epidermal and
parenchyma cells, collapse of sclerenchyma fibres and
disorganization of xylem and phloem vessels (Fig. 6d,j).
In contrast, both Mn treatments promoted an
enhanced integrity of the mesophyll, as demonstrated
by the better structure of xylem and phloem vessels
and well-organized sclerenchyma fibres (Fig. 6e,f,k,l).
The microscopic evaluation of lignin deposition in the
mesophyll revealed a similar pattern of lignification
around the sclerenchyma fibres and bundle sheath in
noninoculated plants across all Mn treatments (Fig. 7a
c). In contrast, the infected plants that received either
Mn
0.5%
or Mn
1%
exhibited more intense lignification
around the sclerenchyma fibres and bundle sheath than
those in the Mn
0%
control treatment (Fig. 7df). Further-
more, comparing noninoculated and inoculated plants, a
lower lignification intensity around the sclerenchyma
fibres and bundle sheath was observed in the diseased
plants for the Mn
0%
treatment.
Figure 2 Effects of foliar application of supplementary manganese
(Mn) to sugarcane plants, inoculated or noninoculated with orange rust
spores, on concentrations of Mn (A) and lignin (B) in the leaves of
plants. Plants were analysed 45 days after inoculation. For comparison
of Mn spray concentrations, columns with different letters within the
same inoculation condition are significantly (P<0.05) different based
on Tukey’s multiple range test. For comparison of disease treatments,
the presence of an asterisk for a given Mn concentration indicates that
noninoculation and inoculation conditions are significantly (P<0.05)
different based on the Ftest. DM, dry mass.
Plant Pathology (2019)
Manganese and resistance to orange rust 5
Antioxidant response in leaves with symptoms
The protein and MDA concentrations were not signifi-
cantly (P>0.05) influenced by the study factors, show-
ing average values of 7.70 mg per g FW and 42.1 nmol
MDA per g FW, respectively. However, a significant
(P<0.05) interaction between Mn and disease treat-
ments was found for the enzyme activities and the con-
centration of H
2
O
2
(Table 1; Fig. 8AD).
Treatment with Mn promoted a pronounced increase
in SOD activity in the presence of the fungus, whereas
much lower variation was found in noninoculated plants
(Fig. 8A). The activity of this enzyme was greater in the
diseased plants, with the greatest difference between
inoculated and noninoculated treatments occurring under
the highest application of Mn (=41%). In contrast, CAT
and APX activities were lower in inoculated plants than
in noninoculated plants (Fig. 8B,C). The activities of
these enzymes were also reduced under Mn treatment
compared with Mn
0%
and this was more evident in dis-
eased sugarcane.
Foliar Mn application did not affect the H
2
O
2
concen-
tration in healthy plants, but in those plants infected
with orange rust, the concentration was reduced by 23%
and 30% under Mn
0.5%
and Mn
1%
, respectively, com-
pared with Mn
0%
(Fig. 8D). Moreover, Mn application
reduced the difference in the H
2
O
2
concentration
between inoculated and noninoculated plants; e.g. in the
Mn
0%
treatment, the H
2
O
2
concentration was 50%
higher in infected than in noninoculated sugarcane, but
no variation in concentration was detected between inoc-
ulated and noninoculated plants treated with Mn
1%
.
Leaf gas exchange and chlorophyll afluorescence of
symptomless top visible dewlap leaves
The effects of foliar Mn on the parameters of gas
exchange varied in sugarcane plants in the presence or
absence of orange rust (Table 1; Fig. 9AC). Under both
conditions, the plants sprayed with Mn exhibited greater
values of A(1225%) and g
s
(2341%) relative to plants
that did not receive micronutrient application, with
plants treated with Mn
1%
showing the greatest differ-
ences from the Mn
0%
control (Fig. 9A,B). The occur-
rence of the disease reduced Aby 19%, 31% and 37%
in the Mn
0%
,Mn
0.5%
and Mn
1%
treatments, respec-
tively. The g
s
values were also lower in the presence of
orange rust, except in the case of Mn
0%
plants. The
Figure 3 Effect of spraying sugarcane
leaves with supplementary manganese (Mn)
prior to inoculation with orange rust. The
youngest fully expanded leaves that
exhibited the symptoms were visually
evaluated 45 days after inoculation. Foliar
Mn treatments consisted of applying spray
solutions containing Mn concentration of
0.5% (Mn
0.5%
) or 1% (Mn
1%
), in addition to a
control with exclusive application of
deionized water (Mn
0%
).
Figure 4 Estimation of orange rust severity at the whole-canopy level
(ratio of leaf area with symptoms to leaf area without symptoms) of
sugarcane plants sprayed with supplementary foliar manganese (Mn)
and inoculated with orange rust spores. Plants were assessed 45 days
after inoculation. Columns with different letters are significantly
(P<0.05) different based on Tukey’s multiple range test.
Plant Pathology (2019)
6G. L. Mesquita et al.
highest value of kin noninoculated plants was observed
under Mn
0%
, and this parameter was reduced in plants
treated with foliar Mn (Fig. 9C). In the presence of the
fungus, the value of kwas higher in the plants treated
with Mn
1%
than with Mn
0%
or Mn
0.5%
, whereas in
healthy plants, treatment with Mn reduced k. Photosyn-
thetic water use efficiency and transpiration were not
influenced by the treatments, with average values corre-
sponding to 7.9 lmol CO
2
(mmol H
2
O)
1
and
5.2 lmol m
2
s
1
, respectively.
Effective quantum efficiency of photosystem II, ETR
and qP exhibited significant (P<0.05) interactions with
the Mn rates and inoculation conditions (Table 1;
Fig. 9DF). The greatest values of these parameters in
either the presence or the absence of the disease were
observed for Mn
1%
plants. Except for the effective quan-
tum efficiency of photosystem II in the Mn
0.5%
treatment
and ETR in the Mn
0%
treatment, orange rust caused
reductions in the values of the studied chlorophyll afluo-
rescence parameters under all other conditions and such
decreases ranged from 15% to 29%.
Discussion
In a challenging scenario demanding more sustainable
agricultural production, there is an urgent need to reduce
the damage from biotic stresses to crops, without aggra-
vating environmental pollution through intensive use of
synthetic pesticides. In this context, the results of this study
are promising for assisting the control of orange rust in
sugarcane. The plants inoculated with the pathogen that
received prior foliar Mn application exhibited improved
biomass production compared to those that were infected
but were not sprayed with Mn. For example, the Mn
0.5%
and Mn
1%
treatments increased whole-plant DM by 17%
and 34%, respectively, compared to Mn
0%
. Furthermore,
the relative growth of the infected plants compared to
those without inoculation or treatment with Mn showed
the following order: Mn
1%
>Mn
0.5%
>Mn
0%
. The bene-
ficial outcome of treatment with Mn products ameliorat-
ing the effects of diseases on crop plant performance
supports the potential use of Mn for disease control man-
agement (Simoglou & Dordas, 2006). To the best of the
authors’ knowledge, this is the first study to reveal that
foliar application of Mn improves the resistance of a sus-
ceptible sugarcane variety to orange rust fungus.
Although it has been consistently demonstrated that
Mn-deficient plants are more susceptible to fungal dis-
eases (Rengel et al., 1994), the findings reported here
indicate that the positive influence of the foliar treat-
ments on sugarcane resistance to orange rust could not
be directly related to correction of Mn deficiency. This
view is indeed supported by the results of leaf Mn con-
centration in the Mn
0%
plants, which presented values in
the range of properly nourished sugarcane (25
250 mg kg
1
; Raij et al., 1997). Moreover, despite the
significant increments in leaf Mn concentration, no
increase in plant DM production was found in noninocu-
lated plants sprayed with Mn compared to Mn
0%
.
Instead, there was a decline in plant growth under the
highest Mn rate, most probably because the leaf Mn con-
centration reached the threshold for toxicity and dis-
turbed leaf metabolism through disorganization of the
mesophyll (Zambrosi et al., 2016). According to these
results, it seems that more research is required to deter-
mine the optimum concentration of foliar Mn applica-
tion for disease management in field-grown plants.
Given that the foliar Mn spray was applied to Mn-suf-
ficient plants, how could the greater resistance to orange
rust be explained? Based on the well-defined negative
relationship between symptom severity and sugarcane
performance when inoculated with orange rust fungal
Figure 5 Surfaces of leaves of sugarcane sprayed with supplementary
foliar manganese (Mn) and inoculated with orange rust fungus spores,
observed via scanning electron microscopy. Arrows indicate loss of
the regular shape of spores. Foliar Mn treatments consisted of
applying spray solutions containing Mn concentrations of 0.5%
(Mn
0.5%
) or 1% (Mn
1%
), in addition to a control with exclusive
application of deionized water (Mn
0%
).
Plant Pathology (2019)
Manganese and resistance to orange rust 7
spores (Zhao et al., 2011), the improved growth of
infected plants after treatment with Mn might have been
a primary consequence of the decline in disease intensity.
Based on SEM images, this decrease in orange rust sever-
ity with Mn spray was associated with direct damage to
fungal structures at the leaf surface, as suggested by the
Figure 6 Cross-sections of leaves of sugarcane plants treated with supplementary foliar manganese (Mn) spray, followed by inoculation or not with
orange rust fungus spores. Leaves were sampled 43 days after inoculation and viewed by light microscopy (af) and scanning electron microscopy
(gl). Arrows indicate a disruption in the connection between the sclerenchyma fibres in the vascular bundle. Foliar Mn treatments consisted of
applying spray solutions containing Mn concentration of 0.5% (Mn
0.5%
) or 1% (Mn
1%
), in addition to a control with exclusive application of deionized
water (Mn
0%
). X, xylem; P, phloem; BS, bundle sheath; SF, sclerenchyma fibres.
Plant Pathology (2019)
8G. L. Mesquita et al.
presence of irregularly shaped spores. In addition to such
direct effects to fungal structures, Mn exerts inhibitory
action on the activities of enzymes produced by
microorganisms that are essential for fungal growth and
the invasion process, such as aminopeptidase and methy-
lesterase (Dordas, 2008). The observed reduction in
Figure 7 Lignin deposition around bundle sheath and sclerenchyma fibres in the leaf mesophyll of sugarcane plants treated with supplementary
foliar manganese (Mn) spray and inoculated or not with orange rust fungus spores. Leaves were sampled 43 days after inoculation and cross
sections were treated with phloroglucinol and hydrochloric acid and viewed under a light microscope. Foliar Mn treatments consisted of applying
spray solutions containing Mn concentration of 0.5% (Mn
0.5%
) or 1% (Mn
1%
), in addition to a control with exclusive application of deionized water
(Mn
0%
). X, xylem; P, phloem; BS, bundle sheath; SF, sclerenchyma fibres.
Figure 8 Activities of the enzymes (A)
superoxide dismutase (SOD), (B) catalase
(CAT) and (C) ascorbate peroxidase (APX)
and (D) the concentration of hydrogen
peroxide (H
2
O
2
) in the leaves of sugarcane
plants treated with manganese (Mn) spray
and inoculated or not with orange rust
fungus spores. Plants were sampled 43 days
after inoculation. For comparison of Mn
treatments, columns with different letters
within the same inoculation condition are
significantly (P<0.05) different based on
Tukey’s multiple range test. For comparison
of disease treatments, the presence of an
asterisk for a given Mn concentration
indicates that noninoculation and inoculation
conditions are significantly (P<0.05)
different based on the Ftest.
Plant Pathology (2019)
Manganese and resistance to orange rust 9
orange rust severity under Mn treatment might have also
been associated with augmented lignification of the bun-
dle sheath compared to Mn
0%
plants. Increased lignin
deposition confers efficient mechanical protection against
occurrence of diseases by restricting the action of patho-
gen enzymes during host cell wall degradation, limiting
the diffusion of resources from the host and functioning
as a barrier against the transport of toxins produced by
the pathogen (Vance et al., 1980; Barros et al., 2015).
This greater abundance of lignin after application of
foliar Mn is in accordance with the well-known partici-
pation of this metal in the activation of enzymes involved
in lignin synthesis pathways (Marschner, 1995). Such a
response under the additional supply of Mn, even in Mn-
sufficient sugarcane, suggests that internal demand for
this nutrient for the synthesis of lignin is greater in plants
inoculated with orange rust fungus spores, most proba-
bly because pathogens have the ability to oxidize Mn
and induce local deficiency (Thompson et al., 2006).
Considering that higher generation of reactive oxygen
species (ROS) compromises leaf functioning (Apel &
Hirt, 2004), it may be possible that the improved resis-
tance to orange rust after Mn treatment is linked to
more efficient antioxidant metabolism. This view is sup-
ported by the disease reduction being accompanied by a
lower oxidative stress, as revealed by a decrease in the
concentration of H
2
O
2
with foliar Mn application and
similar H
2
O
2
concentrations between inoculated and
noninoculated plants under Mn
1.0%
. The reduction in
oxidative stress with foliar Mn treatment was mostly
explained by higher SOD activity, as this enzyme acts in
the first line of defence against ROS by dismutating
excess superoxide ions produced under stressful condi-
tions and producing H
2
O
2
(Grat~
ao et al., 2005). Given
that the activities of CAT and APX were not increased
following Mn application, the observed reduction in
H
2
O
2
(despite an eventual increase in H
2
O
2
due to the
increased activity of SOD) was most probably associated
Figure 9 Effects of foliar manganese (Mn)
spray and inoculation or not with orange rust
spores on parameters of gas exchange and
chlorophyll afluorescence in sugarcane
plant leaves. (A) Net CO
2
assimilation (A),
(B) stomatal conductance (g
s
), (C)
instantaneous carboxylation efficiency (k),
(D) effective quantum efficiency of
photosystem II (ΦPSII), (E) apparent
electron transport rate (ETR) and (F)
photochemical quenching (qP).
Measurements were taken at 45 days after
inoculation. For comparison of Mn
treatments, columns with different letters
within the same inoculation condition are
significantly (P<0.05) different based on
Tukey’s multiple range test. For comparison
of disease treatments, the presence of an
asterisk for a given Mn concentration
indicates that noninoculation and inoculation
conditions are significantly (P<0.05)
different based on the Ftest.
Plant Pathology (2019)
10 G. L. Mesquita et al.
with the action of other peroxidases (Grat~
ao et al.,
2005).
The protective role played by Mn against distur-
bances of leaf function and its further contribution to
augmented disease resistance was also supported by
anatomical study of the leaves. For example, inoculated
plants treated with Mn
0.5%
or Mn
1%
exhibited the
absence of injuries to sclerenchyma fibres and an
improved structural organization of xylem and phloem
vessels compared to inoculated Mn
0%
plants. Indeed,
the structural damage observed in the mesophyll of
these Mn
0%
plants has been linked to hampered move-
ment of water and solutes in leaf tissues and subse-
quent impairments in overall plant performance
(Mesquita et al., 2016; Zambrosi et al., 2017).
Finally, it should also be noted that, despite the
enhanced resistance of sugarcane to orange rust
observed in plants treated with Mn, it remained evident
that the gains in plant growth did not occur in the
same proportion as the reduction in symptom severity.
Such lack of a close relationship between a decrease in
leaf area with symptoms and improvement in sugarcane
performance might occur because, under pathogen
attack, plants demand more energy to activate defence
mechanisms and there is a reduction of available sub-
strates for growth (Huot et al., 2014). Moreover,
despite the ability of Mn to improve the photosynthetic
efficiency of diseased plants compared to the unsprayed
control, the values of the leaf gas exchange and chloro-
phyll afluorescence parameters of the top visible dew-
lap leaves of infected plants were lower than those of
noninoculated plants. Indeed, disturbed carbon metabo-
lism in symptomless leaves and its further contribution
to an overall hampered performance of diseased plants
has been reported (Petit et al., 2006).
In conclusion, this study revealed that the application
of supplementary foliar Mn, even in Mn-sufficient plants,
could contribute to improving the resistance of a suscep-
tible sugarcane variety to orange rust. Such a response,
triggered by Mn spray, is probably associated with anti-
fungal activity and augmented lignin deposition. Supple-
mentation with Mn contributed to reduction of
symptoms and benefited the functioning of infected
leaves by reduction of oxidative stress and improvement
of the structural organization of the mesophyll. Further
investigation into the enhancement of lignification after
spraying with supplemental Mn is warranted to under-
stand how this response is regulated. Taken together,
these results suggest an important role for Mn as a pro-
tective agent for sugarcane against infection with orange
rust, providing a more environmentally friendly alterna-
tive for pathogen control in crop production areas.
Acknowledgements
This research was funded by the S~
ao Paulo State
Research Foundation (FAPESP-Brazil, grant 2016/
14058-9).
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... This effect was accompanied by increased sugarcane biomass production, and, in that system, Mn treatment caused direct damage to the fungal spores and improved lignin deposition in the mesophyll of the plants. During pathogenesis, Mn-sprayed leaves exhibited lower levels of oxidative stress, in addition to an improved structural organization of xylem and phloem vessels, as compared to the untreated control [39]. Since induced resistance may have a role in disease suppression by nutritional elements, further research should be directed towards this mode of action in various plant species. ...
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