382
AJCS 18(07):382-387 (2024) ISSN:1835-2707
https://doi.org/10.21475/ajcs.24.18.07.pne-29
Physiological and sanitary quality of sorghum seeds under the effect
of oxalic and salicylic acid
Gemerson Machado de Oliveira
1
, Jardel da Silva Souza
2
*, Alex Sandro
Bezerra de Sousa
3
, Mirelly Miguel Porcino
4
, Luciana Cordeiro do
Nascimento
5
, Eduardo Felipe da Silva Santos
5
, Matheus Siqueira de
Oliveira
2
, Sandra Helena Unêda-Trevisoli
2
1
Universidade Federal Rural de Pernambuco (UFRPE), Rua Dom Manuel
de Medeiros, s/n - Dois Irmãos, Recife - PE, 52171-900, Brasil
2
Universidade Estadual Paulista (UNESP)- Faculdade de Ciências
Agrárias e Veterinárias (FCAV) - Campus de Jaboticabal- Via de
acesso Prof. Paulo Donato Castellane s/ n - C.P. 14.870-900 -
Jaboticabal, SP- Brasil
3
Empresa de Assistência Técnica e Extensão Rural do Ceará, Brasil
4
Universidade Federal do Rio Grande do Norte, Escola Agrícola de
Jundiaí EAJ, RN 160, KM 03 - Distrito de Jundiaí CEP 59280-000 |
Macaíba/RN - Brasil
5
Universidade Federal da Paraíba (UFPB)- Centro de Ciências Agrárias
(CCA) - 12 Rodovia, PB-079, 58397-000- Areia - PB, Brasil
Abstract: Sorghum is one of the world's most
important cereal crops, but it is susceptible to several
diseases, particularly fungal diseases, with seeds
being the main vehicle for dissemination. The
Submitted:
28/09/2023
Revised:
25/04/2024
Accepted:
09/05/2024
Full Text PDF
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objective of this study was to evaluate the effects of
salicylic acid (SA) and oxalic acid (OA) on the
physiological and sanitary quality of sorghum seeds.
The seeds
of Sorghum bicolor
(L.) were immersed for
1 hour in solutions of OA (0.5, 1.0, 1.5, and 2.0 mM)
and SA (0.5, 1.0, 1.5, and 2.0 mM). A treatment with
Captan® fungicide was also used, and the control
group was immersed in distilled H2O for 1 hour.
Variables from seed vigor tests and fungal incidence
tests were evaluated. The control group showed the
highest germination rate. However, among the
treatments, the doses of 1 mM OA and 0.5 mM SA
resulted in higher germination in the first
germination count. The application of 1 mM OA and
both doses of SA (0.5 mM and 1 mM) promoted a
greater seedling vigor index (GVI). Seeds treated with
1 mM OA and doses of 0.5, 1, 1.5, and 2.0 mM SA, as
well as those treated with fungicide, had the highest
emergence rates, and with 1.5 mM SA, the highest
length of the longest root (LVEI). Seedlings from
seeds treated with 0.5, 1, and 1.5 mM SA and with
0.5 and 1.5 mM had the highest coleoptile length
(CPA). The 0.5 mM dose of SA reduced the incidence
of
Aspergillus flavus
, and the doses of 2 mM OA and
1, 1.5, and 2 mM SA reduced the incidence of
Rhizopus stolonifer
to 0%. SA provided the best
physiological and health effects for sorghum seeds,
making it the best treatment.
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Keywords: seeds, endophytic fungi, resistance induction.
Abbreviation: SA_Salicylic Acid; OA_Oxalic Acid; LAFIT_Laboratory Of
Phytopathology; EIV_Emergence Velocity Index; GVI_Germination
Velocity Index; APL_Aerial Part Length; RL_Root Length; FG_First
Germination Count.
Introduction
Sorghum,
Sorghum bicolor
(L.), a prominent member of the Poaceae
family, ranks as the fifth most important cereal crop globally and is
emerging as a promising feedstock for biofuel production (Zheng et
al., 2011). This crop is renowned for its adaptability to arid and semi-
arid environments where other crops struggle to thrive (Nida et al.,
2019). It is also notable for its growth potential and significant role in
agricultural systems in Brazil (Maia et al., 2010).
Sorghum is susceptible to a wide range of diseases, which can restrict
its cultivation and diminish yields. Among the diseases affecting the
crop in Brazil, the most significant are anthracnose (
Colletotrichum
sublineolum
), downy mildew (
Peronosclerospora sorghi
),
helminthosporium leaf blight (
Exserohilum turcicum
), rust (
Puccinia
purpurea
), ergot or sugar disease (Claviceps africana), and dry rot
(
Macrophomina phaseolina
) (Cota et al., 2010; Flavio et al., 2014).
Seeds are considered significant vectors for the dissemination and
transmission of numerous microorganisms, with fungi being the most
prevalent (Flavío et al., 2014; Cruz et al., 2018). The impact of seed-
associated microorganisms varies considerably, primarily depending
on the pathogens involved, the amount of initial inoculum, the specific
crop grown, and prevailing climatic conditions (Souza et al., 2011).
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Seed treatments are essential for controlling pathogens associated
with seeds, as well as those inhabiting the soil, storage of fungi, and
early leaf pathogens. Under field conditions, effective seed treatment
can ensure an adequate plant stand, promote vigorous plant growth,
delay the onset of disease epidemics, and increase yield (Moraes,
2010). To reduce the use of chemicals in managing phytopathogens,
research has focused on developing efficient alternatives that
minimize the harmful effects associated with conventional chemical
controls (Moura et al., 2018). Inducing resistance has emerged as a
promising alternative, seeking to manage diseases by applying biotic
and abiotic agents capable of activating the plants' innate defense
mechanisms (Iurkiv, 2008).
Pre-treatment with a non-lethal dose of oxalic acid has been shown to
activate defense mechanisms and significantly inhibit fungal growth
(Lehner et al., 2008). Jayaraj et al. (2010) demonstrated that the
application of oxalic acid enhanced rice resistance to
Rhizoctonia
solani
by increasing the accumulation of phenolics and defense-
related proteins, thereby offering new strategies for managing this
disease in rice crops.
Salicylic acid (SA) is an endogenous plant growth regulator and is part
of a diverse group of plant phenolics (Pandey et al., 2013). It was the
first plant-derived phenolic compound proven to induce systemic
acquired resistance (Araujo et al., 2005). Most phytohormones,
including SA, play crucial roles as defensive molecules within signaling
pathways, signaling pathogen recognition and activating defense
pathways that extend from the site of infection to distal tissues, thus
inducing systemic acquired resistance (Vicente & Plasencia, 2011; War
et al., 2011; An and Mou, 2011).
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Therefore, the objective of this study was to evaluate the effects of
salicylic acid and oxalic acid on the physiological and sanitary quality
of sorghum (Sorghum bicolor L.) seeds.
Results
Seed germination protocols and conditions
Sorghum seeds untreated (control) exhibited the highest germination
percentage at 84%. Lower doses of oxalic acid (OA) at 0.5, 1, and 2
mM, and salicylic acid (SA) at 0.5, 1, 1.5, and 2 mM enhanced seed
germination compared to the fungicide treatment and higher
concentrations of 1.5 mM OA and 2 mM SA (refer to Table 1). The
treatments with 1 mM OA and 0.5 mM SA achieved the highest
percentage of seed germination at the first count (FG), recording 43%
and 48% respectively, which were comparable to the control. Higher
concentrations of OA (1.5 and 2.0 mM) and SA (1, 1.5, and 2 mM)
were observed to decrease FG compared to their lower concentrations.
The fungicide treatment resulted in the lowest FG (see Table 1).
Additionally, the application of 1 mM OA and 0.5 mM SA facilitated a
higher initial germination speed (GVI) in sorghum seeds, comparable
to the control. In contrast, treatments with fungicide and 2 mM SA
resulted in the lowest GVI (refer to Table 1).
Percentage of seedling emergence
Sorghum seeds treated with 1 mM OA and with varying concentrations
of SA0.5 mM (47%), 1 mM, 1.5 mM (62%), and 2.0 mM (45%)along
with those treated with fungicide, exhibited the highest percentages
of seedling emergence. Notably, the 1.5 mM concentration of SA
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achieved the highest emergence velocity index (EVI), with seeds
treated with 1 mM SA recording the second-highest EVI (see Table 2).
Seedlings from sorghum seeds treated with SA at concentrations of
0.5 mM (5.41 cm), 1 mM (6.31 cm), and 1.5 mM (5.65 cm) and OA at
concentrations of 0.5 mM (6.36 cm) and 1.5 mM (5.35 cm) exhibited
the greatest lengths of the aerial parts (APL). The root lengths of the
seedlings showed no significant differences across the treatments
applied to the seeds (refer to Table 2).
Assessment of fungal incidence on seedlings
The 0.5 mM dose of SA reduced the incidence of
Aspergillus flavus
in
sorghum seeds but did not significantly differ from the fungicide
treatment. The doses of 2 mM OA and 1, 1.5, and 2 mM SA reduced
the incidence of
Rhizopus stolonifer
to 0%, which was comparable to
the results achieved with the fungicide treatment. Additionally, seeds
treated with 2 mM OA and doses of 0.5, 1, 1.5, and 2 mM SA showed
a reduced incidence of
Penicillium
sp., with no significant difference
from the fungicide treatment. No concentration of the regulators
proved effective in reducing the incidence of
Aspergillus niger
in the
seeds. The application of fungicide was the only treatment that
successfully reduced the incidence of this fungus.
Statistical analysis using ANOVA for experimental data
All treatments had significant effects on most of the dependent
variables, achieving significance at the 1% level (p < 0.01). This
indicates a very low probability that the observed differences in the
means of each treatment occurred by chance. The variable 'APL' (Aerial
Part Length) was significant at the 5% level (p < 0.05), which still
386
provides strong evidence against the null hypothesis that all
treatments have equal effects.
Discussion
Germination is influenced by seed vigor, as observed in seeds treated
with 1 mM OA (oxalic acid) and 0.5 mM SA (salicylic acid), which
characterize a well-developed plant. The longer it takes for seedlings
to emerge from the soil, the greater their exposure to adverse
conditions such as the presence of pathogens or inadequate substrate
temperatures (Gazola et al., 2013). Additionally, rapid germination is a
key component of seed vigor, generally correlating with faster
seedling emergence in the field (Marcos Filho, 2015).
The fungicide acts indirectly to increase seedling emergence and the
emergence velocity index (EVI); it is not intended to increase seed
viability. However, if low emergence is due to a fungal attack, efficient
treatment with fungicides will increase these characteristics (Pinto,
2002). Seedlings that emerge rapidly from seeds treated with 1 mM
OA, 0.5, 1.5, and 2 mM SA, and fungicides are more prominent than
other seedlings with lower development, as they better utilize the
resources of the medium (Gustafson et al., 2004).
Seeds treated with SA (salicylic acid) at concentrations of 0.5, 1.0, and
1.5 mM, and OA (oxalic acid) at 0.5 and 1.5 mM
Table 1. Germination (%), first count and germination velocity index (GVI) of sorghum (Sorghum bicolor) seedlings treated with
different concentrations of oxalic acid (0.5 - OA0.5; 1.0 - OA1.0; 1.5 - OA1.5; and 2 - OA2.0 mM) and salicylic acid (0.5 - SA0.5; 1.0 -
SA1.0; 1.5 - SA1.5; and 2mM - SA2.0 )
Trataments
Germination (%)
FG (%)
GVI
Witness
84.00±3.46a
42.50±2.60a
16.79±0.82a
Fungicide
60.00±2,89c
17.00±2.89d
10.64±0.84c
OA0.5
67.50±3,75b
34.00±3.46b
13.70±0.84b
OA1.0
75.00±0,58b
43.00±2.31a
15.59±0.36a
OA1.5
58.00±1,15c
32.00±1.15b
12.05±0.26c
OA2.0
71.00±4,62b
28.00±3.46b
13.45±1.05b
384
SA0.5
76.50±0,29b
48.00±1.73a
15.78±0.58a
SA1.0
72.50±3,75b
27.50±2.60b
14.08±0.83b
SA1.5
69.00±2,89b
32.00±0.00b
13.77±0.52b
SA2.0
59.00±0,00c
24.50±2.02c
11.44±0.25c
Means followed by the same letter in the column do not differ significantly from each other by the Scott-Knott test up to 5%
probability. Means ± standard error.
Table 2. Emergence (%), emergence velocity index, aerial part length (APL-cm), root length (RL-cm) of sorghum (Sorghum bicolor)
seedlings treated with different concentrations of oxalic acid (0.5 - OA0. 5; 1.0 - OA1.0; 1.5 - OA1.5; and 2mM - OA2.0) and salicylic
acid (0.5 - SA0.5; 1.0 - SA1.0; 1.5 - SA1.5; and 2 - SA2.0 mM)
Emergence (%)
EVI
APL (cm)
RL (cm)
30.00±1.9b
2.75±0.323c
4.51±0.25b
8.63±1.22a
49.00±1.65a
2.68±0.17c
4.55±0.28b
9.18±0.65a
31.00±1.01b
3.02±0.10c
6.36±0.41a
11.11±1.15a
51.00±0.40a
4.98±0.20c
4.61±0.23b
9.80±0.85a
43.00±1.01b
4.15±0.30c
5.35±0.37a
9.88±0.73a
41.00±0.77b
3.08±0.10c
4.44±0.33b
10.08±0.77a
47.00±0.77a
5.31±0.27c
5.41±0.32a
11.26±1.00a
41.00±3.99b
6.36±0.74b
6.31±0.56a
9.71±0.97a
62.00±1.90a
9.62±0.42a
5.65±0.40a
7.44±0.29a
45.00±2.30a
3.41±0.15c
4.61±0.40b
9.31±0.63a
Means followed by the same letter in the column do not differ significantly from each other by the Scott-Knott test up to 5%
probability. Means ± standard error.
exhibit increased vigor due to the induction of resistance. This
resistance enables them to mobilize reserves from storage tissues to
the embryo axis more efficiently, a capability that is reflected in the
growth of the seedlings, resulting in higher aerial part lengths (APL)
(Marcos Filho, 2015).
The fungi detected in this study are commonly reported in sorghum,
as noted by Flavio (2014). The application of SA triggers the induction
of response proteins (NPR1) and other defense genes, which enhance
plant resistance to pathogens (Yang et al., 2016). This mechanism
reduces the incidence of fungal infections. Sub-lethal concentrations
of oxalic acid (3 mM) can act as a pre-treatment to combat fungi, with
the 2.0 mM dose of OA being notably effective (Lehner et al., 2008).
Overall, fungicide treatments are expected to be highly efficient in
reducing pathogen presence (Moura et al., 2018). However,
alternatives that produce effects similar to fungicides, such as
treatments with oxalic acid (2 mM) and salicylic acid (0.5, 1, 1.5, and
384
2.0 mM), show efficacy against specific fungal species like
Aspergillus
flavus, Rhizopus stolonifer
, and
Penicillium
sp.
Fungal attack on seeds reduces vigor by affecting germination (EL-
DAHAB et al. 2016). In this study, seeds with high disease incidence
(control) had higher germination than
seeds with lower disease incidence (treated with fungicides),
indicating that the attack by these fungi did not affect seed vigor.
Materials and methods
Plant material
The sorghum (
Sorghum bicolor
) seeds used in this study were
acquired from small-scale producers in the municipality of Prata,
Paraíba (7°41'35" S, 37°05'22" W), Brazil. These seeds hold cultural
significance for the local farmers, often referred to as 'seeds of
passion', and have been passed down through generations, from
father to son. Following the acquisition, the seeds were transported to
the Laboratory of Phytopathology (LAFIT) at the Center for Agrarian
Sciences, Federal University of Paraíba. At LAFIT, the seeds underwent
a sanitation process involving a 2-minute treatment with 1% sodium
hypochlorite. This procedure is critical as it removes surface
microorganisms without affecting the seed's internal structure, thus
preventing any external fungal contamination from influencing the
outcomes of the study focused on seed-borne pathogens.
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Figure 1. Fungal incidence in sorghum (Sorghum bicolor) seeds treated with different concentrations of oxalic acid (0.5 - OA0.5; 1.0
- OA1.0; 1.5 - OA1.5; and 2 - OA2.0 mM) and salicylic acid (0.5 - SA0.5; 1.0 - SA1.0; 1.5 - SA1.5; and 2mM - SA2.0). Columns with the
same letter do not differ significantly by the Scott-Knott test up to 5% probability. Vertical bars correspond to the standard error of
the mean. Test: Witness; Fung: fungicide
Table 3. Anova of sorghum (Sorghum bicolor) seedlings treated with different concentrations of oxalic acid (0.5 - OA0.5; 1.0 -
OA1.0; 1.5 - OA1.5; and 2 - OA2.0 mM) and salicylic acid (0.5 - SA0.5; 1.0 - SA1.0; 1.5 - SA1.5; and 2mM - SA2.0).
FV
GL
EVI
GVI
FG
APL
RL
Emerg %
EVI-S
FG-S
RL-S
Treatment
9
15.4871**
283.1667**
356.2333**
4.0018*
5.4871**
352.0**
18.8579**
2.7318**
3.0414*
Error
30
1.8929
31.9667
23.8333
4.1573
4.7392
86.4
2.7765
0.8582
7.399
Mean
-
13.7283
59.7867
44.9783
7.275
8.7517
68.35
4.5353
5.1813
9.6381
CV (%)
-
16.3368
30.4543
27.1093
24.5783
24.275
30.06
56.161
23.327
26.3837
F (**) 1% probability, (*) 5% probability
387
Experimental design
The experimental design was completely randomized and included 10
treatments. These consisted of four doses of oxalic acid (0.5, 1.0, 1.5,
and 2.0 mM), four doses of salicylic acid (0.5, 1.0, 1.5, and 2.0 mM),
one fungicide treatment, and one control treatment where seeds were
immersed solely in H2O.
Application of treatments
Sorghum seeds were treated with solutions of oxalic acid (0.5, 1.0,
1.5, and 2.0 mM) and salicylic acid (0.5, 1.0, 1.5, and 2.0 mM) for 1
hour, following a modified protocol from Yang et al. (2016). Seeds
were also treated with Captan® fungicide at a concentration of 240 g
per 100 kg of seed. The control involved immersing seeds in distilled
water for 1 hour.
Germination, First count, and germination speed index
For the germination tests, 200 sorghum seeds were divided into four
replicates of 50 seeds each and placed between double layers of
germination paper, covered with a third sheet, and rolled up. The
paper was moistened with water equivalent to 2.5 times its dry weight
and placed in a Biochemical Oxygen Demand (BOD) chamber set at an
alternating temperature of 20-30°C with an 8-hour photoperiod. Daily
counts were performed from the second to the tenth day (Brasil,
2009). Germination results were expressed as percentages. The first
germination count (FG) was recorded on the second-day post-sowing,
and data were expressed as percentages. The Germination Speed
Index
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(GVI) was calculated daily from the second to the tenth day using
Maguire's (1962) formula.
Emergence, emergence speed index, aerial part, and root length
To determine seedling emergence and the Emergence Velocity Index
(EIV), 100 seeds were sown in four replicates of 25 seeds each in
plastic trays (47x27x8 cm) at a depth of 1 cm, filled with sterilized
washed sand. The sand was moistened daily to maintain 60% of its
water retention capacity. The trays were placed randomly in a
greenhouse, maintained at 25-30°C, and monitored for 10 days (Cruz
et al., 2018). Emergence counts were conducted from the fourth to the
tenth day, and results were expressed as the percentage of seedlings
that emerged by day 10. The Emergence Speed Index was calculated
using an adapted formula from Maguire (1962). Measurements of
aerial part and root lengths were taken from four seedlings per
replicate, totaling 16 seedlings, and 10 days post-sowing.
Fungal incidence
For the fungal incidence study, 100 sorghum seeds were distributed
across 10 replicates. Each replicate was placed in a 15 cm diameter
Petri dish containing a double layer of moistened filter paper. All
materials used were sterilized in an autoclave at 121°C to 134°C for 45
minutes. The Petri dishes were maintained at a constant temperature
of 25 ± 2 °C for 7 days. Fungal identification was conducted using
stereoscopic microscopy (Cruz et al., 2018), with species verification
against specialized literature. Results were expressed as the
percentage of infected seeds (Michereff, 2001).
Statistical analysis
387
Data from the various tests were analyzed using variance analysis
(Table 3), and treatment means were compared using the Scott-Knott
test at a 5% significance level (Jelihovschi et al., 2014). Analyses were
conducted using RStudio version 3.5.1 and Python.
Conclusion
The induction of resistance in sorghum seeds was most effectively
achieved with the use of salicylic acid (SA). SA demonstrated superior
health and physiological effects on sorghum seeds compared to oxalic
acid (OA). We recommend using doses of 0.5 and 1.0 mM of SA for
optimal physiological and sanitary quality in sorghum seeds. For OA, a
dose of 1.0 mM is advised for enhancing physiological quality, while a
dose of 2.0 mM is recommended for reducing fungal incidence.
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