Proteção bacillus subtilis

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Journal of Phytopathology. 2020;00:1–10. wileyonlinelibrary.com/journal/jph  |  1© 2020 Wiley-VCH GmbH
 
Received: 14 July 2020  |  Revised: 9 November 2020  |  Accepted: 10 November 2020
DOI: 10.1111/jph.12968 
O R I G I N A L A R T I C L E
Integrating a chemical fungicide and Bacillus subtilis BIOUFLA2 
ensures leaf protection and reduces ear rot (Fusarium 
verticillioides) and fumonisin content in maize
Rafaela Araújo Guimarães1  | Edgar Zanotto2  | Paul Esteban Pherez Perrony1  | 
Lidia Almeida Salum Zanotto2  | Leonardo José da Silva3  | José da Cruz Machado1  | 
Felipe Augusto Moretti Ferreira Pinto4  | Henrique Novaes Medeiros1 | 
Renzo Garcia von Pinho5  | Itamar Soares de Melo3  | Júlio Carlos Pereira da Silva6  | 
Fernanda Carvalho Lopes de Medeiros4  | Flávio Henrique Vasconcelos de Medeiros1
1Department of Phytopathology, 
Universidade Federal de Lavras, Lavras, 
Brazil
2Vittia Group, São Joaquim da Barra, Brazil
3Embrapa Meio Ambiente, Jaguariuna, Brazil
4Empresa de Pesquisa Agropecuária e 
Extensao Rural de Santa Catarina, São 
Joaquim, Brazil
5Department of Agriculture, Universidade 
Federal de Lavras, Lavras, Brazil
6Departament of Phytosanitary Defense, 
CCR, Universidade Federal de Santa Maria, 
Santa Maria, Brazil
Correspondence
Flávio Henrique Vasconcelos de Medeiros, 
Department of Phytopathology, 
Universidade Federal de Lavras, Lavras, MG 
37200-000, Brazil.
Email: flaviomedeiros@ufla.br
Funding information
Coordenação de Aperfeiçoamento de 
Pessoal de Nível Superior; Conselho 
Nacional de Desenvolvimento Científico e 
Tecnológico
Abstract
Fungicides in maize production under tropical conditions reduce losses from foliar 
diseases, but only a few reduce ear rot incidence or mycotoxin contamination in ker-
nels. Biocontrol agents (BCAs) may reduce postharvest losses but their efficacy has 
not been demonstrated in field conditions. Here, we evaluated the use of bacterial 
isolates in tandem with fungicides on Fusarium verticillioides incidence and fumonisin 
content. After an early screening, Bacillus subtilis and Streptomyces araujoniae isolates 
were used in field trials. Maize plants were sprayed twice: at the end of the veg-
etative stage (V9) and at the beginning of the reproductive stage (R1). Sprays were 
made by applying water, B. subtilis strain BIOUFLA2, S. araujoniae strain ASBV-1T, 
or fungicide (cyproconazole + azoxystrobin) in different combinations, totalling nine 
treatments. Ten days later, all maize ears were inoculated with F. verticillioides. Plants 
were assessed for foliar diseases, grain yield, F. verticillioides incidence and fumonisin 
content in kernels. The treatment with two fungicide sprays reduced most of the 
foliar diseases but not F. verticillioides incidence in kernels. Twice-sprayed B. subtilis 
and S. araujoniae reduced F. verticillioides, but did not protect leaves against other 
pathogens. All treatments encompassing a fungicide followed by one of the BCAs 
reduced F. verticillioides incidence compared to control. Twice-sprayed fungicide in-
creased fumonisin by 50% compared to water control, while fungicide followed by 
B. subtilis decreased the fumonisin content by 40%. Replacing the second chemical 
spray with S. araujoniae did not reduce the fumonisin content but provided a higher 
yield than a twice-sprayed fungicide. Exclusive use of chemical fungicides may not 
ensure higher grain quality and yield, but the integration with B. subtilis BIOUFLA2 
can accomplish both.
K E Y W O R D S
biological control agents, foliar diseases, integrated disease management, Mycotoxin, Zea 
mays
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mailto:flaviomedeiros@ufla.br
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2  |     ARAÚJO GUIMARÃES Et Al.
1  | INTRODUC TION
Several species of Fusarium spp. are pathogens of grain crops world-
wide. They cause both quantitative and qualitative losses by decreasing 
yield and by producing mycotoxins (Atanasova-Penichon et al., 2016; 
Vanara et al., 2018). Fusarium verticillioides and Fusarium graminearum 
cause ear rot in maize (Zeae-maydis) and produce significant amounts 
of mycotoxins, mostly fumonisin and deoxynivalenol, respectively 
(Zhou et al., 2018). However, F. verticillioides is the dominant pathogen 
on maize crops in tropical regions (Adejumo et al., 2007).
The mycotoxins produced by F. verticillioides are grouped into three 
types of fumonisin: FB1, FB2 and FB3 (Desjardins, 2006). Currently, 
fumonisin B1 (FB1) is the most common in F. verticillioides-infected 
kernels and potentially the most carcinogenic to animals and humans 
(Fallahi et al., 2019). Infection by F. verticillioides occurs during the 
cropping season, but mycotoxin accumulation is further augmented 
by delaying the harvest or by poor grain storage conditions (Adetunji 
et al., 2014; Lerda, 2017). To prevent contamination of maize-based 
products by fumonisins, management strategies must be implemented 
before the pathogen infection, that is, soon after flowering (Gromadzka 
et al., 2019), since reducing F. verticillioides incidence is tantamount to 
reducing fumonisin contamination of kernels (Lerda, 2017). Good grain 
quality is closely related to fumonisin reduction that also improves the 
yield obtained on the farm (Ferrigo et al., 2016).
Fungicides such as pyraclostrobin reduce the severity of some fo-
liar diseases and increase maize yield (Paul et al., 2011). In contrast, the 
use of chemical fungicides may not reduce F. verticillioides incidence 
in maize and may even increase fumonisin content in the grain (Falcão 
et al., 2011; Miguel et al., 2015). Additionally, reduction of F. verticilli-
oides mycelial growth by fungicides does not necessarily mean a re-
duction of fumonisin production in maize kernels (Alberts et al., 2016).
Biocontrol-based methods of foliar and/or postharvest disease 
management are not readily available to maize growers worldwide, 
although their efficacy has already been demonstrated (Alberts 
et al., 2016; Fig ueroa-López et al., 2016). New strategies based 
on biological control agents (BCA) as potential substitutes for fun-
gicides in F. verticillioides management have intensively been stud-
ied (Cavaglieri et al., 2005; Chulze et al., 2015; Fig ueroa-López 
et al., 2016; Legrand et al., 2017; Sartori et al., 2012).
There have been few attempts to integrate BCA with chem-
ical fungicides in maize cropping systems (Khokhar et al., 2014). 
Therefore, the goal of this work was to screen effective bacterial 
isolates under tropical conditions that control ear rot caused by F. 
verticillioides in maize and reduce grain fumonisin content, while sus-
taining foliar health and grain yield.
2  | MATERIAL AND METHODS
2.1 | Bacterial isolation
The selection of bacterial isolates as potential BCAs was performed 
using a soil bait method (Ghini & Kimati, 1989). Soils were sampled 
from fields under a no-tillage system with soya bean–maize rotation 
in southern Minas Gerais (Brazil). Only fields that showed no inci-
dence of F. verticillioides were sampled. 100 g of each soil was placed 
in Petri dishes (9 cm diameter) and 10 grains of maize previously in-
oculated with F. verticillioides (baits) were added to it. Petri dishes 
were incubated for five days in a growth chamber at 25°C under con-
tinuous light. Baits were washed with distilled and sterilized water 
then transferred to a new Petri dish with 0.5% agar–water medium 
(Kasvi, Brazil) containing thewater restrictor mannitol (−1.0 MPa) 
to improve the hydrophobic activity of the medium and prevent 
seed germination. These dishes were incubated for seven days at 
20°C under continuous light. Grains with no F. verticillioides mycelial 
growth were immersed in a buffer solution (NaCl 0.9%) for 1 min. 
The resulting solution was diluted from 10–1 to 10–5 and the dilutions 
were transferred to Corn Meal Agar (CMA; Himedia, India) in Petri 
dishes with mannitol. Each bacterial colony that grew on CMA was 
transferred to a new Petri dish, and these were incubated for five 
days at 28°C under continuous light. This selection resulted in two 
isolates of Bacillus spp. that we named BIOUFLA2 and BIOUFLA7. 
The isolates were preserved in peptone-glycerol and refrigerated at 
5°C for later use in the BCAs screening tests.
2.2 | Screening test and phylogenetic 
characterization of isolates
Bacillus spp. obtained from the soil screening (BIOUFLA2 and 
BIOUFLA7) and Streptomyces spp. isolates RESISP-3CSB005, 
GYCSC009WALL, GYSBB007WALL and ASBV-1T were tested on 
maize grains against F. verticillioides. The Streptomyces spp. isolates 
had been used to control Botritys cinerea in a previous study (Silva 
et al., 2014). Grains were surface sterilized with ethanol (70% v/v) 
for 1 min, followed by sodium hypochlorite (NaClO; 1% v/v) for 
30 s and then rinsed three times with sterilized water. Next, 45 
grains were inoculated by placing them for 5 min on top of bac-
terial colonies that were grown on nutrient agar medium (NA) in 
Petri dishes for two days. Fifteen grains were transferred to new 
Petri dishes containing NA, which were then incubated for seven 
days at 28°C in the dark. The control consisted of surface-disin-
fested grains in NA medium. After that, grains were sprayed with 
1.5 ml of a conidial suspension (105 conidia ml−1) of F. verticillioides. 
Seven days later, the presence of mycelial growth was assessed. 
The severity of mycelial growth of F. verticillioides was recorded 
as follows: 0 = absence of mycelium on the surface of the grain, 
1 = 1%–25% of the grain covered by mycelium, 2 = 26%–50% of 
the grain covered by mycelium, 3 = 51%–75% of the grain covered 
by mycelium, and 4 = 76%–100% of the grain covered by mycelium 
(Machado et al., 2013). Finally, grades were converted to disease in-
dexes according to the McKinney index formula (McKinney, 1923). 
Measurement of F. verticillioides mycelial growth was performed 
for seven consecutive days. Finally, we calculated the area under 
the disease progress curve (AUDPC) for each treatment (Campbell 
& Madden, 1990).
     |  3ARAÚJO GUIMARÃES Et Al.
The phylogenetic characterization of BIOUFLA2 was per-
formed by 16S rRNA gene sequence analysis. Genomic DNA was 
isolated from a pure colony using a PureLink Genomic DNA Mini Kit 
(Invitrogen; Thermo Fisher Scientific, California, United States). The 
16S rRNA gene was amplified by polymerase chain reaction (PCR) 
using the primer set 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 
1492R (5′-TACGGCTACCTTGTTACGAC-3′) (Silva et al., 2013). The 
16S rRNA gene sequence (1,489 bp) was aligned using CLUSTALW 
and trimmed (sequence data matrix with a 1,208 bp length) using 
MEGA 7.0 software (Kumar et al., 2016) against corresponding se-
quences of the genus Bacillus, retrieved from the GenBank data-
base using the EzBioCloud server (Yoon et al., 2017). Phylogenetic 
trees were inferred by the methods of maximum likelihood 
(Felsenstein, 1981), maximum parsimony (Fitch et al., 1971) and 
neighbour-joining (NJ) (Saitou & Nei, 1987) and implemented by the 
MEGA software, version 7.0. Phylogenetic trees were drawn using 
the neighbour-joining (NJ) (Saitou & Nei, 1987) and the Tamura 
3-parameter model of sequence evolution with gamma-distrib-
uted evolutionary rates (T92 + G) (Tamura, 1992), selected by the 
Bayesian Information Criterion. The topologies of the evolutionary 
trees were assessed by bootstrap analysis (Felsenstein, 1981) of the 
NJ method based on 1.000 replicates. BIOUFLA2 was further char-
acterized by biochemical tests. Eight different carbon sources and 
arginine were used to analyse its anaerobic decomposition (Gatson 
et al., 2006).
2.3 | Efficacy in field conditions
Bacillus subtilis (BIOUFLA2) and Streptomyces araujoniae (ASBV-1T) 
were selected for efficacy testing under field conditions, alone or 
in combination with a chemical fungicide. Field trials were carried 
out in a randomized block design with four plot replicates. Each plot 
consisted of four 5m-rows spaced 0.6 m apart planted with the hy-
brid DKB390 PRO2 and sown to yield a final population of 70,000 
plants per hectare. The experiment was repeated four times using 
the same design: field trials F1 and F2 were carried out in the same 
location (“−21.204242N, −44.980322W”) in two consecutive years 
(2013 and 2014), field trials F3 and F4 were carried out in different 
locations in 2013 (−21.337645N, −45.126954W and −21.337628N, 
−45.128748W, respectively). Soils were fertilized with 450 kg/ha 
NPK (8-28-16) upon sowing followed by top dressing fertilizations 
with 250 kg/ha NPK (20-00-20) when plants reached the third leaf 
collar (V3) and the sixth leaf collar stages (V6). Foliar treatments 
were sprayed at two phenological stages, once at the ninth leaf 
collar (V9) and once again at silking (R1). Treatments consisted of 
different combinations of water, biocontrol agents (B. subtilis or S. 
araujoniae) and the fungicide cyproconazole (80 g/L) + azoxystrobin 
(200 g/L) (Priori Xtra™, Syngenta, Paulinia, SP, Brazil). The following 
treatments were evaluated: Water Control (V9 + R1); Fungicide (V9) 
+ Water (R1); Fungicide x2 (V9 + R1); B. subtilis x2 (V9 + R1); S. arau-
joniae x2 (V9 + R1); Fungicide (V9) + B. subtilis (R1); Fungicide (V9) 
+ S. araujoniae (R1); B. subtilis (V9) + Fungicide (R1) and S. araujoniae 
(V9) + Fungicide (R1). B. subtilis and S. araujoniae isolates were grown 
in liquid yeast peptone dextrose (YPD) medium for 72 hr and diluted 
in water at a 1:1 ratio (108 CFU/ml). Spray volumes were 200 L/ha 
and 250 ml/ha for the biological and chemical applications, respec-
tively. A mineral oil solution (0.5%) was added to all spray solutions.
After both foliar treatment applications, each maize ear was in-
oculated with 5 ml of a 105 conidia ml−1 suspension of F. verticillioides 
strain F425 (Lanza et al., 2014), placed directly on the corn silk with a 
syringe. Inoculation was performed 10 days after stigma-style emis-
sion (Mendes et al., 2012).
2.4 | Foliar disease assessments
Disease assessment started one week after treatment application. 
Assessments were made on the first leaf just below the spike in three 
plants per plot according to validated disease scales. We performed 
five evaluations over five weeks. Five common maize diseases, in-
cluding common rust (Puccinia sorghi) (Dudienas et al., 2013), grey 
leaf spot (Cercospora Zeae-maydis) (Lazaroto et al., 2012), Diplodia 
leaf streak (Stenocarpella sp.) (Bradley et al., 2010), anthracnose 
(Colletotrichum graminicola) (Trojan & Pria, 2018) and maize white 
spot (Pantoea ananatis) (Malagi et al., 2011) were quantified. Finally, 
we calculated the AUPDC based on the severity of each disease and 
treatment (Campbell & Madden, 1990).
2.5 | Postharvest assessments
Assessment included grain yield, ear rot incidence and fumonisin 
content. The maize was harvested when kernels achieved 18% hu-
midity and they were immediately transferred to a grain dryer with 
an airflow of 60-m3. min-1m-3 and 90°C until kernels reached 13% 
moisture (BRASIL, 2009). Yield was determined by harvesting the 
two central rows of each plot and presented as tonnes ha-1. The inci-
dence of F. verticillioides on kernels of each treatment was evaluated 
by the blotter test (Michail et al., 1985) with 200 grains per treat-
ment (25 kernels per replicate).). First, 25 grains were surface steri-
lized (as described in the screening test), then allowed to dry for 2 hr 
in alaminar flow hood, and distributed over Water–Agar (WA; 2% 
w/v) in a 15-cm-diameter Petri dish. The dishes were kept in growth 
chambers at 25°C for seven days. F. verticillioides incidence was as-
sessed using a stereoscopic microscope. The percentage of mouldy 
grains was also estimated using the same grains.
Based on previous experience of interfield variation of fu-
monisin content but low variation within each field (Guimarães 
et al., 2020), for FB1 and FB2 content analysis, all four replicates 
from each field trial were combined into one sample and each of 
such composite sample per trial was considered to be a block. 
Therefore, kernels randomly sampled right after harvest were 
ground and a sub-sample of ten grams per treatment was used for 
mycotoxin extraction. FB1 and FB2 extraction was performed by 
the free fumonisins analysis method. Fumonisins were detected 
4  |     ARAÚJO GUIMARÃES Et Al.
according to Oliveira et al. (2015). Ground samples were passed 
through a 2.0 mm screen and extracted with 50 ml water/ace-
tonitrile (1:1 v/v) for 5 min in a high-speed blender. The extract 
was then filtered. An aliquot of 20 μl was diluted in a 1% formic 
acid acetonitrile/water solution (1:1, v/v) before liquid chroma-
tography–mass spectrometry analysis. The column used was C18 
(150 × 4.6 mm × 5.5 μm). The primary transition was used for peak 
quantification.
2.6 | Statistical analysis
The postharvest and in vitro experimental designs were a completely 
randomized design with four to eight replicates depending on the 
experiment. All data were tested for normality and homoscedastic-
ity before analysis of variance (ANOVA). When treatments were sig-
nificant, the means were compared using Tukey's test (p < .05). The 
correlation between yield, F. verticillioides incidence, the severity 
of foliar diseases, and fumonisin content was analysed by Principal 
Coordinates Analysis (PCA). Data were transformed using the equa-
tion [(x-mean)/stdev)] and the correlation matrix between variables 
was obtained using PAST software.
3  | RESULTS
3.1 | Screening for promising biocontrol agents
We obtained two Bacillus spp. isolates (BIOUFLA2 and BIOUFLA7) 
from the soil bait method. In screening tests, BIOUFLA2, RESISP-
3CSB005 and ASBV-1T reduced AUDPC by 45% (p < .01) (Figure 1). 
The two most promising isolates (BIOUFLA2 and ASBV-1T) were 
further tested in field trials.
3.2 | Molecular identification of BIOUFLA2
A phylogenetic tree based on 16S rRNA gene sequences showed 
that BIOUFLA2 clustered into the Bacillus tequilensis KCTC 13622T 
and B. subtilis subsp. inoquosorum KCTC 13429T clade (Figure S1). 
Biochemical tests were performed to discriminate between species. 
BIOUFLA2 did not produce acid from the tested carbon sources and 
did not decompose arginine under anaerobic conditions, therefore it 
cannot be described as B. tequilensis (Gatson et al., 2006) and likely 
belongs to the B. subtilis group (Table S1).
3.3 | Foliar disease control under field conditions
Anthracnose AUDPC (Table 1) was only significantly different in 
field trial F1 (p < .01). In F1, treatments fungicide x2, fungicide + S. 
araujoniae and S. araujoniae + fungicide significantly reduced disease 
incidence compared to control.
Regarding common rust (P. sorghi), although inoculum pressure 
was higher in field trials F2 and F3 than in the others, we observed a 
significant effect of the foliar sprays in all trials, except in F1 (Table 2). 
The treatments fungicide x2, fungicide + B. subtilis and fungicide + S. 
araujoniae reduced the AUDPC in trials F2, F3 and F4 compared to 
control. Treatments fungicide + water and B. subtilis + fungicide re-
duced the AUDPC in F2 and F3. S. araujoniae + fungicide reduced 
the AUDPC in F3 and F4. Also, B. subtilis x2 only performed better 
than control in treatment F4. Overall, when the first fungicide spray 
was replaced by either B. subtilis or S. araujoniae, the reduction of 
AUDPC ranged from 23% to 83% compared to control, statistically 
similar to fungicide x2 (p < .01).
Grey leaf spot AUDPC was also higher in field trials 2 and 3 than 
in the others (Table 3). None of the treatments significantly reduced 
the disease in all four fields. The most consistent disease reductions 
(p < .01) were achieved when fungicide was applied twice (Fungicide 
x2) for fields F1 (41.1%), F2 (40%) and F3 (50.7%).
Although we observed a significant effect of treatments to the 
previous fungal diseases, this was not the case for Diplodia leaf 
streak. There were no significant differences in AUDPC between 
treatments in any field trial (F1: p = .08; F2: p = .10; F3: p = .055; F4: 
p = .09) (data not shown).
Maize white spot (Pantoea ananatis) behaved similarly to the fun-
gal diseases, with the inoculum pressure being higher in field trials 
F2 and F3 than in F1 and F4 (Table 4). However, we only found a 
significant effect in F2 (p = .02). All treatments that included at least 
one fungicide application except B. subtilis + fungicide significantly 
reduced the AUDPC. B. subtilis x2 did not reduce the AUDPC, while 
S. araujoniae x2 significantly reduced the disease.
F I G U R E 1   Screening of bacterial isolates against Fusarium 
verticillioides in maize grains measured by the area under the 
disease progress curve (AUDPC). AUDPC was calculated 
by assessing grains colonized with F. verticillioides for seven 
consecutive days. BIOUFLA isolates are Bacillus spp., while other 
isolates are Streptomyces spp. Means followed by the same letter do 
not differ according to Tukey's post hoc test (p < .05). Bars indicate 
the standard error of the mean
     |  5ARAÚJO GUIMARÃES Et Al.
3.4 | Postharvest assessments
Regarding grain yield, there was a significant difference between 
treatments (p < .05) in all field trials except F3 (p = .06) (Table 5). 
Grain yield increased when S. araujoniae was sprayed in the first or 
the second application in fields F1, F2 and F4. In F1, the highest yield 
was obtained with treatments fungicide x2 and S. araujoniae + fungi-
cide (respectively, 11.53 and 11.98 tonnes ha−1) (p = .03). In F3, fun-
gicide + B. subtilis, fungicide + S. araujoniae and B. subtilis x2 resulted 
in the highest yields (4.44, 4.68 and 4.56 tonnes ha-1, respectively). 
Finally, in field F4, fungicide + S. araujoniae and S. araujoniae x2 re-
sulted in the highest yields (7.06 and 7.23 tonnes ha-1, respectively) 
(p = .02) (Table 5).
In F1, the incidence of F. verticillioides was reduced regardless 
of treatment compared to control (Table 6) (p < .01). In the other 
fields, most treatments using at least one bacterial application re-
duced the pathogen incidence (fungicide + B. subtilis, fungicide + S. 
First (V9) + second (R1) spray 
treatments
AUDPC
F1 F2 F3 F4
Water Control 1,014.0 a 190.0 ns 45.5 ns 11.0 ns
Fungicide + Water 819.0 a 117.0 ns 107.0 ns 12.0 ns
Fungicide x2b  456.0 b 65.0 ns 164.0 ns 11.0 ns
Fungicide + B. subtilis 883.0 a 190.0 ns 192.0 ns 14.0 ns
Fungicide + S. araujoniae 647.0 b 115.0 ns 145.0 ns 13.0 ns
B. subtilis + Fungicide 966.0 a 81.0 ns 148.0 ns 7.5 ns
S. araujoniae + Fungicide 622.0 b 74.0 ns 97.0 ns 7.0 ns
B. subtilis x2 784.0 a 71.0 ns 98.0 ns 6.5 ns
S. araujoniae x2 814.0 a 116.0 ns 109.0 ns 13.5 ns
aV9 – ninth leaf collar stage; R1- silking stage. 
bx2 - Fungicide or microorganism sprayed twice (V9 and R1). Means followed by the same letter do 
not differ according to Tukey's post hoc test (p < .05). 
cF1: p < .05; F2,F3 and F4: p = not significant - ns. 
TA B L E 1   Area under the 
disease progress curve (AUDPC) of 
anthracnose (Colletotrichum graminicola) 
on maize leaves sprayed with 
different combinations of fungicide 
(cyproconazole + azoxystrobin), Bacillus 
subtilis BIOUFLA2 and Streptomyces 
araujoniae ASBV-1T in four field trials 
(F1-F4)
TA B L E 2   Area under the disease progress curve (AUDPC) 
of common rust (Puccinia sorghi) on maize leaves sprayed with 
different combinations of fungicide (cyproconazole + azoxystrobin),Bacillus subtilis BIOUFLA2 and Streptomyces araujoniae ASBV-1T in 
four field trials (F1-F4)
First (V9) + second (R1) 
spray treatments
AUDPC
F1 F2 F3 F4
Water Control 34.0 
ns
284.0 a 366.0 a 47.0 a
Fungicide + Water 35.0 
ns
188.0 b 37.0 b 52.0 a
Fungicide x2b  32.0 
ns
47.5 b 32.5 b 38.0 b
Fungicide + B. subtilis 39.0 ns 160.2 b 89.0 b 43.0 b
Fungicide + S. araujoniae 30.0 
ns
126.0 b 76.0 b 39.0 b
B. subtilis + Fungicide 37.0 ns 132.5 b 195.0 b 47.0 a
S. araujoniae + Fungicide 34.0 
ns
219.0 a 99.0 b 43.0 b
B. subtilis x2 37.0 ns 262.0 a 470.0 a 41.0 b
S. araujoniae x2 38.0 
ns
357.0 a 400.0 a 51.0 a
aV9 - ninth leaf collar stage; R1- silking stage. 
bx2 - Fungicide or microorganism sprayed twice (V9 and R1). Means 
followed by the same letter do not differ according to Tukey's post hoc 
test (p < .05). 
cF2, F3 and F4: p < .05; F1: p = not significant - ns. 
TA B L E 3   Area under the disease progress curve (AUDPC) of 
grey leaf spot (Cercospora Zeae-maydis) on maize leaves sprayed with 
different combinations of fungicide (cyproconazole + azoxystrobin), 
Bacillus subtilis BIOUFLA2 and Streptomyces araujoniae ASBV-1T in 
four field trials (F1-F4)
First (V9) + second (R1) 
spray treatments
AUDPC
F1 F2 F3 F4
Water Control 14.0 a 582.5 a 523.0 a 33.0 a
Fungicide + Water 10.5 a 412.0 a 449.0 a 35.0 a
Fungicide x2b  5.0 b 233.0 b 265.0 b 32.0 a
Fungicide + B. subtilis 8.0 a 409.0 a 471.0 a 26.0 b
Fungicide + S. araujoniae 8.0 a 413.0 a 375.0 b 30.5 a
B. subtilis + Fungicide 8.0 a 291.0 b 294.0 b 17.0 c
S. araujoniae + Fungicide 9.5 a 243.0 b 165.5 b 25.0 b
B. subtilis x2 15.0 b 542.0 a 522.0 a 35.0 a
S. araujoniae x2 15.0 b 576.0 a 339.0 b 25.0 b
aV9 - ninth leaf collar stage; R1- silking stage. 
bx2 - Fungicide or microorganism sprayed twice (V9 and R1). Means 
followed by the same letter do not differ according to Tukey's post hoc 
test (p < .05). 
cF1, F2, F3 and F4: p < .05. 
6  |     ARAÚJO GUIMARÃES Et Al.
araujoniae, B. subtilis x2 and S. araujoniae x2), while treatments S. 
araujoniae + fungicide and S. araujoniae x2 reduced the incidence of 
F. verticillioides in all four fields (F1-F4). Fungicide + water, fungicide 
x2 and B. subtilis + fungicide did not consistently reduce F. verti-
cillioides incidence in kernels when compared to control (p < .01) 
(Table 6).
Fungicide + B. subtilis reduced the fumonisin content by almost 
40% (1.5 µg/g) compared to control (2.3 µg/g). Fungicide x2, S. arau-
joniae + fungicide and S. araujoniae x2 increased fumonisin content 
by around 78% (5.1 µg/g) compared to control (p = .02). For the other 
treatments, fumonisin content was similar to control (Table 7). No 
significant difference in the percentage of mouldy grains was found 
(p = .97).
To identify patterns among the variables, a principal component 
analysis was carried out (Table S1). Components 1 and 3 had the 
highest Eigenvalue and explained most of the variance between 
groups: 48.29% for Component 1 (Yield) and 15.18% for Component 
3 (Fumonisin content). Treatments that result in disease reduction 
would ensure higher grain yield. Additionally, the severity of the 
other foliar diseases (grey leaf spot, common rust and white spot) 
correlated with F. verticillioides incidence but was not related to grain 
contamination with fumonisins.
4  | DISCUSSION
In this study, the fungicide was efficient, although control efficacy 
varied with the disease evaluated (Tables 1–4). When fungicides 
are used as the sole disease management strategy, they may select 
for resistance in the pathogens and result in the loss of fungicide 
efficacy (Georgopoulos & Skylakakis, 1986; Kluge et al., 2017). In 
our work, fungicide applied twice resulted in higher grain yield com-
pared to a single application. However, the higher yield did not trans-
late into less mouldy grains and/or fumonisins (Figure S2). Fumonisin 
reduction guarantees a good grain quality, a condition as important 
as a good yield for the food industry (Vanara et al., 2018).
The introduction of either a biocontrol agent or the replace-
ment of the second fungicide spray by a biocontrol agent (except S. 
araujoniae x2) resulted in similar yield and lower fumonisin content 
than treatments just with fungicides. Although the combination of 
cyproconazole + azoxystrobin reduces most foliar diseases (Juliatti 
et al., 2007), in our work the incidence of F. verticillioides in kernels 
was higher than water control. These fungicides are not registered 
for use against ear rot caused by F. verticillioides. Fungicides are 
effective in controlling maize foliar diseases, leading to consistent 
yield even at high disease pressure (Paul et al., 2011). However, 
previous reports have demonstrated that cyproconazole + azox-
ystrobin is ineffective against diseases caused by Fusarium spp. 
(Falcão et al., 2011; Miguel et al., 2015). In preliminary in vitro 
trials, we confirmed that mycelial growth of F. verticillioides was 
not inhibited even when cultivated with 100 µg/g of this fungicide 
(Figure S3).
Two applications of the fungicide or the S. araujoniae isolate 
reduced F. verticillioides incidence but did not reduce fumoni-
sin content. Some species of Fusarium do not produce high levels 
of mycotoxin, but compete for nutrients and space with fumoni-
sin-producing species, possibly reducing fumonisin content in ker-
nels (Moussa et al., 2017). It is known that S. araujoniae competes 
with F. verticillioides (Kinkel et al., 2012). Considering that fumonisin 
TA B L E 4   Area under the disease progress curve (AUDPC) 
of white spot (Pantoea ananatis) on maize leaves sprayed with 
different combinations of fungicide (cyproconazole + azoxystrobin), 
Bacillus subtilis BIOUFLA2 and Streptomyces araujoniae ASBV-1T in 
four field trials (F1-F4)
First (V9) + second (R1) 
spray treatments
AUDPC
F1 F2 F3 F4
Water Control 4.0 ns 416.0 a 127.0 ns 2.0 ns
Fungicide + Water 2.0 ns 332.0 b 112.0 ns 1.0 ns
Fungicide x2b  3.0 ns 208.0 b 173.0 ns 1.0 ns
Fungicide + B. subtilis 3.5 ns 285.0 b 154.0 ns 1.0 ns
Fungicide + S. araujoniae 2.5 ns 317.0 b 169.0 ns 0.5 ns
B. subtilis + Fungicide 1.5 ns 458.0 a 71.0 ns 2.0 ns
S. araujoniae + Fungicide 5.0 ns 176.0 b 112.0 ns 1.0 ns
B. subtilis x2 4.0 ns 483.0 a 77.0 ns 1.0 ns
S. araujoniae x2 6.0 ns 301.0 b 127.0 ns 2.0 ns
aV9 - ninth leaf collar stage; R1- silking stage. 
bx2 - Fungicide or microorganism sprayed twice (V9 and R1). Means 
followed by the same letter do not differ according to Tukey's post hoc 
test (p < .05). 
cF2: p < .05; F1,F3 and F4: p = not significant - ns. 
TA B L E 5   Yield (tonnes ha−1) of maize sprayed with different 
combinations of fungicide (cyproconazole + azoxystrobin), Bacillus 
subtilis BIOUFLA2 and Streptomyces araujoniae ASBV-1T in four 
field trials (F1-F4)
First (V9) + second (R1) 
spray treatments
Yield (tonnes ha−1)
F1 F2 F3 F4
Water Control 9.99 c 3.15 ns 3.55 b 5.26 c
Fungicide + Water 10.98 b 3.55 ns 4.00 b 6.18 b
Fungicide x2b  11.53 a 4.03 ns 3.60 b 6.55 b
Fungicide + B.subtilis 10.99 b 3.70 ns 4.44 a 5.68 c
Fungicide + S. araujoniae 10.99 b 3.99 ns 4.68 a 7.06 a
B.subtilis + Fungicide 10.95 b 3.51 ns 3.42 b 6.52 b
S. araujoniae + Fungicide 11.98 a 4.14 ns 3.81 b 6.31 b
B.subtilis x2 10.98 b 3.64 ns 4.56 a 5.38 c
S. araujoniae x2 11.10 b 3.70 ns 3.40 b 7.23 a
aV9 - ninth leaf collar stage; R1- silking stage. 
bx2 - Fungicide or microorganism sprayed twice (V9 and R1). Means 
followed by the same letter do not differ according to Tukey's posthoc 
test (p < .05). 
cF1, F3 and F4: p < .05; F2: p = not significant - ns. 
     |  7ARAÚJO GUIMARÃES Et Al.
has a toxic effect against Fusarium competitors (Bush et al., 2004), 
the competition for nutrients with S. araujoniae in the kernels may 
stimulate F. verticillioides to produce fumonisin in order to gain a 
more exclusive nutrient source from the plant (Medina et al., 2017). 
Therefore, competition as the sole mode of action against F. verticil-
lioides may notbe enough to reduce fumonisin content in kernels, 
even if it reduces the incidence of the fungi (Table 6).
A reduction of F. verticillioides population in combination with 
low fumonisin content (FB1 and FB2) after Bacillus treatment were 
reported under laboratory (Pereira et al., 2007) and field condi-
tions (Guimarães et al., 2020). In this study, all treatments with B. 
subtilis application had a fumonisin content in kernels lower than 
the acceptable level for the international market of maize (4 µg/g) 
(Romer Labs, 2019; Vanara et al., 2018). Furthermore, we evaluated 
the timing of fungicide and bacteria applications that not only re-
sulted in the protection of kernels from F. verticillioides colonization 
and fumonisin content but also against foliar diseases (Tables 1–4).
Despite its use in agriculture to control soil-borne diseases, B. 
subtilis also performs as a biopesticide when applied to foliar tissue 
(Kloepper et al., 2004). Bacillus species not only are antagonistic to 
pathogenic fungi but can also induce plant defences and promote 
plant growth (Kloepper et al., 2004; Pereira et al., 2007). Those com-
binations of modes of action ensure an advantage when compared 
to Streptomyces as a BCA to reduce fumonisin in maize kernels. We 
found that the spray of the BCA strains at the earlier phenological 
stage (V9) played a role in the control of some foliar diseases such 
as grey leaf spot but did not reduce fumonisin, while BCA sprays de-
creased fumonisin production when applied at the later phenological 
stage (R1) or applied twice (except S. araujoniae x2). Studies have 
suggested a negative impact of chemical fungicides on nonpatho-
genic organisms (native antagonists), and consequently, they may af-
fect the emergence and competitiveness of these organisms against 
pathogenic species (Gardner et al., 1998). Therefore, the substitu-
tion of some fungicide sprays with BCAs may have had a positive 
effect on the functional diversity of the microorganisms colonizing 
the leaves (Meyer & Leveau, 2012).
Even though fungicides modify the abundance of antagonistic 
native microbial communities (Gu et al., 2010; Moulas et al., 2013), 
their tandem application with B. subtilis strain BIOUFLA2, which 
may act through different modes of action, resulted in a reduction of 
most foliar diseases, ensured increased yield and decreased kernel 
contamination by F. verticillioides and fumonisin content. Therefore, 
BCAs and chemical fungicides could be adopted in crop manage-
ment programs together with other disease management practices, 
such as resistant hybrids and crop rotation, to ensure sustainable 
crop production and reductions in fumonisin contamination of maize 
and maize-derived products.
First (V9) + second (R1) spray 
treatments
Fusarium verticillioides incidence (%)
F1 F2 F3 F4
Water Control 73.75 b 56.75 b 85.00 c 48.50 b
Fungicide + Water 54.25 a 52.25 b 66.50 a 51.75 b
Fungicide x2b  58.50 a 58.75 b 71.75 b 49.00 b
Fungicide + B. subtilis 57.00 a 43.25 a 73.50 b 38.00 a
Fungicide + S. araujoniae 50.25 a 49.25 a 57.75 a 46.75 b
B. subtilis + Fungicide 57.00 a 52.00 b 71.50 b 47.50 b
S. araujoniae + Fungicide 50.75 a 42.75 a 65.00 a 39.50 a
B. subtilis x2 54.25 a 43.75 a 71.25 b 37.75 a
S. araujoniae x2 56.00 a 47.00 a 65.75 a 43.00 a
aV9 - ninth leaf collar stage; R1- silking stage. 
bx2 - Fungicide or microorganism sprayed twice (V9 and R1). Means followed by the same letter do 
not differ according to Tukey's post hoc test (p < .05). 
cF1, F2, F3 and F4: p < .05. 
TA B L E 6   Postharvest incidence of 
Fusarium verticillioides on maize grains 
under different combinations of fungicide 
(cyproconazole + azoxystrobin), Bacillus 
subtilis BIOUFLA2 and Streptomyces 
araujoniae ASBV-1T in four field trials 
(F1-F4)
TA B L E 7   Total fumonisin content (FB1 + FB2) in maize grains 
and percentage (%) of mouldy grains in relation of treatments under 
different combinations of fungicide (cyproconazole + azoxystrobin) 
and biological control agents, Bacillus subitilis or Streptomyces 
araujoniae
First (V9) + second (R1) spray 
treatments
Total fumonisins 
(µg/g)
Mouldy 
grains (%)
Water Control 2.34 b 15.07 ns
Fungicide + Water 2.80 b 11.42 ns
Fungicide x2** 4.49 a 12.91 ns
Fungicide + B.subtillis 1.50 c 11.41 ns
Fungicide + S. araujoniae 2.38 b 12.58 ns
B.subtillis + Fungicide 2.53 b 12.47 ns
S. araujoniae + Fungicide 2.83 a 9.52 ns
B.subtillis x2 2.48 b 13.56 ns
S. araujoniae x2 5.01 a 10.85 ns
aV9 - ninth leaf collar stage; R1- silking stage. 
bx2 - The same treatment sprayed twice. Means followed by the same 
letter do not differ according to the Tukey post hoc test (p ≤ .05). 
c(Total amount fumonisins: p = .02; Mouldy grains: p = not significant 
- ns). 
8  |     ARAÚJO GUIMARÃES Et Al.
ACKNOWLEDG EMENTS
We thank Dr Pablo Schulman for the English review.
CONFLIC TS OF INTERE S T
The authors declare no conflict of interest.
PEER RE VIE W
The peer review history for this article is available at https://publo 
ns.com/publo n/10.1111/jph.12968.
DATA AVAIL ABILIT Y S TATEMENT
The data that support the figures of this study are available from the 
corresponding author upon reasonable request.
ORCID
Rafaela Araújo Guimarães https://orcid.
org/0000-0003-4238-0745 
Edgar Zanotto https://orcid.org/0000-0002-8791-8361 
Paul Esteban Pherez Perrony https://orcid.
org/0000-0001-5326-514X 
Lidia Almeida Salum Zanotto https://orcid.
org/0000-0002-5869-694X 
Leonardo José da Silva https://orcid.org/0000-0002-7625-9460 
José da Cruz Machado https://orcid.org/0000-0003-4515-8736 
Felipe Augusto Moretti Ferreira Pinto https://orcid.
org/0000-0002-9717-3324 
Renzo Garcia von Pinho https://orcid.org/0000-0001-5276-2806 
Itamar Soares de Melo https://orcid.org/0000-0003-2785-6725 
Júlio Carlos Pereira da Silva https://orcid.
org/0000-0002-1961-6695 
Fernanda Carvalho Lopes de Medeiros https://orcid.
org/0000-0003-3142-1652 
Flávio Henrique Vasconcelos de Medeiros https://orcid.
org/0000-0003-0993-796X 
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SUPPORTING INFORMATION
Additional supporting information may be found online in the 
Supporting Information section.
How to cite this article: Guimarães RA, Zanotto E, Perrony 
PEP, et al. Integrating a chemical fungicide and Bacillus subtilis 
BIOUFLA2 ensures leaf protection and reduces ear rot 
(Fusarium verticillioides) and fumonisin content in maize. 
J Phytopathol. 2020;00:1–10. https://doi.org/10.1111/
jph.12968
https://doi.org/10.3390/toxins10090357
https://doi.org/10.3390/toxins10090357https://doi.org/10.1099/ijsem.0.001755
https://doi.org/10.3390/toxins10020090
https://doi.org/10.3390/toxins10020090
https://doi.org/10.1111/jph.12968
https://doi.org/10.1111/jph.12968