Keywords:

Bacillus

Combined control

Fungicide

Fusarium culmorum

Wheat seedlings

Study of synergistic eect of combined application of tebuconazole with two biocontrol agents

for management of Fusarium crown rot in durum wheat

Estudio del efecto sinérgico de la aplicación combinada de tebuconazol con dos agentes de biocontrol

para el manejo de la pudrición de la corona por Fusarium en trigo duro

Estudo do efeito sinérgico da aplicação combinada de tebuconazol com dois agentes de biocontrole

no manejo da podridão-da-colheita por Fusarium em trigo duro

Abdelmalek Oulmi

Amor Bencheikh

Walid Mamache

Asma Gharzouli

Meriem Barkahoum Daichi

Noureddine Rouag

Rev. Fac. Agron. (LUZ). 2023, 40(3): e234025

ISSN 2477-9407

DOI: https://doi.org/10.47280/RevFacAgron(LUZ).v40.n3.03

Crop Production

Associate editor: Professor Beltrán Briceño

University of Zulia, Faculty of Agronomy

Bolivarian Republic of Venezuela

Department of plant Biology and Ecology, Faculty of

Nature and Life Sciences, Valorization of Natural Biological

Resources Laboratory, Ferhat ABBAS University-Setif

19000, Algeria.

Department of Microbiology, Faculty of Nature and Life

Sciences, Laboratory of applied Microbiology, Ferhat

ABBAS University - Setif 19000, Algeria.

Department of Biochemistry, Faculty of Nature and Life

Sciences, Laboratory of applied phytotherapy to chronic

diseases, Ferhat ABBAS University - Setif, Algeria.

Department of Agronomy, Faculty of Nature and Life

Sciences, Laboratory of applied Microbiology Ferhat

ABBAS University - Setif, 19000, Algeria.

Received: 30-03-2023

Accepted: 27-06-2023

Published: 18-07-2023

Abstract

The in vitro and growth chamber, tests were conducted in order to

assess the eects of Bacillus amyloliquefaciens B18 and Bacillus subtilis

S8 strains each alone and in combination with tebuconazole against

Fusarium culmorum (FC) isolate responsible of Fusarium crown rot (FCR)

in durum wheat. The in vitro growth of B18 and S8 strains was unaected

by 30 µg.mL

-1

tebuconazole. The Bacillus strains (at 10

CFU.mL

-1

) and

tebuconazole, each alone, reduced the mycelial growth, this eect was

signicantly improved when they were combined (inhibition of more than 92

%). In growth chamber experiments, ecacy against FCR was signicantly

higher when integrating Bacillus strains and tebuconazole than by either

alone; control ecacy of tebuconazole at 30 µg.mL

-1

in combination with

S8 and B18 strains reached 90.91 and 95.45 %, respectively. The obtained

results indicated that combination of tebuconazole with the biocontrol agents

B18 and S8 synergistically improved control eciency of the fungicide

against FCR of wheat.

This scientic publication in digital format is a continuation of the Printed Review: Legal Deposit pp 196802ZU42, ISSN 0378-7818.

Rev. Fac. Agron. (LUZ). 2023, 40(3): e234025. July-September. ISSN 2477-9407.2-6 |

Resumen

Se realizaron experimentos in vitro y en cámara de crecimiento

para evaluar los efectos de las cepas Bacillus amyloliquefaciens B18 y

Bacillus subtilis S8, cada una sola y en combinación con tebuconazol,

contra el aislado de Fusarium culmorum (FC) responsable de

la pudrición de la corona por Fusarium (FCR) en trigo duro. El

crecimiento in vitro de las cepas B18 y S8 no se vio afectado por

30 µg.mL

-1

de tebuconazol. Las cepas de Bacillus (10

UFC.mL

-1

) y

tebuconazol, cada una sola, redujeron el crecimiento micelial, este

efecto mejoró signicativamente cuando se combinaron (inhibición

de más del 92 %). En experimentos en cámaras de crecimiento,

la ecacia contra FCR fue signicativamente mayor cuando se

integraron cepas de Bacillus y tebuconazol que cuando se integraron

cualquiera de las dos solas; la ecacia de control de tebuconazol a 30

µg.mL

-1

en combinación con las cepas S8 y B18 alcanzó el 90,91 y el

95,45 %, respectivamente. Los resultados obtenidos indicaron que la

combinación de tebuconazol con los agentes de biocontrol B18 y S8

mejoró sinérgicamente la eciencia de control del fungicida contra

FCR de trigo.

Palabras clave: Bacillus, control combinado, fungicida, Fusarium

culmorum, plántulas de trigo.

Resumo

Os experimentos in vitro e em câmara de crescimento

foram realizados para avaliar os efeitos das cepas de Bacillus

amyloliquefaciens B18 e Bacillus Subtilis S8 isoladamente e em

combinação com tebuconazol contra o isolado de Fusarium culmorum

(FC) responsável pela podridão da coroa de Fusarium (FCR) em

trigo duro. O crescimento in vitro das cepas B18 e S8 não foi afetado

por 30 ug.mL

-1

de tebuconazol. As cepas de Bacillus (10

CFU.mL

) e tebuconazol, cada uma isoladamente, reduziram o crescimento

micelial, este efeito foi signicativamente melhorado quando foram

combinados (inibição de mais de 92 %). Em experimentos de câmara

de crescimento, a ecácia contra FCR foi signicativamente maior

ao integrar cepas de Bacillus e tebuconazol do que por qualquer um

deles sozinho; a ecácia de controle do tebuconazol a 30 µg.mL

-1

em combinação com as cepas S8 e B18 atingiu 90,91 e 95,45 %,

respectivamente. Os resultados obtidos indicaram que a combinação

de tebuconazol com os agentes de biocontrole B18 e S8 melhorou

sinergicamente a eciência de controle do fungicida contra FCR de

trigo.

Palabras-chave: Bacillus, controle combinado, fungicida, Fusarium

culmorum, mudas de trigo.

Introduction

Triticum turgidum var. durum L., also known as durum wheat,

is a crucial crop for the economy crop globally after rice and corn

(Food and Agriculture Organization [FAO], 2018). In Algeria, about

4 million tons of wheat were harvested in 2019, an increase of 1.2

million tons compared to 2017 (FAO, 2019). Despite this, Algeria

remains one of the important importing countries for this important

crop, which is due to the weak yield and the increasing consumer

needs in parallel with the increasing demographic growth (Bellout et

al., 2020; FAO, 2018).

Every year, most regions that produce wheat suer large losses

due to one of the most economically signicant diseases of wheat,

the FCR. On average, it caused losses in yield, between 24 and 52

% in durum wheat elds per year (Chekali et al., 2013; Hollaway et

al., 2013).

For decades, the primary method of preventing the spread of

fungal phytopathogens has been the use of synthetic fungicides

(Ceiro-Catasú et al., 2022; Palmieri et al., 2022). Despite this, the

control ecacy of azole fungicides has been signicantly decreased

due to the developed resistance in FCR agents resulting from the

excessive use of fungicides (De Chaves et al., 2022; Hellin et al.,

2018). For this reason, more eective and environmentally friendly

methods for controlling FCR agents must be found. Tebuconazole is

a potent multifunctional systemic fungicide that rapidly penetrates all

plant parts. It works by preventing the sterol C-14 -demethylation of

2,4-methylenedihydrolanosterol, which is the precursor to ergosterol

in fungi. This inhibits the formation of the cell membrane, which

results in the pathogen’s death (Odds et al., 2003; Shishatskaya et al.,

2018). It is proved to be very ecient in decreasing deoxynivalenol

(DON) amounts when is using in the control of Fusarium Head Blight

(Sun et al., 2014). Unfortunately, given the harmful impact of synthetic

fungicides, such as the impact on ecosystems and human well-being,

it is required to seek to develop alternative control methods. Instead

of synthetic fungicides, employing biological control agents (BCAs)

to control FCR is viewed as a promising option (Khedher et al., 2020;

Lee et al., 2017).

Bacillus amyloliquefaciens and Bacillus subtilis are widely

recognized for their antifungal activity and they can be promising

BCAs against several plant diseases (Khedher et al., 2020; Lee et

al., 2017; Zhao et al., 2013; Zhu et al., 2020). BCAs can minimize

the frequency and amount of fungicide employed, which reduces the

danger of residues and resistance development, but their eciency

is often lower than that of chemical fungicides since it is frequently

unstable in eld circumstances and may be aected by a variety of

factors (Ji et al., 2019; Yu et al., 2017). The combination of Bacillus-

tebuconazole could lead to a practical method for controlling F.

culmorum in wheat elds. This study aimed to 1- evaluation of the

compatibility of tebuconazole and two antagonistic Bacillus strains

(B18 and S8) and then 2- evaluation of the ecacy of tebuconazole

and Bacillus strains (B18 and S8) each alone, and in combination,

for the control of F. culmorum in in vitro experiments (eect on

mycelium growth) and nally, in growth chamber experiments (eect

on induced FCR on durum wheat seedlings).

Material and methods

Plant material

The wheat seed sample namely: Boutaleb (Harvest season 2020-

2021), was kept at room temperature pending use.

Fungicides

Tebuconazole under its commercial formulation (Raxil 60FS®:

active ingredient content: 60 g.L

-1

), was purchased from CASAP

company specializes in agricultural products, Algiers - Algeria, which

it imports from Bayer CropScience. Stock solutions of tebuconazole

(7.5, 15, and 30 µg.mL

-1

) were made with distilled water and kept at

a temperature of 4 °C until use.

Antifungal bacteria

The bacterial strains Bacillus amyloliquefaciens (B18) and

Bacillus subtilis (S8) previously obtained from the wheat rhizosphere

from Bordj Bou Arreridj and Setif province respectively, Algeria.

Through macro and micro-morphological, biochemical, and

 =

( − ) × 100



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Oulmi et al. Rev. Fac. Agron. (LUZ). 2023 40(3): e234025

3-6 |

molecular assessment, the bacterial strains were identied (Bencheikh

et al., 2022). Bacterial strains were stored in 30 % glycerol at −20 °C.

Nutrient agar (NA) medium (Liolchem)

was used as the standard

growth medium for bacteria. They were cultured at 35 °C until

colonies appeared.

The bacterial suspension was prepared by the inoculation of the

bacteria in the Nutrient Broth (NB) medium for 18 hours. After that,

the suspension was diluted in physiological water, until the obtention

of a nal concentration of 10

colony forming units (CFU) per mL.

FCR agent, Fusarium culmorum

Using morphological and molecular characterization, the

previously obtained F. culmorum (FC) isolate, which was acquired

from durum wheat seeds, was identied (Bencheikh et al., 2022). The

F. culmorum isolate was maintained at 25 °C on Potato Sucrose Agar

(PSA). According to Bouanaka et al. (2021) procedure, the conidial

suspension was established, with some modications, according to

the following steps:

1-sterilized distilled water (10 mL) was added to the aerial

mycelium of a 15-day-old fungal colony. 2- The colony was carefully

scraped in order to retrieve all spores present within the aerial

mycelium. 3- The concentration of spores was estimated using the

Malassez counting cell (hemocytometer). 4- The appropriate dilutions

were made to obtain a nal concentration of 10

conidia.mL

-1

Tebuconazole and Bacillus strain compatibility

After the bacterial suspension was prepared, a tenfold dilution was

made in physiological water, then 100 µl from the 10

-3

, 10

-4

, and 10

-5

dilutions were strewn throughout Petri plates containing NA medium

(used as control) or NA medium added by 7.5, 15, and 30 µg.mL

-1

of tebuconazole fungicide. The highest concentration used was taken

from the concentrations suggested by the manufacturer, while the

other concentrations represent half and a quarter of this concentration.

Three plates were used for each test, the plates were incubated at 35

°C for 24 hours and then the colonies were enumerated.

In vitro tebuconazole tests for preventing the mycelial

development of F. culmorum in conjunction with Bacillus strains

Plugs of 6 mm-diameter from F. culmorum 7-days-old colony were

put on the center of fresh PDA plates containing tebuconazole alone

(mixed with the medium), Bacillus strain alone (streaked parallelly

at a distance of 2.5 cm from either side of the F. culmorum plug), or

both tebuconazole and Bacillus strain. Doses of tebuconazole were

7.5, 15, and 30 µg.mL

-1

, and the concentration of Bacillus strains was

CFU.mL

-1

PDA plates without tebuconazole and Bacillus strains were

used as controls. Each treatment received three dishes, which were

incubated at 25 °C for 5-7 days (until the mycelium in the control

dishes reaches the edge of the dish). The F. culmorum colonies’

diameters were measured. Using the Vincent (1947) formula, which

is given below, the percentages of mycelium growth inhibition by

tebuconazole and/or Bacillus strains in comparison with the control

were estimated.

Where: I= percent inhibition of mycelium growth; C= diameter

colony of the control,

T= diameter colony in the presence of tebuconazole and/or

Bacillus strain.

Growth chamber experiments

Pot experiments were performed in a controlled growth chamber

(22 °C ± 3, 12 h/12 h photoperiod of light and dark and 90 % relative

humidity, consecutively), in circular 8-cm-diameter pots containing

sterilized potting soil (FLORAVA

). In tubes containing NB, at 28

°C for 48 hours, Bacillus strain inoculum was prepared. To acquire

a nal concentration of 10

CFU.mL

-1

, the resulting suspension was

diluted with sterile physiological water. Tebuconazole was applied

with or without Bacillus strains at 7.5, 15, and 30 µg.mL

-1

Three seeds (cv. Boutaleb), surface sterilized (Bencheikh et al.,

2022) and submerged in the suspension of bacterial strain for 3h,

were displayed in each pot. Each seed received one 6 mm diameter

plug from a 7-day-old F. culmorum culture. The seeds and inocula

were carefully buried in sterile soil. The seeds without bacteria and

Fusarium plugs served as the negative control, whereas the direct

contact of a 6 mm-diameter Fusarium plug with each seed without

bacteria served as the positive control.

Concerning the chemical control, the tebuconazole solution was

applied to the surface-sterilized wheat seeds before soaking (with

the concentrations cited above) for 15 min. Each seed was in contact

with one 6-mm-diameter plug from a culture of F. culmorum that was

seven days old. On the other hand, seeds soaked in tebuconazole but

without Fusarium plugs were used as control.

Relating to the combined control, surface-sterilized wheat seeds

were submerged in the bacterial suspension supplemented with

tebuconazole solution (the same concentrations as the chemical

control test) for 3 h. One 6-mm diameter plug from the Fusarium

7-days-old culture was put in contact with each surface-sterilized

seed.

Three duplicate pots were employed in a factorial totally

randomized design, and the plants received two weekly waterings. The

seedlings were carefully removed after 21 days, and measurements of

the plant height and root weight were taken. For the evaluation of the

FCR, a disease scale from 0 to 3 was used (Grey and Mathre, 1984),

where:

0 = no discoloration in crown, 1 = 1–25 % of browning in crown,

2 = 25–50 % of browning in crown, 3= more than 50 % of browning

in crown. In accordance with the method, the McKinney (1923) index

was used to estimate the disease severity (DS):

Where: a= disease class, b= frequency, n =number of observations,

and N = greatest value of the empirical scale adopted (class 3).

The control ecacy of the FCR was calculated using the formula

proposed by Ji et al. (2019): Disease control ecacy (%) = ((C −

T)/C) × 100, where C= disease severity of control; T= disease severity

of treatment.

Statistical analysis

The IBM SPSS Statistics V25 program was used to conduct

the statistical analysis. With the exception of the growth chamber

experiment, all tests employed the one-way ANOVA instead of the

two-way ANOVA. The means were compared using the Duncan post

hoc test at the 0.05 threshold of risk.

Results and discussion

Compatibility of tebuconazole with Bacillus strains

After 24 hours of incubation, the growth of the two tested Bacillus

strains was not aected by tebuconazole using the NA medium

containing 7.5, 15, and 30 µg.mL

-1

of tebuconazole, and the number

of CFU.mL

-1

was not statistically dierent compared with the number

of CFU.mL

-1

of the untreated control (table 1).

 =

( − ) × 100



 =

(∑( × )/ × ) × 100

This scientic publication in digital format is a continuation of the Printed Review: Legal Deposit pp 196802ZU42, ISSN 0378-7818.

Rev. Fac. Agron. (LUZ). 2023, 40(3): e234025. July-September. ISSN 2477-9407.4-6 |

Table 1. Tebuconazole’s impact on Bacillus strain growth in NA

medium.

Concentration of tebuconazole

(µg.mL

-1

)

Number of CFU

.mL

-1

(x10

)

S8 B18

Control 0 47.97±2.11a

50.4±2.3a

F1/4 7.5 50.3±3.4a 48.3±1.4a

F1/2 15 48.47±1.05a 49.7±2.6a

F1 30 49.2±1.6a 51.33±1.7a

CFU: colony-forming unit; * Means in same column followed by the same letter

are not signicantly dierent at p < .05 as determined by Duncan test.

The attempt to combine antagonistic bacteria with synthetic

fungicides depends on their compatibility, which is usually dicult

to achieve. In this study, the growth of the two tested Bacillus strains

(S8 and B18) on the NA medium was not aected by tebuconazole at

a concentration of 30 µg.mL

-1

(table 1). This result indicates the full

compatibility of Bacillus strains with tebuconazole.

In vitro tests of tebuconazole in combination with Bacillus

strains for preventing F. culmorum’s mycelial expansion

Tebuconazole and the two tested Bacillus strains substantially

(p> .05) reduced the radial mycelial development of F. culmorum

on PDA dishes and when treated separately (table 2). Combining

tebuconazole with Bacillus strains signicantly increased the rate of

inhibition. As an example, the inhibition rate was 65.1 % with B18

strain alone, and 87.84 % with tebuconazole alone at 30 µg.mL

-1

(F1), but it increased to 93.73 % when the two treatments were

combined (table 2). Using Colby’s equation (Colby, 1967), each of

the anticipated inhibition rates were greater than those obtained when

combined with the Bacillus strains with the tebuconazole (table 2).

These results conrm that the combination of the tested Bacillus

strains and the tebuconazole gives a synergetic eect.

Table 2. Tebuconazole and other Bacillus strains individually and

in combination inuenced Fusarium culmorum growth

on PDA plates

Traitement

Diameter

(mm)

Inhibition

observed (%)

Inhibition

expected

(%)

Dierence

8.5f - - -

3.03e 64.31a - -

B18

2.94e 65.10a - -

1.03b 87.84d - -

F1/2

1.33c 84.31c - -

F1/4

1.67d 80.39b - -

S8_F1

0.53a 93.73

95.65 +1.93

S8_F1/2

0.57a 93.33

94.41 +1.07

S8_F1/4

0.6a 92.94

93.00 +0.06

B18_F1

0.53a 93.73

97.81 +4.08

B18_F1/2

0.57a 93.33

97.67 +4.34

B18_F1/4

0.6a 92.94

97.54 +4.60

The means in the same column that are separated by the same letter are not

considerably dierent p < .05 (Duncan test);

Each gure represents the average

of three separate trials;

Dierences, shown by a plus sign, indicate synergistic

eects (the percentage decrease seen minus the percentage reduction anticipated).

Growth chamber experiment

In the growth chamber experiments, tebuconazole at 7.5, 15,

and 30 µg.mL

-1

in combination with the Bacillus strains (B18 or

S8) signicantly reduced FCR severity and displayed a noticeable

control eect with rates of control ecacies of 59.09, 77.27, and

90.91 % respectively with S8 strain, and of 72.73, 81.82, and 95.45

% respectively with B18 strain (table 3). On the other hand, the

results showed that each of the tebuconazole (with all the tested

concentrations) and the Bacillus strains, can reduce the FCR severity,

but with greater eectiveness of the fungicide compared to the

Bacillus strains, where control ecacies rates were between 27.27

and 81.82 % for the tebuconazole, and between 9.09 and 13.64 % for

S8 and B18 strains respectively (table 3).

Through the previous results, it was found that the process of

combining tebuconazole with one of the two Bacillus strains gave

the desired results, which is to provide the best protection against the

FCR damages. In addition, the combination (Bacillus-tebuconazole)

increased signicantly both the weight of the fresh roots and plant

height in comparison to each treatment separately. On the contrary,

no signicant dierences have been observed between the control

ecacy rates of the S8 and the B18 strain each alone or in combination

with tebuconazole 15 and 30 µg.mL

-1

Table 3. Eect of tebuconazole and Bacillus strains against

Fusarium crown rot (FCR) in the growth chamber,

alone and in combination.

Traitement Seedlings

hight (cm)

Root fresh

weight (g)

Disease

severity (%)

Control

ecacy (%)

Control

11.82±0.44a

0.081±0.002a 81.48g -

F1/4 16.17±1.21bc 0.095±0.005bc 59.26f 27.27ab

F1/2 17.04±1.42c 0.108±0.01c 44.44e 45.45bc

F1 20.12±0.34d 0.155±0.01ef 14.81abc 81.82de

S8 14.50±1.78b 0.107±0.009c 74.07g 9.09a

S8 + F1/4 16.17±1.21bc 0.095±0.008bc 33.33de 59.09cd

S8 + F1/2 19.37±1.12d 0.145±0.011de 18.52bc 77.27de

S8 + F1 23.61±1.11e 0.197±0.025g 7.41ab 90.91e

B18 12.02±1.6a 0.090±0.012ab 70.37fg 13.64a

B18 + F1/4 17.04±1.42c 0.108±0.09c 22.22cd 72.73de

B18 + F1/2 20.12±0.34d 0.133±0.01d 14.81bc 81.82de

B18 + F1 21.49±1.01d 0.162±0.02f 3.70a 95.45e

Untreated durum wheat seeds with FC1 plug

;

The values represent the 9

replicates’ means and standard errors. At p < .05 (Duncan test), values in the same

columns that are followed by the same letter are not statistically dierent.

With the very intensive use of the fungicides, the resistance of

Fusarium isolates against these latter was highly increased (Yin et

al., 2009; Zhang et al., 2013). Therefore, it has become necessary

to look for more eective ways to control these pathogens in cereal

production. Tebuconazole has been used for controlling FHB in

several countries (Sun et al., 2014) and proved its very eectiveness

in decreasing the FHB methylenedihydrolanosterol and production of

deoxynivalenol (DON). Akgül and Erkiliç (2016), found that wheat

seed covered by tebuconazole decreased disease severity (DS) in

wheat seedlings’ crown compared to the non-treated seeds and they

concluded that it was the most eective fungicide with an ecacy rate

This scientic publication in digital format is a continuation of the Printed Review: Legal Deposit pp 196802ZU42, ISSN 0378-7818.

Oulmi et al. Rev. Fac. Agron. (LUZ). 2023 40(3): e234025

5-6 |

of 47.8 %. These ndings are in accordance with the obtained in vitro

and growth chamber results proving the eectiveness of tebuconazole

in the limitation of the FCR, where both the in vitro growth inhibition

rate and control ecacy were more than 80 %. Tebuconazole’s

mechanism of action was examined, and the ndings revealed that it

inhibits one of the ergosterol precursors in fungus (DMI’s fungicides),

the pathogen dies as a result of this activity because it prohibits the

establishment of the cell membrane (Odds et al., 2003).

The obtained results proved that the two Bacillus strains were

eective in reducing the radial growth of the F. culmorum, where

the inhibition was more than 60 %. Via a number of processes,

the species of the genus Bacillus can decrease the growth of

phytopathogenic fungi and even eradicate them. The most signicant

of these mechanisms is the release of antifungal compounds like

antibiotics, cyanides, and gas products like ammonia (Fira et al.,

2018; Lugtenberg et al., 2009; Zhao et al., 2013), and by generating

hydrolytic enzymes including cellulase, glucanase, chitinase, and

protease that demolish the cell wall (Brzezinska et al., 2020; Yanti et

al., 2021), or by enhancing plant development (Huang et al., 2020;

Kalam et al., 2020). B. amyloliquefaciens and B. subtilis have been

considered to be promising biocontrol agents with diverse capabilities.

Wang et al. (2016) reported that the B. amyloliquefaciens W19 strain

can produce bioorganic fertilizers “BIO6”, which could eectively

suppress FCR disease in bananas and promote plant growth. On the

other hand, B. subtilis was able to produce three natural substances

called lipopeptides, namely: fengycin, surfactin, and mycosubtilin.

These latter have shown an interesting antifungal activity each alone

or in combination compared to tebuconazole in the in vitro control

of two strains of Venturia inaequalis, the responsible agent of apple

scab (Desmyttere et al., 2019). Previous research revealed that the

B. subtilis S8 strain and B. amyloliquefaciens B18 were potential

agents in biocontrol of F. culmorum isolates (FC1 and FC2), by

producing several hydrolytic enzymes (amylase, pectinase, cellulase,

protease, and chitinase) and by producing antifungal metabolites like

siderophore and ammonia (Bencheikh et al., 2022). Based on all the

aforementioned advantages of the antagonistic bacteria and their anti-

fungal metabolites, they can be relied upon as ideal alternatives to

chemical fungicides (Desmyttere et al., 2019; Ji et al., 2019). While

being safe for the environment and eective against FCR, B. subtilis

and B. amyloliquefaciens strains eectiveness is often unstable in the

eld and may be aected by a variety of factors (Ji et al., 2019; Yu

et al., 2017). This nding is conrmed by the obtained results in the

growth chamber experiments where low control ecacy rates were

obtained when each Bacillus strain was used alone. Moreover, the

combination of Bacillus-tebuconazole revealed a synergistic eect in

inhibition of mycelium growth (table 2). Rotolo et al. (2018), declared

that the integration of synthetic fungicides and biocontrol agents

might be a successful plan of action more than the use of each alone.

The obtained results demonstrated also that control of Fusarium

crown rot was signicantly improved by combining tebuconazole

with Bacillus strains S8 or B18.

Conclusion

Through this study, it was proven that Bacillus strains (S8 and

B18) were completely compatible with tebuconazole and that they

can be combined without aecting each other’s growth. Furthermore,

a synergistic eect was obtained in the laboratory and growth chamber

experiments.

The control ecacy of the combination B18-tebuconazole, at half

of the concentration suggested by the manufacturer, was too close

to that of the combination B18-tebuconazole, at the concentration

suggested by the manufacturer, with no signicant dierences

with the combination B18-tebuconazole, at the quarter of the

concentration suggested by the manufacturer, or with the combination

S8-tebuconazole at half of the concentration suggested by the

manufacturer. These ndings showed that the amount of fungicide

suggested by the manufacturer can be reduced by half if combined

with the S8 strain or even by a quarter if combined with the B18

strain.

To determine the optimal application method of these biocontrol

agents (B18 and S8 strain) to control FCR in durum wheat production,

more research must be carried out on the environmental destiny and

behavior of Bacillus strains in the eld.

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