Biological control of plant diseases makes extensive use of the antagonists. The effectiveness of the control depends on the choice of an effective antagonist strains from criteria of implying a good knowledge of biological peculiarities of the material used. In this study, the antagonistic activities of nine isolates of Bacillus amyloliquefaciens and one isolate of Trichoderma harzianum were tested in vitro against seven strains of Colletotrichum acutatum, the causal agent of strawberry anthracnose. It was found that T. harzianum and B. amyloliquefaciens strains showed the ability to inhibit the mycelial growth of the pathogen by dual culture technique by more than 50 percent. The nonvolatile substances produced by the antagonists showed high inhibition percentages that are more than 99 percent, meanwhile their volatile compounds inhibited the mycelial growth of C. acutatum strains with low inhibition percentages starting from 30 percent except the substances produced by B. amyloliquefaciens Bc2 that showed a high inhibition percentage of more than 70 percent.
Dissemination of phytopathogens has been detected from nurseries to plantations through asymptomatic plants. Consumers require less chemical residues on products and many fungi have developed resistance to commonly used fungicides [
The antagonistic microorganisms such bacteria and fungi provide an alternative source of control of these pathogens. Biological control is an alternative to the use of chemical pesticides with the benefits of greater consumer acceptance and reduced environmental impact.
In addition, microbial diversity in soil is considered important for maintaining the sustainability of agricultural production systems. The quantity and activity of microorganisms is a determining factor for the productivity of all types of soil [
The selection of antagonists for biological control of plant diseases usually involves collecting and examining a large number of microbial isolates to increase the likelihood of finding a highly effective strain. Natural antagonists on host surfaces are a promising component of organic crop protection [
Natural compounds produced by bacteria and fungi antagonists are an important source of biopesticides and special conditions are required for the extraction and optimization of secondary metabolites that have an antibiotic effect [
In Morocco, the control of anthracnose in cultivated strawberry uses chemical pesticides in spite of their harmful effects. Many pathogenic microorganisms have developed resistance against chemical fungicides. This seriously hampers the management of phytopathologies. Considering the deleterious effects of chemical phytosanitary products on the maintenance of life systems, it is urgent to find an alternative for the management of pathogenic microorganisms. The present work aims to evaluate the antagonistic potential of Bacillus amyloliquefaciens and Trichoderma harzianum against Colletotrichum acutatum isolates.
Seven isolates of Colletotrichum acutatum (Ca1, Ca2, Ca3, Ca4, Ca5, Ca6 and Ca7) from naturally infected strawberries with anthracnose symptoms of strawberry field from Loukkos region (Larache, Morocco) were isolated and purified at the laboratory of plant biotechnology in Faculty of Science of Tetouan. They were grown on PDA (Potato Dextrose Agar) medium 7 to 10 days at 25˚C in the dark. Successive subcultures were carried out until complete purification. Their identification was performed by macroscopic and microscopic observations of the isolates using determination keys [
Nine bacterial strains were isolated by serial dilution from the soil of rhizosphere and strawberry plant roots of the Loukkos region (Larache, Morocco), and identified by [
The strain of Trichoderma harzianum (TR) was isolated from the soil in Petri dishes containing PDA using a spreading plate technique. The litter materials were grown in PDA culture medium for isolation. The TR strain was isolated in pure culture on PDA.
Trichoderma strain was evaluated against the seven isolates of Colletotrichum by the technique of dual culture [
L = [ ( C − T ) / C ] × 100
L: Percent inhibition of radial mycelial growth,
C: Radial growth of the control pathogen,
T: Radial growth of the pathogen in the presence of Trichoderma [
Strain’s name | Code ofstrain after identification | Similarity Percent % | Reference of strain |
---|---|---|---|
I1 | B. amyloliquefaciens Bc1 | 99.8% (1014/1016 pb) | LMG 22478 |
I2 | B. amyloliquefaciens Bc2 | 99.8% (1033/1035 pb) | CR-502 |
I3 | B. amyloliquefaciens Bc3 | 100% (1030/1030 pb) | CR-502 |
I18 | B. amyloliquefaciens Bc4 | 100% (1035/1035 pb) | CR-502 |
B3 | B. amyloliquefaciens Bc5 | 99.9% (1020/1022 pb) | LMG 22478 |
B12 | B. amyloliquefaciens Bc6 | 99.9% (1021/1022 pb) | LMG 22478 |
B24 | B. amyloliquefaciens Bc7 | 99.9% (1019/1020 pb) | LMG 22478 |
RA9 | B. amyloliquefaciens Bc8 | 99.9% (778/779 pb) | LMG 22478 |
RA12 | B. amyloliquefaciens Bc9 | 99.9% (1035/1036 pb) | CR-502 |
The dual culture was performed using a mixed culture: a 5-mm diameter mycelial disc from culture of Colletotrichum acutatum was placed at one side of a Petri dish containing PDA medium and a bacterial isolate was seeded at 3.5 cm from the fungal strain in the opposite way. The dishes were incubated for 7 days at 26˚C ± 2˚C. Each treatment was repeated three times. The percent inhibition of radial growth (ICRP) was measured according to the following formula:
PICR = Fungal growth control − Fungal growth near the antagonist Control fungal growth × 100
This method consists in inoculating the antagonist and the pathogenic, in two plates separated thereafter, an assembly is carried out by the superposition of two plates, antagonist in bottom and the pathogenic one in top, the junction between the two plates is ensured by layers of Parafilm in order to avoid all loss of volatile substances [
To study the effect of the volatile substances of Trichoderma harzianum on the growth of pathogenic fungi on PDA, two disks (8 mm in diameter), one of the antagonist agent (Trichoderma harzianum) and the other of the pathogen,were placed in the center of two Petri dishes containing the PDA medium. The lids were removed aseptically, and then the bottom of the plate containing Trichoderma was placed below the one containing the phytopathogen, and the two juxtaposed plates were unsured by layers of Parafilm. The cultures were incubated for 4 days at 25˚C. The test was repeated three times [
The test of volatile antifungal compounds was carried out according to the method used by [
Each Erlenmeyer flask containing 15 ml PDB (Potato Dextrose Broth) was inoculated with a 5 mm diameter disc from the Trichoderma harzianum culture and incubated for 12 days at 25˚C ± 2˚C with shaking at 200 rpm. After this time, the suspension was filtered to remove the mycelium and spores then adjust concentration to 108 conidia/ml by using Malassez cell.
The bacterial strain was grown on Luria Bertani Agar (LBA) 24 h at 28˚C. Then, using a sterile loop, a bacterial culture is prepared by inoculating 50 ml of the liquid medium Luria Bertani Broth with a colony of the strain studied, in a 250 ml Erlenmeyer flask. The cultures were incubated at room temperature with 125 rpm shaking for 2 days. After incubation, a volume of 5 ml of bacterial suspension is centrifuged at 4000 rpm for 10 min. The cells were washed, in the same volume of 5 ml of sterile distilled water, twice, using the centrifuge at the same conditions mentioned above [
The bacterial cells were then suspended in 5 ml of sterile physiological saline, and the suspension was adjusted to 3 × 108 cell/ml according to the Mac Farland scale [
The phytopathogenic fungi (C. acutatum) were inoculated (106 conidia) and cultured in 50 ml of PDB medium for one week at 28˚C at 200 rpm with presence (1% (v/v)) of the antagonist suspensions (B. amyloliquefaciens and TR) already prepared previously. For the control, distilled water was used instead of the antagonists.
The biomass was separated, dried 24 h at 100˚C and weighed to obtain the dry weight. Values were expressed as a percentage:
% inhibition = 1 − Dry matter in the presence of the antagonist Dry matter of control × 100
Three repetitions were performed.
In vitro comparison showed that Trichoderma harzianum (
Direct challenge of Trichoderma with C. acutatum isolates resulted in up to 86.64% inhibition of the Ca1 isolate (
All bacterial isolates produce volatile substances inhibiting mycelial growth of C.
acutatum isolates. The percentage of inhibition did not exceed 42% for the Bc1, Bc3, Bc4, Bc5, Bc6, Bc7, Bc8 and Bc9 isolates, but in the case of the Bc2 isolate, the inhibition of the Ca5 isolate reached up to at 72% (
The volatile substances of Trichoderma harzianum have a variable inhibitory effect depending on the degree of resistance of the isolates of C. acutatum, the percentage inhibition of Ca3, Ca4 and Ca6 exceeding 46% whereas for the other isolates the inhibition is less than 44% (
Bacillus amyloliquefaciens filtrates have a strong influence on the growth of Colletotrichum acutatum isolates in liquid medium and the percentage inhibition varies from one bacterial isolate to another insignificantly (
In this study, Bacillus amyloliquefaciens and Trichoderma harzianum were found to have inhibitory effects against the pathogen Colletotrichum acutatum
Ca1 | Ca2 | Ca3 | Ca4 | Ca5 | Ca6 | Ca7 | |
---|---|---|---|---|---|---|---|
Bc1 | 76.21 ± 2.53b | 77.00 ± 1.14c | 76.86 ± 0.92b | 77.49 ± 0.30bc | 76.29 ± 2.61c | 76.97 ± 0.60bc | 70.38 ± 0.38b |
Bc2 | 88.40 ± 0.68a | 88.23 ± 0.29a | 82.31 ± 1.03a | 83.63 ± 0.59a | 92.33 ± 0.66a | 81.73 ± 0.10a | 79.80 ± 0.21a |
Bc3 | 64.07 ± 0.85c | 64.20 ± 1.33f | 61.26 ± 0.15e | 64.62 ± 1.46d | 82.07 ± 0.40b | 59.25 ± 0.75f | 55.23 ± 1.23d |
Bc4 | 84.62 ± 2.16a | 71.14 ± 0.45e | 77.76 ± 1.83b | 75.77 ± 0.33bc | 82.02 ± 1.32b | 71.85 ± 0.94de | 65.34 ± 1.34c |
Bc5 | 58.94 ± 1.05c | 64.81 ± 0.71f | 64.01 ± 1.05e | 65.93 ± 1.02d | 75.31 ± 1.63cd | 72.23 ± 1.32de | 65.93 ± 2.07c |
Bc6 | 75.65 ± 0.21b | 73.68 ± 0.46de | 76.27 ± 0.34b | 74.56 ± 0.88c | 73.47 ± 1.54cd | 75.71 ± 1.16bcd | 80.82 ± 0.82a |
Bc7 | 72.46 ± 0.53b | 84.04 ± 0.44b | 72.17 ± 0.06c | 80.12 ± 1.17ab | 71.53 ± 1.35d | 73.78 ± 1.05cd | 67.70 ± 1.70bc |
Bc8 | 84.05 ± 1.59a | 74.46 ± 2.05cd | 68.18 ± 0.34d | 66.38 ± 3.22d | 81.19 ± 0.49b | 77.98 ± 1.62b | 71.72 ± 0.28b |
Bc9 | 75.35 ± 0.10b | 77.00 ± 0.56cd | 74.77 ± 0.70bc | 67.25 ± 2.34d | 72.08 ± 0.14cd | 69.13 ± 1.79e | 68.67 ± 1.33bc |
Ca1 | Ca2 | Ca3 | Ca4 | Ca5 | Ca6 | Ca7 | |
---|---|---|---|---|---|---|---|
Bc1 | 37.50 ± 2.50b | 36.50 ± 0.50b | 35.00 ± 0.00c | 35.00 ± 2.00b | 40.00 ± 0.00b | 40.00 ± 0.00b | 41.00 ± 4.00b |
Bc2 | 71.00 ± 1.00a | 70.00 ± 1.00a | 71.00 ± 2.00a | 71.50 ± 1.50a | 72.00 ± 2.00a | 70.00 ± 1.00a | 70.50 ± 0.50a |
Bc3 | 37.00 ± 1.00b | 36.50 ± 0.50b | 36.00 ± 1.00bc | 37.50 ± 2.50b | 36.00 ± 2.00b | 36.00 ± 4.00b | 38.00 ± 2.00b |
Bc4 | 38.00 ± 1.00b | 37.50 ± 2.50b | 37.00 ± 0.00bc | 37.50 ± 0.50b | 36.00 ± 0.00b | 38.50 ± 1.50b | 38.50 ± 1.50b |
Bc5 | 37.50 ± 2.50b | 38.50 ± 1.50b | 38.50 ± 1.50bc | 37.50 ± 1.50b | 36.50 ± 1.50b | 38.50 ± 2.50b | 38.50 ± 1.50b |
Bc6 | 33.50 ± 1.50b | 39.00 ± 1.00b | 39.50 ± 0.50b | 39.00 ± 1.00b | 38.00 ± 2.00b | 38.00 ± 3.00b | 39.00 ± 2.00b |
Bc7 | 37.00 ± 2.00b | 38.00 ± 1.00b | 39.00 ± 1.00b | 38.50 ± 1.50b | 36.00 ± 1.00b | 35.00 ± 0.00b | 36.50 ± 1.50b |
Bc8 | 37.50 ± 2.50b | 38.50 ± 1.50b | 38.50 ± 1.50bc | 36.00 ± 1.00b | 37.00 ± 2.00b | 36.00 ± 1.00b | 36.50 ± 0.50b |
Bc9 | 41.00 ± 5.00b | 38.00 ± 1.00b | 36.00 ± 1.00bc | 36.00 ± 1.00b | 35.50 ± 1.50b | 38.50 ± 3.50b | 39.50 ± 2.50b |
Ca1 | Ca2 | Ca3 | Ca4 | Ca5 | Ca6 | Ca7 | |
---|---|---|---|---|---|---|---|
Bc1 | 99.16 ± 0.09a | 99.39 ± 0.02a | 99.35 ± 0.04a | 99.15 ± 0.08a | 99.20 ± 0.04a | 99.27 ± 0.06a | 99.19 ± 0.13a |
Bc2 | 99.44 ± 0.06a | 99.58 ± 0.02a | 99.66 ± 0.04a | 99.46 ± 0.03a | 99.64 ± 0.02a | 99.69 ± 0.02a | 99.61 ± 0.08a |
Bc3 | 99.22 ± 0.03a | 99.41 ± 0.00a | 99.35 ± 0.04a | 99.28 ± 0.05a | 99.18 ± 0.02a | 99.27 ± 0.06a | 99.09 ± 0.03a |
Bc4 | 99.16 ± 0.09a | 99.36 ± 0.05a | 99.29 ± 0.02a | 99.20 ± 0.03a | 99.14 ± 0.02a | 99.40 ± 0.06a | 99.17 ± 0.10a |
Bc5 | 99.29 ± 0.03a | 99.39 ± 0.02a | 99.27 ± 0.04a | 99.25 ± 0.03a | 99.22 ± 0.06a | 99.31 ± 0.02a | 99.04 ± 0.03a |
Bc6 | 99.16 ± 0.09a | 99.34 ± 0.07a | 99.33 ± 0.02a | 99.15 ± 0.08a | 99.11 ± 0.04a | 99.35 ± 0.02a | 99.14 ± 0.08a |
Bc7 | 99.10 ± 0.16a | 99.31 ± 0.10a | 99.27 ± 0.04a | 99.30 ± 0.08a | 99.24 ± 0.08 a | 99.44 ± 0.10a | 99.22 ± 0.16a |
Bc8 | 99.13 ± 0.12a | 99.44 ± 0.02a | 99.38 ± 0.06a | 99.18 ± 0.05a | 99.18 ± 0.02a | 99.25 ± 0.08a | 99.01 ± 0.05a |
Bc9 | 99.19 ± 0.06a | 99.36 ± 0.05a | 99.38 ± 0.06a | 99.36 ± 0.13a | 99.26 ± 0.11a | 99.48 ± 0.15 a | 99.11 ± 0.05 a |
causing anthracnose in cultivated strawberry (Fragaria x ananassa Duch.). Biological control is therefore an alternative to the use of chemical plant protection products.
The sensitivity of the isolates of Colletotrichum acutatum appears variable for both bacterial and fungal antagonists. All bacterial isolates and Trichoderma harzianum reduce mycelial growth of C. acutatum isolates. The growth of Trichoderma harzianum seems more marked than that of Colletotrichum acutatum.
The Bc2 isolate has superior antagonistic activity: the Bc2 isolate inhibits the growth of Strain Colletotrichum acutatum from 79.8% to 92.33% in direct confrontation and its volatile substances by 70% to 72% the mycelial growth. The nonvolatile substances of all isolates indiscriminately inhibit the development of Colletotrichum acutatum isolates.
Several studies have been made to evaluate the in vitro antagonism of Bacillus amyloliquefaciens with respect to the development of different phytopathogenic agents: [
The results of this study revealed that Trichoderma harzianum has a high inhibitory effect against different isolates of C. acutatum at three life stages. Its growth during the confrontation is faster than that of the pathogen (
The use of products based on Trichoderma harzianum and Bacillus amyloliquefaciens in the fight against phytopathogenic fungi is safe for farmers and consumers and good for the environment. However, much remains to be done
to develop a stable, cost-effective formulation that is easy to produce and apply.
The authors declare no conflicts of interest regarding the publication of this paper.
Es-Soufi, R., Tahiri, H., Azaroual, L., El Oualkadi, A., Martin, P., Badoc, A. and Lamarti, A. (2020) In Vitro Antagonistic Activity of Trichoderma harzianum and Bacillus amyloliquefaciens against Colletotrichum acutatum. Advances in Microbiology, 10, 82-94. https://doi.org/10.4236/aim.2020.103008