The effect of nine isolates of Bacillus amyloliquefaciensand one strain of Trichoderma harzianum, TR, on mycelial growth and germination of Colletotrichum acutatum were studied. The nine isolates were identified as Bacillus amyloliquefaciens. The efficacy of isolates was tested, at different concentrations. Results showed that one Bacillus isolates (Bc2) and TR were more effective at the lower concentration tested (3 × 10 5 CFU/ml and 10 5 conidia/ml).
Like all living organisms, plants have to cope with infections and diseases following the attacks of a mass of plant pathogens. These diseases may be causing only minor reduction in plant growth and capacity or they can cause much more serious damage leading to plant death in the worst case [
The root zone is surrounded by many microorganisms producing molecules; they contain some volatile compounds that can affect the growth via different mechanisms, such as biochemical signals causing local defense reactions or systemic resistance [
Bacillus is a genus of bacteria forming endospores shaped Gram positive rod [
The objective of this work is the evaluation of the effectiveness of suspensions of Trichoderma harzianum and Bacillus amyloliquefaciens at various concentrations to inhibit the growth of Colletotrichum acutatum.
The conidial suspension of the plant pathogen Colletotrichum acutatum (Ca6) was obtained by adding 10 ml of sterile distilled water in Petri dishes containing cultures of C. acutatum of 10 to 15 days old on PDA [
Each Erlenmeyer flask containing 15 ml of PDB (Potato Dextrose Broth) medium was inoculated with a disc of 5 mm diameter from the culture of Trichoderma harzianum and incubated for 12 days at 25˚C ± 2˚C with stirring at 200 rpm. After this period, the suspension was filtered using Whatman paper (150 mm) to remove the spores and mycelium. The suspension was adjusted to 108 spores/ml using a Malassez cell.
Antagonist each bacterial isolate was grown on PCA for 24 h at 37˚C. After, and by using a sterile loop, a bacterial culture was prepared by inoculating 50 ml of the LBB, by a colony of the isolate studied; in an Erlenmeyer flask of 100 ml. Cultures were incubated at room temperature with stirring at 125 rpm for two days. After incubation, a volume of 5 ml of bacterial suspension was centrifuged at 3000 rpm for 15 min. Cells were washed twice in the same volume of sterile physiological water used, using the centrifuge under the same conditions cited in the above instructions. The bacterial cells were then suspended in 5 ml of sterile physiological water and the suspension was adjusted to 3 × 108 CFU/ml depending on the scale of Mac Farland [
To evaluate the effect of the antagonist on the pathogenic agent in different concentrations, a disc 5 mm PDA culture medium diameter already prepared and poured into Petri dishes was cut with a prevails sterile in order to perform well in the middle of the dish [
Percent inhibition was calculated according to following formula:
P I = ( D t − D i ) D t × 100
PI: Percentage inhibition.
Dt: diameter of the mycelial colony of C. acutatum in the absence of antagonist (control).
Di: diameter of the mycelial colony of Cacutatum in the presence of the antagonist.
The number of repetitions is three dishes for each concentration combination-strain, and the test was repeated three times.
The effect of the antagonists on spore germination of Colletotrichum acutatum was tested in sterile PDB (Potato Dextrose Broth). A volume of 200 µl of antagonist suspension already prepared and adjusted to various concentrations is mixed with 200 µl of Ca6 of suspension into 10 ml tubes containing 5 ml of PDB liquid medium. The tubes are incubated for 24 h at 25˚C.
To estimate whether there is a difference in the activity of the antagonists over time on the implementation of the spores germination of C. acutatum, this activity was evaluated either by mixing the two suspensions (antagonist and pathogen) simultaneously t0, adding the suspension conidial suspensions after four hours of incubation of antagonists t4 [
The results of the determination and the effect of different concentrations of antagonists on mycelial growth of Colletotrichum acutatum gave several percentage grades of development inhibiting this pathogen. The comparison of the percentages of inhibition of mycelial growth based bacterial concentration generally shows, for all isolates that inhibition of mycelial growth decreases with decreasing bacterial concentrations (
At 3 × 104 CFU/ml, mycelial growth was inhibited to 86.67% by Bc2 isolate; the percentages of inhibition obtained from other isolates are less than 50%.
At 3 × 103 CFU/ml, the efficiency of isolates begin to decrease at lower inhibitions to 30% apart from the Bc2 isolate that gave an inhibition of 68.33%.
At 3 × 102 CFU/ml, the percentages of inhibition obtained by Bc1 isolates, Bc3, Bc4 Bc6, Bc7, and Bc8 Bc9 is below 10%, the Bc2 isolate showed an inhibition of 29.50%, Isolate Bc5 showed no activity on the pathogen growth.
At the low concentration of 3 × 101 CFU/ml, all strains have a minimum inhibition of 0 to 0.33%, while the strain Bc2 inhibition 18.33.
Bacterial strains | Concentration CFU/ml | |||||||
---|---|---|---|---|---|---|---|---|
3 × 108 | 3 × 107 | 3 × 106 | 3 × 105 | 3 × 104 | 3 × 103 | 3 × 102 | 3 × 101 | |
Bc1 | 100.00 ± 0.00a | 100.00 ± 0.00a | 100.00 ± 0.00a | 58.33 ± 2.01bc | 47.50 ± 1.44c | 15.00 ± 1.23c | 8.33 ± 0.20cd | 0.00 ± 0.00b |
Bc2 | 100.00 ± 0.00a | 100.00 ± 0.00a | 100.00 ± 0.00a | 100.00 ± 0.00a | 86.67 ± 1.00a | 68.33 ± 0.41a | 29.50 ± 1.61a | 8.83 ± 1.92a |
Bc3 | 100.00 ± 0.00a | 100.00 ± 0.00a | 100.00 ± 0.00a | 56.67 ± 1.64bc | 46.50 ± 2.20c | 15.83 ± 1.35c | 8.00 ± 1.32cd | 0.00 ± 0.00b |
Bc4 | 100.00 ± 0.00a | 100.00 ± 0.00a | 100.00 ± 0.00a | 49.17 ± 0.83c | 47.50 ± 1.63c | 19.17 ± 0.83bc | 11.67 ± 1.67bc | 0.33 ± 0.18b |
Bc5 | 100.00 ± 0.00a | 73.83 ± 0.73b | 50.83 ± 1.20b | 34.67 ± 0.85d | 13.50 ± 1.00e | 0.67 ± 0.44d | 0.00 ± 0.00f | 0.00 ± 0.00b |
Bc6 | 100.00 ± 0.00a | 100.00 ± 0.00a | 100.00 ± 0.00a | 20.00 ± 0.33e | 20.00 ± 1.44de | 15.00 ± 0.25c | 5.00 ± 1.44de | 0.50 ± 0.35b |
Bc7 | 100.00 ± 0.00a | 100.00 ± 0.00a | 100.00 ± 0.00a | 32.50 ± 1.44de | 23.67 ± 0.73d | 15.85 ± 0.20c | 9.42 ± 1.54cd | 0.17 ± 0.21b |
Bc8 | 100.00 ± 0.00a | 100.00 ± 0.00a | 100.00 ± 0.00a | 53.33 ± 0.46c | 21.67 ± 2.2d | 12.50 ± 1.44c | 2.17 ± 0.44ef | 0.00 ± 0.00b |
Bc9 | 100.00 ± 0.00a | 100.00 ± 0.00a | 100.00 ± 0.00a | 68.33 ± 0.41b | 62.83 ± 4.33b | 25.83 ± 0.83b | 15.00 ± 1.44b | 0.00 ± 0.00b |
The averages of the same column with the same letter do not significantly differ from each other at the 5% threshold.
Trichoderma harzianum has completely inhibited mycelial growth of C. acutatum 108, 107 and 106 spores/ml. Its percent inhibition at 105 spores/ml reached 99.33% and 95.66%; 63.66%; 30.00% and 10.27% at 104, 103, 102 and 101 spores/ml respectively (
The results allowed us to infer three minimum inhibitory concentrations of bacterial suspensions ie 3 × 108 CFU/ml (Bc5 isolate), 3 × 106 CFU/ml (Bc1, BC3, Bc4, Bc6, BC7, BC8 and Bc9) and weak inhibitory concentration 3 × 105 CFU/ml represented by Bc2 (
Antagonist | Minimum inhibitory concentration | Unit |
---|---|---|
Bacillus bc1 | 3 × 106 | |
Bacillus Bc2 | 3 × 105 | |
Bacillus BC3 | 3 × 106 | |
Bacillus Bc4 | 3 × 106 | CFU/ml |
Bacillus Bc5 | 3 × 108 | |
Bacillus bc6 | 3 × 106 | |
Bacillus BC7 | 3 × 106 | |
Bacillus BC8 | 3 × 106 | |
Bacillus Bc9 | 3 × 106 | |
Trichoderma harzianum | 106 | Spores/ml |
The results of this study show the percentage of inhibition of germination by B. amyloliquefaciens strains varies depending on the concentration of the bacterial suspension and the application time (t0, t4 and t8) (Tables 3-5).
The minimum inhibitory concentrations of bacterial isolates remain the same in every application time but there are some isolates that are more effective at t4 (Bc2, BC3, Bc4, Bc6 and BC7) other at t0 (Bc1, Bc5, BC8 and Bc9). The application at t8 reduces the effectiveness of the isolates to inhibit the germination of spores (Tables 3-5).
At 3 × 108 CFU/ml and 3 × 107 CFU/ml all bacterial isolates completely inhibit spore germination of Ca6 in all application time except Bc5 isolate that presented inhibition of 75.33%, 55.67% and 30.00% in t8, t4 and t8 respectively 3 × 107 CFU/ml (Tables 3-5).
Bacterial strains | Concentration CFU/ml | |||||||
---|---|---|---|---|---|---|---|---|
3 × 108 | 3 × 107 | 3 × 106 | 3 × 105 | 3 × 104 | 3 × 103 | 3 × 102 | 3 × 101 | |
Bc1 | 100.00 ± 0.00a | 100.00 ± 0.00a | 96.67 ± 0.88a | 72.33 ± 0.88d | 60.00 ± 0.58f | 46.30 ± 2.02d | 29.67 ± 1.48b | 0.00 ± 0.00b |
Bc2 | 100.00 ± 0.00a | 100.00 ± 0.00a | 100.00 ± 0.00a | 95.67 ± 2.03a | 83.33 ± 1.07a | 68.00 ± 1.15a | 54.00 ± 2.31a | 17.00 ± 1.53a |
Bc3 | 100.00 ± 0.00a | 100.00 ± 0.00a | 92.67 ± 1.45b | 63.00 ± 1.73e | 58.00 ± 1.15b | 16.00 ± 1.15b | 3.33 ± 0.88e | 0.33 ± 0.33b |
Bc4 | 100.00 ± 0.00a | 100.00 ± 0.00a | 86.33 ± 1.83c | 56.00 ± 1.53f | 52.00 ± 1.22d | 3.00 ± 0.58b | 2.00 ± 1.00e | 0.00 ± 0.00b |
Bc5 | 100.00 ± 0.00a | 75.33 ± 1.45b | 37.33 ± 1.20d | 27.33 ± 1.45g | 10.00 ± 1.54f | 2.67 ± 0.33d | 2.00 ± 0.58e | 0.00 ± 0.00b |
Bc6 | 100.00 ± 0.00a | 100.00 ± 0.00a | 60.67 ± 1.74e | 52.67 ± 1.2f | 52.33 ± 1.85e | 24.00 ± 1.00c | 19.67 ± 0.88c | 0.00 ± 0.00b |
Bc7 | 100.00 ± 0.00a | 100.00 ± 0.00a | 92.67 ± 1.45b | 89.33 ± 1.2b | 75.33 ± 1.45c | 66.00 ± 0.58b | 22.33 ± 1.45c | 0.00 ± 0.00b |
Bc8 | 100.00 ± 0.00a | 100.00 ± 0.00a | 88.67 ± 1.45c | 82.33 ± 1.45c | 39.33 ± 1.76e | 19.00 ± 0.65d | 5.33 ± 1.96e | 0.00 ± 0.00b |
Bc9 | 100.00 ± 0.00a | 100.00 ± 0.00a | 88.00 ± 1.53c | 72.00 ± 1.15d | 28.67 ± 0.88d | 29.33 ± 1.45c | 14.33 ± 1.2d | 0.00 ± 0.00b |
The averages of the same column with the same letter do not significantly differ from each other at the 5% threshold.
Bacterial strains | Concentration CFU/ml | |||||||
---|---|---|---|---|---|---|---|---|
3 × 108 | 3 × 107 | 3 × 106 | 3 × 105 | 3 × 104 | 3 × 103 | 3 × 102 | 3 × 101 | |
Bc1 | 100.00 ± 0.00a | 100.00 ± 0.00a | 90.00 ± 0.58cd | 17.33 ± 1.45d | 10.67 ± 1.20c | 5.67 ± 0.88b | 4.67 ± 1.45c | 0.00 ± 0.00c |
Bc2 | 100.00 ± 0.00a | 100.00 ± 0.00a | 100.00 ± 0.00a | 100.00 ± 0.00a | 95.33 ± 1.76a | 85.66 ± 1.20a | 35.66 ± 0.88a | 18.33 ± 0.88a |
Bc3 | 100.00 ± 0.00a | 100.00 ± 0.00a | 92.00 ± 2.08c | 91.67 ± 1.20e | 66.00 ± 1.15c | 43.33 ± 0.88e | 29.33 ± 2.04ab | 4.00 ± 1.15b |
Bc4 | 100.00 ± 0.00a | 100.00 ± 0.00a | 91.67 ± 1.20c | 60.67 ± 1.08f | 56.66 ± 1.45d | 14.00 ± 1.15f | 7.67 ± 0.88c | 0.00 ± 0.00c |
Bc5 | 100.00 ± 0.00a | 55.67 ± 1.45b | 25.00 ± 1.15g | 14.67 ± 1.92g | 4.33 ± 0.88g | 3.00 ± 1.15f | 0.67 ± 0.33d | 0.00 ± 0.00c |
Bc6 | 100.00 ± 0.00a | 100.00 ± 0.00a | 72.00 ± 2.31e | 36.67 ± 0.63f | 36.67 ± 1.45d | 24.33 ± 0.88d | 4.33 ± 1.45c | 0.00 ± 0.00c |
Bc7 | 100.00 ± 0.00a | 100.00 ± 0.00a | 96.00 ± 1.15b | 96.33 ± 0.77b | 92.00 ± 1.53b | 67.33 ± 1.20b | 26 ± 2.31b | 0.00 ± 0.00c |
Bc8 | 100.00 ± 0.00a | 100.00 ± 0.00a | 66.00 ± 1.15f | 36.67 ± 0.67c | 15.33 ± 1.76e | 5.00 ± 1.15e | 5 ± 1.15c | 0.00 ± 0.00c |
Bc9 | 100.00 ± 0.00a | 100.00 ± 0.00a | 86.33 ± 0.88d | 75.33 ± 0.33d | 69.33 ± 1.20f | 35.33 ± 1.20c | 2.33 ± 1.86c | 0.00 ± 0.00c |
The averages of the same column with the same letter do not significantly differ from each other at the 5% threshold.
Bacterial strains | Concentration CFU/ml | |||||||
---|---|---|---|---|---|---|---|---|
3 × 108 | 3 × 107 | 3 × 106 | 3 × 105 | 3 × 104 | 3 × 103 | 3 × 102 | 3 × 101 | |
Bc1 | 100.00 ± 0.00a | 100.00 ± 0.00a | 75.00 ± 1.53c | 13.33 ± 2.40f | 6.33 ± 1.45f | 3.67 ± 0.68e | 1.00 ± 0.57b | 0.00 ± 0.00b |
Bc2 | 100.00 ± 0.00a | 100.00 ± 0.00a | 100 ± 0.00a | 92.87 ± 1.45a | 81.67 ± 1.68a | 61.00 ± 1.15a | 25.00 ± 1.53a | 10.33 ± 0.88a |
Bc3 | 100.00 ± 0.00a | 100.00 ± 0.00a | 85.33 ± 0.88b | 70.33 ± 1.34b | 23.00 ± 1.15bc | 6.66 ± 0.66d | 1.33 ± 0.88b | 0.00 ± 0.00b |
Bc4 | 100.00 ± 0.00a | 100.00 ± 0.00a | 75.00 ± 1.15c | 49.33 ± 1.72c | 24.33 ± 1.45b | 2.33 ± 0.79ef | 0.00 ± 0.00c | 0.00 ± 0.00b |
Bc5 | 100.00 ± 0.00a | 30.00 ± 0.58b | 24.67 ± 1.45f | 12.00 ± 1.15f | 2.00 ± 1.15g | 1.33 ± 0.14g | 0.00 ± 0.00c | 0.00 ± 0.00b |
Bc6 | 100.00 ± 0.00a | 100.00 ± 0.00a | 45.33 ± 1.20e | 19.00 ± 1.73e | 17.33 ± 1.77d | 8.33 ± 0.85c | 0.00 ± 0.00c | 0.00 ± 0.00b |
Bc7 | 100.00 ± 0.00a | 100.00 ± 0.00a | 83.67 ± 2.33b | 76.00 ± 2.31a | 58.67 ± 2.41ab | 10.67 ± 0.03b | 0.00 ± 0.00c | 0.00 ± 0.00b |
Bc8 | 100.00 ± 0.00a | 100.00 ± 0.00a | 59.67 ± 1.20d | 20.33 ± 0.88e | 6.67 ± 1.20e | 3.33 ± 1.45e | 0.00 ± 0.00c | 0.00 ± 0.00b |
Bc9 | 100.00 ± 0.00a | 100.00 ± 0.00a | 71.67 ± 0.88c | 45 ± 2.64d | 12.67 ± 1.76c | 6.33 ± 1.76d | 0.00 ± 0.00c | 0.00 ± 0.00b |
The averages of the same column with the same letter do not significantly differ from each other at the 5% threshold.
Inhibition of spore germination of Ca6 decreases for other decreasing concentrations tested from 3 × 106 CFU/ml. The Bc2 isolate remains effective with inhibitory percent of 95.67%, 100% and 92.87 in t0, t4 and t8 respectively at 3 × 105 CFU/ml (Tables 3-5) and Bc5 is the least effective to inhibit the growth of C. acutatum in all the application times.
T. harzianum completely inhibits the germination of spores of Ca6 at 108, 107, 106 and 105 spores/ml in all the application time. Its inhibition percent at 104 spores/ml reached 97.33; 98.68 and 99.45% in t0, t4 and t8 respectively, since this concentration is decreasing, the inhibition decreases slightly in all the application time but remains important in the t8 (
Inhibition of spore germination of bacterial isolates decreased from 3 × 106 CFU/ml except inhibition Bc5 isolate that starts to decrease from 3 × 107 CFU/ml and isolate Bc2 retains its power inhibitor at 3 × 106 CFU/ml in all the application times (
And the minimal inhibitory concentration of T. harzianum is 105 spores/ml (
The microorganisms that develop in the rhizosphere are ideal for use as biological control agents, by what the rhizosphere provides as the first defense against the attack of plant pathogens [
In recent years, various Bacillus spp. such as B. amyloliquefaciens, B. subtilis, B. atrophaeus, B. amyloliquefaciens, B. thuringiensis, and B. pumilis were used as potential biocontrol agents against different Colletotrichum spp. T [
In this study, the effect of different concentrations of inoculums of nine isolates of B. amyloliquefaciens and an isolate of T. harzianum was tested in vitro
bacterial strains | Application time | Concentration | ||
---|---|---|---|---|
simultaneous t0 | preventive | |||
t4 | t8 | |||
bc1 | 3 × 107 | 3 × 107 | 3 × 107 | |
Bc2 | 3 × 106 | 3 × 106 | 3 × 106 | |
BC3 | 3 × 107 | 3 × 107 | 3 × 107 | |
Bc4 | 3 × 107 | 3 × 107 | 3 × 107 | CFU/ml |
Bc5 | 3 × 108 | 3 × 108 | 3 × 108 | |
bc6 | 3 × 107 | 3 × 107 | 3 × 107 | |
BC7 | 3 × 107 | 3 × 107 | 3 × 107 | |
BC8 | 3 × 107 | 3 × 107 | 3 × 107 | |
Bc9 | 3 × 107 | 3 × 107 | 3 × 107 | |
Trichoderma harzianum | 106 | 106 | 106 | Spores/ml |
vis-à-vis a mycelial growth and spores germination of phytopathogenic agent to find the potential of biological control at a low minimal inhibitory concentration against C. acutatum.
We found all the studied antagonists can inhibit or reduce mycelial growth and spore germination of C. acutatum Ca6. The effectiveness of B. amyloliquefaciens isolates to control against C. acutatum isolate differs from one to another, and we deduce that Bc2 submitted a total inhibition of mycelial growth of plant pathogen to MIC = 3 × 105 CFU/ml. And the minimal inhibitory concentration of T. harzianum obtained for inhibiting the mycelial growth of Ca6 is 106 spores/ml.
Comparing the minimal inhibitory concentrations of spore germination of plant pathogen antagonistic, we observe that the majority of isolates give maximum inhibition at a concentration of 3 × 107 CFU/ml within three days of application, except for the isolate Bc2 to 3 × 106 CFU/ml and Bc5 at 3 × 108 CFU/ml. These concentrations are higher than those obtained in inhibiting mycelial growths which are 3 × 105 CFU/ml for Bc2 and 3 × 106 CFU/ml for the other isolates, except for Bc5 isolate that remains the same. Hamdache et al., [
T. harzianum has a greater efficacy against the development of Ca6, even with the variation of its minimal inhibitory concentration of the mycelial growth and sporesgermination of the plant pathogen.
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) Effect of Different Concentrations of the Suspensions of B. amyloliquefaciens and T. harzianum on the Development of C. acutatum. Advances in Microbiology, 10, 72-81. https://doi.org/10.4236/aim.2020.103007