The objective of the study was to evaluate the effects of nine plant growth-promoting rhizobacteria (PGPR) alone or in combination on maize seed germination and seedling growth under laboratory and greenhouse conditions. The germination and growth tests were carried out in square petri dishes and pots. Maize seeds were inoculated with suspension of 10 8 CFU/ml of rhizobacteria. The experimental device was a random block of 16 treatments with four repetitions. Germination test results showed that seeds inoculated with PGPR including the control induced good germination in the range of 93.75% to 100%. The vigor index and root length of the seeds treated with <i> Bacillus panthothenicus </i> were significantly improved by 76.64% and 58.86%, respectively, while the maximal lengths of the seedlings were obtained with <i> Pseudomonas cichorii </i> with an increase of 118.95%. In greenhouse experience, data demonstrated that <i> Serratia marcescens </i> better improved the leaf area, height and underground biomass, respectively by 58.83%, 108.43%, and 59.16% as compared to the control. The highest fresh aerial biomass and air dry matter was obtained with plants treated only with <i> Pseudomonas putida </i> . These results show the potential to use such rhizobacteria as biofertilizers to improve maize productivity in Benin.
Maize (Zea mays L.) is one of the most important cereal crops in the world [
Corn Seeds: The maize seeds used are those of the variety EVDT 97 STR C1 from the South Agricultural Research Centre (CRA-Sud) of the National Agricultural Research Institute of Benin (INRAB). This variety has good resistance to american rust, streak, blight, curvulariose, and drought [
Strains of PGPR: Nine (9) strains of PGPR namely Bacillus polymyxa, Bacillus anthracis, Bacillus thuringiensis, Bacillus circulans, Bacillus panthothenicus, Pseudomonas cichorii, Pseudomonas putida, Pseudomonas syringae and Serratia marcescens were used. These strains were isolated and characterized from the rhizosphere of maize from the different agroecological zones of central and northern Benin by [
The method described by [
The device adopted was a complete random block with sixteen (16) treatments with four (04) repetitions. The treatments were defined as: CTL: Control without bacteria, B1: Bacillus polymysa, B2: Bacillus anthracis, B3: Bacillus circulans, B4: Bacillus thuringiensis, B5: Bacillus panthothenicus, P1: Pseudomonas cichorii, P2: Pseudomonas putida, P3: Pseudomonas syringae, S. m: Serratia marcescens, B1B2B3B4B5: Bacillus polymysa-Bacillus anthracis-Bacillus circulans-Bacillus thuringiensis-Bacillus panthothenicus, B4B5S.m: Bacillus thuringiensis-Bacillus panthothenicus-Serratia marcescens, P1P2P3: Pseudomonas cichorii-Pseudomonas putid-pseudomonas syringae, P2S.m: Pseudomonas putida-Serratia marcescens, B4P1: Pseudomonas cichorii-Bacillus thuringiensis, P2B4S.m: Pseudomonas putida-Bacillus thuringiensis-Serratia marcescens.
1) Disinfection and inoculation of maize seeds: The seeds were disinfected by soaking for two (02) minutes in a sodium hypochlorite solution (0.024%) and then rinsed abundantly with sterile distilled water under vortex agitation [
2) Germination of maize seed: After inoculation, twelve (12) seeds were dispersed equidistantly on a paper towel previously moistened with 10 ml of sterile distilled water deposited in sterile square petri dishes of 11.8 cm side. Maize seeds were subsequently covered by another paper towel, watering with 10ml and the petridishes were incubated at 30˚C. For seven (07) days [
1) Seedlings, inoculation and maintenance of plants: The ferruginous soil used for the potty test was previously sifted and autoclaved twice at 121˚C with 24 hour time interval for twenty minutes [
The chemical analyses of soil samples were carried out at the laboratory of Soil Sciences, water and Environment (LSSEE) of the National Institute of Agricultural Research of Benin (INRAB). These analyses consisted of pH measurement using an electrode pH meter in a soil/water ratio of 2/5 (g/ml) [
Plant Heigth and collar diameter were measured from the 7th days after seeding (DAS) to up 30 DAS. The leaf area of the seedlings was calculated using the method described by [
Dry (aerial and underground) biomasses were crushed to mortar and digested using the micro-Kjeldahl method and nitrogen n was determined by colorimetry [
The data collected were subjected to a two-factor ANOVA (repeats and treatments) statistical analysis after performing the normality and variance homogeneity test. The concentrations of nitrogen, phosphorus and potassium in the air dry matter and the levels of phosphorus and potassium in the underground dry matter did not verify these conditions. The Kruskal Wallis test was therefore carried out on the data of these variables. Evidence of significant differences between treatments was achieved using the student-Newman-Keuls test at the 5% probability threshold. An ascending hierarchical classification (CHA) was performed on the average of the different parameters by treatment to group them into homogeneous classes. Finally, the principal component (PCA) analysis on the treatment averages allowed us to describe the links between the variables and to characterize each treatment group. These analyses were carried out in the software R version 3.4.3 assistant of the packages graphics, Factoextra and Facto MineR. The graph Pad software Prism version 7.00 allowed us to trace the graphs.
Traitements | Germination rate | Root length (cm) | Shoot length (cm) | Vigor index |
---|---|---|---|---|
CTL | 93.75 ± 4.16a | 17.21 ± 1.13efg | 6.33 ± 0.74h | 2206.87 ± 294.6e |
B1 | 93.75 ± 7.88a | 24 ± 2.18cde | 11.06 ± 2.49cdef | 3286.87 ± 652.7cd |
B2 | 95.83 ± 4.80a | 22.72 ± 1.6cdefg | 9.74 ± 2.00fg | 3110.64 ± 308.5d |
B3 | 93.75 ± 4.16a | 22.56 ± 1.18defg | 9.87 ± 0.66efg | 3040.31 ± 284.9cd |
B4 | 93.75 ± 4.16a | 23.35 ± 2.01cdef | 11.59 ± 0.87bcde | 3275.62 ± 190.2cd |
B5 | 95.83 ± 4.8a | 27.34 ± 1.89a | 13.34 ± 0.44ab | 3898.36 ± 143.9a |
P1 | 97.91 ± 4,16a | 21.67 ± 0.95fg | 13.86 ± 0.78a | 3478.74 ± 139.1bcd |
P2 | 97.91 ± 4.16a | 23.19 ± 1.24cdef | 10.15 ± 1.03defg | 3264.31 ± 93.97d |
P3 | 97.91 ± 4.16a | 23.00 ± 1.30cdef | 10.11 ± 1.63defg | 3241.80 ± 312.5cd |
S.m | 100 ± 0a | 26.46 ± 1.18ab | 11.75 ± 0.70bcd | 3821 ± 150ab |
B1B2B3B4 | 97.91 ± 4.16a | 23.04 ± 1.10cdef | 10.26 ± 0.20defg | 3260.40 ± 59.46cd |
B4B5S.m | 100 ± 0a | 20.76 ± 2.06g | 12.24 ± 1.36abc | 3300 ± 329.5cd |
P1P2P3 | 97,91 ± 4.16a | 24.76 ± 0.49bc | 11.20 ± 0.36cdef | 3520.84 ± 162.5abc |
P2S.m | 100 ± 0a | 22.94 ± 1.78cdef | 8.97 ± 1.09g | 3191 ± 202cd |
B4P1 | 95.83 ± 4.8a | 22.85 ± 0.8cdefg | 10.34 ± 1.28bcd | 3180.59 ± 184.6cd |
B4P2S.m | 100 ± 0a | 24.17 ± 1.50cd | 10.52 ± 1.49cdefg | 3469 ± 267.3bcd |
Probabilité | 0.3385 | 0.000 | 0.000 | 0.000 |
Signification | NS | *** | *** | *** |
*** = P < 0.001 (highly significant), NS = P > 0.05 (not significant), values indicate means, ± indicate standards of calculated deviations of the four replicates. The averages followed by the same letter are not significantly different from the Newman-Keuls test at the 0.05 probability level. CTL: Control without bacteria, B1: Bacillus polymysa, B2: Bacillus anthracis, B3: Bacillus circulans, B4: Bacillus thuringiensis, B5: Bacillus panthothenicus, P1: Pseudomonas cichorii, P2: Pseudomonas putida, P3: Pseudomonas syringae, Sm: Serratia marcescens, B1B2B3B4B5: Bacillus polymysa-Bacillus anthracis-Bacillus circulans-Bacillus thuringiensis + Bacillus panthothenicus, B4B5Sm: Bacillus thuringiensis-Bacillus panthothenicus + Serratia marcescens, P1P2P3: Pseudomonas cichorii-Pseudomonas putida + Pseudomonas syringae, P2Sm: Pseudomonas putida-Serratia marcescens, B4P1: Pseudomonas cichorii-Bacillus thuringiensis, P2B4Sm: Pseudomonas putida-Bacillus thuringiensis-Serratia marcescens.
The assessment of experimental soil chemistry (
Soil | PH | B.E (meq/100g) | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
C | N | C/N | M0 | P .ass | Ca | Mg | K | Na | Som.cat | CEC | |||
Water | Kcl | % | % | % | % | ppm/% | % | meq/100 g | |||||
6.01 | 5.5 | 0.81 | 0.1 | 7.87 | 1.4 | 6.91 | 2.35 | 0.63 | 0.16 | 0.16 | 3.32 | 6.06 |
From the analysis in
At the reading of
Traitements | Height (cm) | Circumference (cm) | Leaf Area (cm2) | |||
---|---|---|---|---|---|---|
m | cv | m | cv | m | cv | |
CTL | 11.37f | 12.82 | 2.40a | 7.53 | 55.13d | 8.65 |
B1 | 13.73de | 4.85 | 2.93a | 12.37 | 73.49c | 3.52 |
B2 | 13.5def | 7.80 | 2.61a | 6.93 | 59.82d | 13.15 |
B3 | 11.83ef | 4.65 | 2.51a | 0.00 | 59.46d | 9.40 |
B4 | 14.56cd | 10.33 | 3.14a | 10.00 | 81.64bc | 6.35 |
B5 | 14.86cd | 5.44 | 2.82a | 19.25 | 94.30b | 3.64 |
P1 | 17.53ab | 2.63 | 3.24a | 22.35 | 85.47bc | 15.38 |
P2 | 16.56abc | 7.55 | 3.45a | 18.18 | 114.62a | 1.45 |
P3 | 16.46abc | 2.45 | 3.24a | 14.78 | 92.10b | 12.63 |
S.m | 18.06a | 2.84 | 3.24a | 27.94 | 114.91a | 1.36 |
B1B2B3B4B5 | 14.63cd | 7.50 | 2.82a | 0.00 | 90.38bc | 12.28 |
B4B5S.m | 13.5def | 11.11 | 2.66a | 17.65 | 93.78b | 7.66 |
P1P2P3 | 14.8cd | 2.44 | 2.82a | 11.11 | 79.57bc | 4.12 |
P2S.m | 14.73cd | 4.36 | 2.82a | 2.83 | 87.27bc | 6.70 |
B4P1 | 15.6bcd | 2.56 | 3.14a | 20.00 | 97.73b | 4.10 |
P2B4S.m | 14cde | 10.57 | 2.82a | 2.83 | 88.28bc | 3.75 |
Probability | <0.001 | 0.578 | 3.721e-12 | |||
Signification | *** | NS | *** |
NS = P > 0.05 (not significant). m = means and cv = coefficient of variation. The averages followed by the same letter are not significantly different by the Newman-Keuls test at the 0.05 probability level.
Figures 3(a) and Figures 3(b) represent the projection on the factorial plane (Dim1 and Dim2) of the data of the variables studied in greenhouse maize plants. The two axes (Dim1 and Dim2) represent 83.16% of the total variance, which is sufficient to guarantee a precision of interpretation for the identification of the main parameters and the discriminant treatments. Variables such as air dry matter (MSA), leaf area (S. Folliaire), fresh aerial biomass (BAF), circumference and height are strongly represented on the first main component (Dim1) while the underground dry matter (MSS) and the fresh underground biomass (BSF) are represented on the second main component (Dim2). With regard to the behaviour of the treatments against the different variables evaluated, four distinct major groups emerge:
- Group 4 includes strains of PGPR with significantly improved the height, circumference, fresh aerial biomass (BAF), dry underground biomass (BSF), material dry air (MSA), and leaf area of the maize plants. This group includes P. putida and S. marcescens which are well represented on the first axis (Dim1).
- Group 3 located opposite the axes of variables includes the stem B. circulans, B. anthracis and the witness with the lowest values of all the evaluated parameters.
- Group 2 brings together eight treatments that stand out particularly because of their performance at the level of the underground dry matter (MSS) developed by corn plants with P. putida-S. marcescens at the top.
- Group 1 includes treatments such as: P. cichorii-P. putida-P. syringae, B. polymysa and B. thuringiensis having mainly negatively as corn plants underground dry matter (MSS) evidenced by their position on the Dim1 axis by report to this variable.
With the exception of the content of nitrogen, the statistical analysis of the results illustrated by
handled by S. marcescens have presented the strongest levels in phosphorus and potassium. These values exceed respectively 80% and 11.29% those obtained at the level of the plants not inoculated. It is the same for plants under influence of P. putida where an increase of 15% and 80% respectively of the potassium and phosphorus content was recorded in comparison with plants witnesses. Phosphorus in the underground biomass of plants has been significantly improved (P < 0.05) with inoculation in comparison with plants witnesses. The highest average phosphorus (0.189 ± 0.01) has been registered with the combination S. marcescens-P. putida. On the other hand, nosignificant difference was observed on the rate of nitrogen and potassium between treatments. However, the best nitrogen levels (2.04 ± 0.39) and potassium (2.38 ± 0.72) were obtained with the inoculated plants.
The results obtained in vitro show that treatment of the seeds of corn with PGPR strains has impacted positively the germinal parameters of the maize seeds. Indeed, good (93.75% to 100%) germination of the seeds was noted whether at the level of the inoculated seed as witnesses thus attesting the good quality of the
seeds used in our study. However, no significant differences (P > 0.05) were observed between treatments. Note even when that seeds inoculated with S. marcescens followed by some combinations of which P. putida-S. marcescens have been best (100%) in comparison with the witnesses seeds germination rates. These observations are consistent with the work of [
All of the maize seeds treated with the PGPR showed highly significant improvements (P < 0.001) about the length of the seedling and roots. Indeed, values the highest length of seedlings were obtained with the application of P. cichorii followed by B. panthothenicus by the respective increases of 118.95% and 110.74% compared to controls. The seeds treated with B. panthothenicus and S. marcescens stimulated a significant elongation of the roots, which exceeded 58.86% and 53.74% relative to the control, respectively. These improvements in our study are confirmed by the work of [
Treatments | Aerial dry matter | Underground dry matter | |||||
---|---|---|---|---|---|---|---|
% N | % P | % K | % N | % P | % K | ||
CTL | 2.63 ± 0.70a | 0.15 ± 0.0cd | 4.16 ± 0.02def | 1.63 ± 0.24a | 0.181 ± 0.05abc | 1.80 ± 0.19a | |
B1 | 2.86 ± 0.55a | 0.13 ± 0.00cd | 3.88 ± 0.11ef | 1.67 ± 0.26a | 0.120 ± 0.01de | 1.80 ± 0.08a | |
B2 | 3.06 ± 0.91a | 0.18 ± 0.05bcd | 4.76 ± 0.25ab | 1.46 ± 0.08a | 0.180 ± 0.01ab | 1.78 ± 0.20a | |
B3 | 2.93 ± 0.85a | 0.11 ± 0.01d | 2.83 ± 0.10f | 1.98 ± 0.18a | 0.112 ± 0.01e | 1.79 ± 0.19a | |
B4 | 3.10 ± 1.15a | 0.18 ± 0.05bcd | 4.30 ± 0.50bcde | 1.60 ± 0.26a | 0.137 ± 0.03cde | 1.74 ± 0.15a | |
B5 | 3.32 ± 1.05a | 0.25 ± 0.04ab | 4.06 ± 0.85cdef | 1.86 ± 0.12a | 0.168 ± 0.00cde | 2.38 ± 0.72a | |
P1 | 3.23 ± 0.78a | 0.23 ± 0.01ab | 4.68 ± 0.25abc | 1.90 ± 0.17a | 0,159 ± 0.00abc | 1.67 ± 0.25a | |
P2 | 3.31 ± 0.91a | 0.27 ± 0.01a | 4.81 ± 0.20a | 1.79 ± 0.31a | 0.138 ± 0.00cde | 1.99 ± 0.52a | |
P3 | 3.07 ± 0.88a | 0.25 ± 0.05ab | 4.44 ± 0.26abcd | 1.84 ± 0.12a | 0.140 ± 0.03cde | 1.75 ± 0.18a | |
S.m | 3.33 ± 0.95a | 0.27 ± 0.01a | 4.63 ± 0.30abcd | 1.98 ± 0.10a | 0.156 ± 0.01abcd | 1.32 ± 0.08a | |
B1B2B3B4B5 | 3.45 ± 0,70a | 0.27 ± 0.01a | 4.75 ± 0.24ab | 2.04 ± 0.39a | 0.149 ± 0.00abcd | 1.75 ± 0.26a | |
B4B5S.m | 3.25 ± 0.89a | 0.27 ± 0.01a | 4.34 ± 0.22bcde | 1.93 ± 0.35a | 0.141 ± 0.01bcde | 1.83 ± 0.05a | |
P1P2P3 | 3.24 ± 0.60a | 0.19 ± 0.03bcd | 4.44 ± 0.26abcde | 1.82 ± 0.22a | 0.133 ± 0.01cde | 1.79 ± 0.09a | |
P2S.m | 2.83 ± 1.12a | 0.21 ± 0.09abc | 4.32 ± 0.37bcde | 1.40 ± 0.26a | 0.189 ± 0.01a | 1.46 ± 0.30a | |
B4P1 | 3,16 ± 1.00a | 0.26 ± 0.01ab | 4.33 ± 0.38bcde | 1.64 ± 0.08a | 0.181 ± 0.05cde | 1.24 ± 0.15a | |
P2B4S.m | 3.22 ± 0.54a | 0.22 ± 0.01abc | 4.23 ± 0.43bcde | 2.03 ± 0.04a | 0.184 ± 0.05abc | 1.93 ± 0.02a | |
P-value | 0.955 | 0.0186 | 0.0233 | 0.059 | 0.036 | 0.089 | |
Significance | NS | ** | ** | NS | ** | NS | |
NS = P > 0.05 (not significant). Value: mean ± standard deviation, **: significant difference (P < 0.05), the means followed by the same letter are not significantly different according to the Newman-Keuls test at P < 0.05. CTL: Control without bacteria, Bacillus polymysa, B2: Bacillus anthracis, B3: Bacillus circulans, B4: Bacillus thuringiensis, B5: Bacillus panthothenicus, P1: Pseudomonas cichori, P2: Pseudomonas putida, P3: Pseudomonas syringae, Sm: Serratia marcescens, B1B2B3B4B5: Bacillus polymysa-Bacillus anthracis-Bacillus circulans-Bacillus thuringiensis-Bacillus panthothenicus, B4B5Sm: Bacillus thuringiensis-Bacillus panthothenicus-Serratia marcescens, P1P2P3: Pseudomonas cichorii-Pseudomonas putida-Pseudomonas syringae, P2Sm: Pseudomonas putida-Serratia marcescens, B4P1: Pseudomonas cichorii-Bacillus thuringiensis, P2B4Sm: Pseudomonas putida-Bacillus thuringiensis-Serratia marcescens.
The effectiveness of the strains of S. marcescens, B. panthothenicus, P. cichorii observed in our study can be attributed to the ability of these isolates to produce the acid indole Acetic (AIA), a hormone that positively affects the growth and development of roots thus increasing absorption of nutrients [
The importance of PGPR strains on crops has been highlighted by [
The higher aerial biomass was observed with the plants treated with P. putida and S. marcescens either respective increases of 161.60 and 94.37% compared to the control. On the underground biomass, best results were incurred with the inoculation of S. marcescens (59.16%) followed by B. panthetonicus (52.08%). The results achieved with the effect of the strain S. marcescens on biomass of the plants are in agreement with those [
As for the rate of the material dry air and underground developed by corn plants, the largest aerial dry matter production were recorded by plants treated with P. putida (78.83%) followed by S. marcescens (78.09%). Their combination has led to the largest underground dry matter (82.64%). This rate would be due to the synergistic effect of combined two strains. Our results are similar to those of [
In our study, the improvement of nutritional status at the level of the inoculated plants would result from a better accumulation of dry matter in the aerial part of the plant maize. Tarafdar et al., [
The results of the present study showed the beneficial role of PGPR inoculation on maize seed germination and seedling growth under laboratory and greenhouse conditions. For the majority of the evaluated parameters, the rhizobacteria S. marcescens, P. putida, and P. cichorii are most effective among those in the study. Furthermore, treatment of seeds with S. marcescens, and P. putida have led to better improvement in the nutritional status of plants including the content in phosphorous and potassium in aerial biomass of corn with a percentage plants improvement between 11.29% and 80% compared to plants not inoculated. These results are very interesting, and thus leave the possibility to exploit all of the strains selected in future experimental studies in order to produce some biofertilizers.
The authors thank the “Centre National de Spécialisation sur le Maïs (CNS-Maïs), the National Fund for scientific research and Innovation Technology (FNRSIT) for theit financial supports.
The authors declare no conflicts of interest regarding the publication of this paper.
Amogou, O., Dagbénonbakin, G., Agbodjato, N.A., Noumavo, P.A., Salami, H.A., Valère, S., Ricardos, A.M., Sylvestre, A.A., Djihal, K.F.A., Adjanohoun, A. and Baba-Moussa, L. (2018) Influence of Isolated PGPR Rhizobacteria in Central and Northern Benin on Maize Germination and Greenhouse Growth. American Journal of Plant Sciences, 9, 2775-2793. https://doi.org/10.4236/ajps.2018.913201