Pseudomonas fluorescens

International Journal of Contemporary Applied Sciences (ISSN: 2308-1365)

Vol. 2, No. 7, July 2015 www.ijcas.net

Multi-Variant Statistical Analysis Evaluating the Impact of Rhizobacteria (Pseudomonas fluorescens) on Growth and Yield Parameters of Two Varieties of Maize (Zea mays. L) DIARRASSOUBA Nafan1*, DAGO Dougba Noel1, SORO Sibirina2, FOFANA Inza Jesus1, SILUE Souleymane1 and COULIBALY Adama1,3 1

UFR Sciences Biologiques Université Péléforo Gon Coulibaly BP 1328 Korhogo, Côte d’Ivoire. 2 UFR agroforesterie Université Jean Lorougnon Guédé BP 150 Daloa, Côte d’Ivoire. 3 UFR Biosciences Université Felix Houphouet Boigny 22 BP 582 Abidjan 22, Côte d’Ivoire. *Corresponding author, E-mail: [email protected] / [email protected] / [email protected]

Abstract The present survey was conducted to study the effects of rhizobacteria Pseudomonas fluorescens (P. fluorescens) bio-fertilizer on growth and yield parameters of two varieties of maize, DMRESR-Y and EV99-MRP. The field experiment was conducted in Korhogo (north of Côte d’Ivoire). For this purpose, we inoculated maize seeds with rhizobacteria P. fluorescens bio-fertilizer at a concentration of 7 ml.kg-1. Statistical analysis based on several correlation tests and on variance principal component analysis (PCA) of R bio-statistical software evidenced that disregarding the varieties of maize used, both analysed growth and yield parameters were differentially influenced by the bio-fertilizer. Moreover, the present study evidenced a strong difference in term of dry biomass, growth and yield parameters between maize plants under treatment T0 (treatment with any rhizobacteria P. fluorescens) with respect to treatment T1 (treatment with rhizobacteria P. fluorescens only), T2 (treatment with rhizobacteria P. fluorescens associated to foliar fertilizer) and T3 (treatment with foliar fertilizer only) suggesting a positive effect of rhizobacteria P. fluorescens bio-fertilizer on maize growing and produce. We also showed that maize plants treated with the rhizobacteria P. fluorescens associated to foliar fertilizer (T2 treatment) yielded a high dry biomass ratio (p-value = 0.006) with respect to the other analysed treatments (T0, T1 and T3). Key words: Bio-fertilizer; Rhizobacterie Pseudomonas fluorescens, Maize (Zea mays. L)

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1. Introduction Maize (Zea mays. L) is called king of cereal because of it productivity potential compared to any other cereal crop (Umesha S. et al., 2014). In Côte d'Ivoire, maize (Zea mays. L) is cultivated in different agro-ecological zones, alone or in combination with most crops. In this country, maize is the most cultivated cereal after rice with an annual production estimated to 600,000 tons. Maize is the staple food for many Ivorian’s. However, it is also involved in animal feed (poultry, pigs, cattle) and used as a raw material in some industries (brewing, soap and oil mill). The traditional production of maize yield around 0.8 tons/hectare (tons/ha) against 2-5 tons/ha in the controlled environment for the selected varieties (Akanvou Louise et al., 2006). Although being a food crop, maize has also become a cash crop. Being an exhaustive crop, it has very high nutrient requirement and its productivity is closely depend on nutrient management system. Under the present trend of the decline of the soil fertility in north of Cote d'Ivoire, maize production in this area facing many constraints including the productivity of the soil. Some of these problems can be tackled by using bio-fertilizers, which are natural, beneficial and ecologically friendly. Among the means available to achieve sustainability in agriculture production, bio-fertilizers plays an important and key role because it processes many desirable soil properties and exerts beneficial effect on the physical, chemical and biological characteristics of the soil. Then bio-fertilizers because of it vital role maintaining long term soil fertility (Hossein S. et al., 2013) could be requested for many agricultural soils in the north of Côte d'Ivoire. Moreover, they are essential for obtaining good yields avoiding depleting the soil. In the north of Côte d'Ivoire, the maize production to ensure food self-sufficiency and substantial income to producers, is still dependent on the intensive use of chemical fertilizers. This approach is not without negative consequences. The intensive use of mineral fertilizers can cause problems as the flow of these fertilizers into rivers, lakes and streams where they constitute a source of pollution (Alalaoui, 2007). It is 207



therefore appropriate to find alternative solutions at the overuse of chemical fertilizers. Biofertilizer are found positive contribution to soil fertility, resulting in an increase in crop yield without causing any environmental, water or soil pollution hazards (Umesha S. et al., 2014). Research work has already been undertaken on the potential effect of bio-fertilizers on crop yields. Hernandez et al. (1995) claim that inoculation of maize seed with rhizobacteria in combination with a dose of 120 kg/ha of nitrogen (N), results in an increase of around 25% of returns compared to those obtained with the same dose of nitrogen but without inoculation with microorganisms. Also, the combination of rhizobacteria raised with a dose of 120 kg/ha of nitrogen can increase over 60% the maize yields in comparison with those obtained in plots without nitrogen application or inoculation of bacteria. Here, basing on these results and observations, we highlighted the effects of rhizobacterie Pseudomonas fluorescens (P. fluorescens) bio-fertilizer on both growth and yield parameters of two varieties of maize through a statistical analysis based on a combination of several tests (Dago et al., 2015) of R software package (R Core Team, 2013) with the aim to promote its future use as bio-fertilizers for maize plants (Zea mays. L) in the north of Côte d'Ivoire.

2. Materials and Methods 2.1. Characteristics of the study site The study was conducted on-farm of the sub prefecture of Napiéledougou in the Department of Korhogo (north of Côte d'Ivoire). The site is located between an average altitude of 392 meters between - 5 ° 34 '31" and - 5 ° 29' 34 '' West longitude and between 9 ° 31 '23'' and 9 ° 31' 32 '' latitude North and 5°38' 83.2'' at an average altitude and is at 40 Km from Korhogo (Supplementary Fig. 1).The climate in this area is characterized by two types Sudanese seasons: a dry season, from November to April, punctuated by the Harmattan (dry wind from the Sahel) and a rainy season from May to November. This area is irrigated by the rivers 208



Bandama and Solomougou. The climate of the area is the maritime sub-equatorial with the temperatures that range between 24 and 33°C and the annual precipitation that vary between 1100mm and 1600 mm (Anonymous, 2006). 2.2. Maize Seed Material The characteristics of the two varieties of maize, DMRESR-Y (yellow colour) and EV99MRP (white colour) have been represented in the supplementary figure 2. The production cycle of DMRESR-Y and EV99-MRP exhibits the same duration (short cycle; 90-95 days) with an annual production comprised between 2 and 4 tons/ha. The variety EV99-MRP contain more proteins with respect to DMRESR-Y variety and displays a relative tolerance to drought. The improve seed of these maize varieties are available at the National Agricultural Research Centre (CNRA of Côte d'Ivoire). 2.3. Experimental Design The experimental design consisted in a block of 4 treatments and 4 repetitions. The two blocks corresponding to each of the two analysed maize varieties were divided into 16 basic plots corresponding to the four different treatments (T0, T1, T2 and T3). The space between the ridges was 75 cm between rows and 40 cm between bunches with 2 plants per hill after thinning. The various treatments have covered each elementary plot of 4m x 4m (16 m²). Each elementary plot contained 4 lines of 4 m long. The four treatments were as follows: T0: plots planted without rhizobacteria P. fluorescens and without foliar fertilizer; T1: subdivision sown with the seed inoculated with rhizobacteria P. fluorescens only; T2: land sown with the seed inoculated with rhizobacteria P. fluorescens bio-fertilizer and foliar fertilizer; T3: subdivision without rhizobacteria P. fluorescens biofertilizer with foliar fertilizer only. Moreover, a regular weeding (once a month) was made for the maintenance of the plot. The present experimental design have been performed in the raining season then no watering were made on the analysed maize plants outside the rain. 209



2.4. Preparation of Inoculums and Seed Inoculation Rizofos Liq Maize Bio fertilizer manufacture requires the strain of bacterium Pseudomonas fluorescens specifically selected for its phosphorus solubilizing ability. If stored under the recommended conditions (cool below 25°C), the product contains 1x109 CFU/ml in manufacturing. The inoculums were prepared from 500 ml Rizofos Liq Maize Premax-R + 200 ml of Premax-R for 100 kg of maize seed. We put the needed amount of Premax-R in a container and then added the required amount of Rizofos Liq Maize. We mix until a homogeneous mixture before inoculation of the required amount of seed. What follows is planting seeds making holes about 3 cm deep, in which two inoculated seed of maize are deposited. 2.5. Growth and Production Measured Parameters During this study, growth and yield parameters were measured, on each plant in the two considered experimental site. 2.5.1. Growth Parameters Six features were evaluated during the growth phase (i) height of the plant, (ii) dry biomass, (iii) number of leaves, (iv) length of leaves, (v) width of the leaves and (vi) the diameter of the collar which has been measured using an electronic sliding calliper. The growth parameters were evaluated using a centimeter as scale of measurement (cm).The height of the plants have been measured from the ground level to the end of the longest petiole. The number of leaves per plant was counted weekly. For the estimation of dry biomass, we first determined the fresh weight of the vegetative material and next we dry the material by using an oven-dried for 48 hours (at a temperature of 60 ° C: time and temperature required for a constant dry weight). The weights were measured using a precision balance 10-4 g (Toledo). Dry biomass was calculated using the following formula:

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Dry biomass = [(Fresh weight of leaves)×( dry weight of the leave sample )/( Fresh weight of the leave sample )]+[(fresh weight rods )x(dry weight of the sample rod )/( wet weight of the sample rods )]. 2.5.2. Yield or Production Parameters. Five features were evaluated during the production phase (i) length of the ears length (ii) width of the ears, (iii) geometric diameter of the ears (iv) number of seed per ear or spike and (v) number of ear per plant. Length, width and the geometric diameter parameters have been measured by using an electronic calliper. 2.6. Statistical Analysis Based on R Software. R is a free software environment for bio-statistical computing and graphics. For the present study and analysis, we processed our own script (R version 3.2.0). Then, we performed several correlation tests (Spearman and Pearson correlation tests) between both growth and yield parameters. We also estimated the variance of 8 analyzed parameters (4 growth and 4 yield parameters) by performing the principal component analysis (PCA) based on R software. The evaluation of the variance estimation, due to the large multivariate datasets have been processed by the principal component analysis to reduces the dimensionality of the analyzed parameter. The PCA facilitates the observation of different behavior among all analyzed parameters evaluating the effect of the bio-fertilizer on plant growth and yield. This analysis have been performed on 41 samples for each considered parameter in the two considered experimental sites. Multi-variant analysis based on boxplot representation have been performed with the aim to estimate the variability of each analyzed parameters (8 parameters) processed by the biofertilizer. In descriptive statistics, boxplot is a convenient way of graphically depicting groups of numerical data through their quartiles. Box plots are non-parametric and they display variation in samples of a statistical population without making any assumptions of the 211



underlying statistical distribution. The spacing’s between the different parts of the box indicate the degree of dispersion (spread) in the data, and show outliers (Rousseeuw, P. J et al., 1999).

3. Results 3.1.Impact of the Rhizobacteria Bio-fertilizer on Both DMRESR-Y and BEV99-MRP Maize Varieties. Correlation test in R statistical analysis (cor.test) have been used to evaluate the association between paired parameters. In order to corroborate the effect of the rhizobacteria bio-fertilizer on the two varieties of maize, we applied a correlation test (i) between the six examined growth parameters (height of the plant, dry biomass, number of leaves, length of leaves, width of leaves and diameter of the collar) and (ii) between the five considered production parameters (ears length, ears width, number of ear per plant, geometric diameter of the ear and number of seed per spike). The present analysis have been accomplished basing on the mean values of each analysed parameters and revealed a high and significant correlation values between all features assessed for (i) growth and (ii) yield parameters. In fact the correlation values obtained for these surveys were higher than 0.75 (R: 0.76-0.98) with an associated p-value that ranged between 4.868e-07 and 8.882e-16. Taking together, these results suggested that the impact of rhizobacteria P. fluorescens bio-fertilizer on the development of the analysed maize plants is relatively associated to the examined varieties. 3.2.Spearman Correlation Evaluating the Effect of Bio-fertilizer Treatment on Both Growth and Yield Parameters. Next, we investigated the effect of each treatment (T0, T1, T2 and T3) on both maize growth and yield parameters. The Spearman correlation analysis showed that disregarding (i) the analysed parameters, (ii) the variety of maize and (iii) the experimental parcel, all maize 212



plants under T0 treatment (treatment without bio-fertilizer) exhibited a very high correlation value among themselves as reported in Fig. 1 (R > 0.95 at a p-value ≤ 0.001). These results presume that growth parameters are good predictors of yield parameters in maize plants under treatment T0 (maize without any bio-fertilizer treatment). In other words, the variability between the different analysed features of both growth and yield parameters under treatment T0 is low. Moreover, the same analysis exhibited a weak correlation between maize plants under treatment T0 and the plants under treatments T1, T2 and T3 (correlation values range from -1 to 0.5; p-value ≤0.0001) as reported in Fig. 1. In view of the foregoing, these results suppose that maize plants under bio-fertilizer treatment (T1, T2 and T3) exhibit a strong difference with maize plants without any bio-fertilizer treatment (T0). However, when compared among them, maize plants under treatment T1, T2 and T3 display a heterogeneous correlation between themselves (Fig. 1) (correlation values range from 0.90 to -1; p-value ≤ 0.0001), suggesting a strong variability between analysed features of both growth and yield parameters. Taking together, these results reveal the strong influence of rhizobactéries P. fluorescens bio-fertilizer on maize growth and yield. 3.3.Evaluation of Growth and Yield Parameters by a Boxplot Multi-Variant Analysis. We processed a multi-variant statistical analysis between 8 features of both growth and yield parameters (these 8 features have been chosen for this survey because of the availability of their detailed values for each plant and treatment) by using a boxplot representation. For this statistical analysis and the others, we developed a script in R environment, highlighting a considerable difference between growth and yield parameters. The boxplot analysis evidenced tow tendencies estimating a relative high dispersion for analysed features associated to growth parameters with respect to those associated to the yield or production parameters. Then, the present boxplot analysis evidenced a relative small variability in yield or production parameters with respect to in growth parameters. These results suppose that the two analysed 213



parameter categories (growth and yield or production parameters) are differentially influenced by the rhizobacteries P. fluorescens bio-fertilizer. Moreover, while plant length parameter exhibits the highest variability among all features of growth parameter, ear length results to be the most variable parameters among the production parameters (Fig. 2). The smallest variability have been observed in the seed number parameter (yield parameter). Taking together, these observations suggest that rhizobacteries P. fluorescens bio-fertilizer strongly reduce the variability of yield parameter and in particular those of seed number (Fig. 2). 3.4.Correlation Test Analysis Between Growth and Yield Parameters. Next, we performed a correlation analysis between both growth and yield parameters to test for their agreement reacting to maize bio-fertilizer stimulus (8 parameters as above have been considered for this analysis). This survey evidenced a high correlation between all analysed growth feature parameters (R: 0.76-0.96; p-value ≤ 0.001) as reported in Table 1, suggesting a comparative effect of rhizobacteries P. fluorescens bio-fertilizer on maize plants growth parameters (collar diameter, plant height, leaves number and leaves length). Moreover, figure 3 displayed a convincing difference between both growth and yield (ear length, ear width, seed number per ear and ear diameter) parameters evaluating the effect of rhizobacteria P. fluorescens on maize plant development. The same analysis exhibited a heterogeneous correlation values between yield parameter features (R: 0.13-0.81).However, the present results suppose that (i) ear diameter (yield parameter) is a good predictor of ear width (yield parameter) (R: 0.81 at a p-value ≤0.001) and that (ii) the seed number per ear and the length of ear parameters displayed a relative good correlation between themselves (Fig. 3). Taking as whole, this analysis suggests that rhizobactéries P. fluorescens bio-fertilizer influences strongly the analysed production parameters. In other words, yield or production parameters are more sensible to bio-fertilizer effect with respect to growth parameters (Fig. 3, Table 1).

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3.5.Comparison Between Ear Seed Number and Ear Length Parameters by PCA Analysis (yield parameters). Because of their relatively good correlation (Fig. 3), we performed a principal component analysis (PCA) comparing both ear length and ear seed number (two yield parameters). The analysis showed a high variability in maize plants under T0 treatment for the two analysed parameters (Fig. 4). However, the PCA survey revealed a strong normalization effect of the rhizobactéries P. fluorescens bio-fertilizer on both analysed ear length and ear seed number parameters (Fig. 4). This tendency could explain the relative good correlation between these two parameters as indicated in figure 3 and table 1. Further, the normalization effect of the bio-fertilizer seem to be relatively less drastic on ear length parameter with respect to in ear seed number parameter. These results could explain a relative high variability of ear length parameter in comparison with those of ear seed number parameter as evidenced in figure 2. 3.6.Comparison Between Ear Diameter and Ear Width Parameters by PCA Analysis (yield parameters). Comparison analysis based on a PCA approach between ear diameter and ear width parameters (yield parameters) have been performed because of the high correlation between these two features, evaluating rhizobacteria P. fluorescens bio-fertilizer impact on maize plants development and yield. This analysis highlighted and confirmed the stabilization effect of the bio-fertilizer on the maize plants production parameters (Fig. 5). It noteworthy to observe that the stabilisation effect of rhizobacteria P. fluorescens bio-fertilizer on these two parameters is less strong in comparison to in ear seed number parameter (Fig. 4). Moreover, rhizobacteria P. fluorescens bio-fertilizer impact on ear width parameter is comparable with those on the ear diameter parameter (Fig. 5). This result is in agreement with the above boxplot analysis and representation which suggested that ear width and ear diameter yield parameters exhibited a comparable data variability (Fig. 2). This result could also explain the 215



high correlation degree between these two parameters assessing bio-fertilizer stimulus effect on the maize plants development. Then, the substantial differences observed between both figures 4 and 5supports the high sensibility of maize plants production (or yield) parameters to the rhizobacteria bio-fertilizer. 3.7.Comparison Between Features of Growth Parameter by PCA Analysis. We next evaluated the effect of rhizobacteria P. fluorescens bio-fertilizer on the maize growth parameters by a PCA analysis showing a moderate stabilisation effect of this bio-fertilizer on features of growth parameter with respect to those of yield parameter (Fig. 6). These results hint a high variability of growth parameters with respect to yield parameters, assessing the effect of rhizobacteria P. fluorescens bio-fertilizer on maize plants development (Fig.2). The profile of the PCA analysis curves associated to growth parameters are comparable among themselves (Panels A and B in Fig. 6). These observations may explain the high correlation value among these parameters evaluating the effect of the analysed bio-fertilizer on maize plants growth and development. It is also noteworthy to observe that the treatment T2 having recommended dose of rhizobacteria P. fluorescens + foliar fertilizer compost recorded the drastic effect on the growth parameters (Fig. 6). This might be due to more availability of nutrient from compost and beneficial effect due to rhizobacteria P. fluorescens associated to organic foliar fertilizer inoculation which provide nitrogen and phosphorus to plant growth. It is interesting to note that treatment T2 impact maize plants growth parameter acting as an intermediary phase between treatments T1 and T3 as expected. Taking together, this analysis evidenced a modest normalization effect of rhizobactéries P. fluorescens bio-fertilizer on maize plant development and support a weak variability of growth parameters assessing the influence of the former on maize plant increasing process with a particular attention to treatment T2.

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3.8.Pearson Correlation Evaluating the Degree of Agreement Between the Mean Value of all Analysed Growth and Yield Parameters. Pearson correlation analysis (Euclidian distance of Pearson correlation) between the mean values of all analysed growth and yield parameters (11 parameters in total) assessing maize plants development by rhizobacteria P. fluorescens bio-fertilizer have been performed and evidenced four tendencies (Fig. 7).This survey evidenced that assessing the mean values of the analysed parameters, yield parameters exhibited a good clustering among themselves (except ear width parameter) with respect to growth parameters. In other words, the analysed growth parameters (appraising their mean values) exhibited a heterogeneous behaviours among themselves in term of Pearson correlation analysis (Fig. 7). This analysis confirmed the moderate stabilisation effect of the bio-fertilizer on growth parameters with respect to yield parameters. It is interesting to note that this survey displays a good agreement between the number of maize ear per plants and the number of leaves per plants (Fig. 7). However, it is also noteworthy to observe that dry biomass parameter exhibits a different behaviour with respect the other’s analysed parameters in term of Euclidean distance of Pearson correlation. This might be due to the diverse effect of rhizobactéries P. fluorescens bio-fertilizer on this parameter in comparison to the other’s one. 3.9.Bio-fertilizer Effect on Maize Dry Biomass Parameter. We performed a statistical t.test based on R software with the aim to evaluate the effect of rhizobacteria P. fluorescens bio-fertilizer on maize dry biomass parameter. As previously showed this parameter exhibited different behaviour compared with the other analysed parameters. This assay emphases a strong difference between dry biomass obtained under T0 treatment and those detected in T2 treatments (Fig. 8 panels A and B). Moreover, our statistical analysis suggested that rhizobacteria P. fluorescens and foliar bio-fertilizer respectively improve the ratio of maize dry biomass (T1and T3 treatments improve the maize 217



dry biomass parameter with respect to treatment T0). It is noteworthy to observed that treatment T2 having recommended dose of rhizobacteria P. fluorescens and foliar fertilizer, recorded the highest test dry biomass compared to treatments T1 and T 3 in both parcels 3 and 4 (Fig. 8 panel B). In the other words, a significant difference assessing the dry biomass parameter has been observed between maize plants under T2 treatment and the other treated plants (p-value ranged from 0.012 and 0.007). These analysis evidenced the good effect of rhizobacteria P. fluorescens combined with foliar fertilizer improving maize dry biomass by providing sufficient nutriment (nitrate and phosphorus) for the maize plants growth and development. 4. Discussion The present study assessed the effects of rhizobacteria P. fluorescens bio-fertilizer on growth and yield parameters of two varieties of maize, DMRESR-Y and EV99-MRP.This investigation have been conducted during rainy season in the north of Cote d’Ivoire and three types of treatments (treatment T1, T2 and T3) based on the rhizobacteria P. fluorescens biofertilizer combined with foliar fertilizer and/or without foliar fertilizer have been processed (see materials and methods). We showed that the two analysed varieties of maize; DMRESRY and EV99-MRP relatively gave the same response to rhizobacteria P. fluorescens biofertilizer stimulus. However, it is emerged that maize plants under treatment T0 (planted without rhizobacteria P. fluorescens and without foliar fertilizer) exhibited a strong difference with respect to maize plants under rhizobacteria P. fluorescens bio-fertilizer treatment (Fig.1). Our finding also evidenced that rhizobacteria P. fluorescens bio-fertilizer carry out a high heterogeneity in term of correlation between both growth and yield parameters suggesting it influence on maize plant development. Moreover, the boxplot multi variant analysis revealed that rhizobacteria bio-fertilizer favours a high variability in growth parameters in comparison to yield parameters. Taking together, these results showed that growth parameters were 218



significantly influenced by the bio-fertilizer treatment. However among the different features examined for growth and yield parameters, significant increase in maize plant height and ear length respectively has been observed assessing bio-fertilizer effect on maize plant growth and yield (Fig. 2). The Pearson correlation analysis demonstrated that both yield (heterogeneous correlation values) and growth (high correlation values) parameters are strongly differentially influenced by the bio-fertilizer evaluating maize plant development (Fig. 3). The principal component analysis (PCA) applied to variance parameter evidenced a strong normalizing effect of the bio-fertilizer on the yield parameters, showing that rhizobacteria P. fluorescens impact the maize plants development reducing the variability of their yield parameters (Fig. 4 and 5). It is interesting to observe that the same investigation highlighted a weak stabilizing effect of the same bio-fertilizer on growth parameters (Fig. 6). Considering as whole, these results evidenced a different impacts (two tendencies) of the rhizobacteria P. fluorescens bio-fertilizer on growth and yield parameters respectively improving maize plants increase and production. However, the development and the good growth and productivity of maize could be attributed to the enhanced nutrient use efficiency in the presence of organic fertilizer (Umesha et al., 2014). Moreover, treatment T2 having recommended dose of rhizobacteria P. fluorescens and foliar fertilizer detailed a strong effect on the growth parameters (Fig. 6), suggesting a high availability of nutrient and beneficial effect due to rhizobacteria P. fluorescens associated to organic foliar fertilizer inoculation which provide nitrogen and phosphorus to plant growth. Several research studies have showed that the composted organic materials release nutrients slowly and may reduce the leaching losses, particularly nitrogen (Naveed et al., 2008). It also emerged from our study that dry biomass parameter (mean value) exhibits a low correlation with the other analysed parameters (Fig.7). In fact the normalization effect of the bio-fertilizer on dry biomass parameter is wholly different with respect to the other’s analysed parameters. Our findings 219



showed that the treatment T2 that results to be the combination of rhizobacteria P. fluorescens and foliar fertilizer, yielded the highest amount of maize dry biomass (more than 70 g) in comparison with the treatments T0 (39 g), T1 (49 g) and T3 (45 g) (Fig. 8 panels A and B) (pvalues: 0.006). However, it is noteworthy to observe that recent study shows that the treatment T2 (bio-fertilizers + foliar fertilizer) is the best recommended in maize farming. Moreover, even if the efficiency difference shown between T1, T2, T3 treatment could be attribute to the bio-fertilizers, the applied period, the amelioration (high dry biomass ratio and strong effect on growth parameters) of the inoculated plants growth could be due to rizobacteria and foliar fertilizer synergic effect. In fact, several assays proved that rhizobacteria P. fluorescens associated to foliar fertilizer impacted positively both maize plant growth and maize yielded seeds on intact soil in the south of Benin (Adjanohoun et al., 2012; Adjanohoun et al., 2011) showing that 87 days after sowing, the inoculated plants of maize with P.fluorescens and P.aeroginosa produce an average value of dry biomass that significantly exceeded respectively 59,57% and 23,40% of the dry biomass value of maize plant without any treatment. However, the homogeneity effect of the bio-fertilizer on the yield parameters and the amelioration of the dry biomass production parameters were obtained by the action of both rhizobacteries and foliar fertilizer. In fact the inoculation of maize seeds by the rizobacteries P. fluoresens bio-fertilizer increases the availability of soil in phosphorus and nitrogen, ameliorates the efficiency of phosphated fertilizer and produces phytohormones which react as growth factor favouring crops growth (Lee et al., 1970, Kang et al., 2010). Shaharoona et al. (2006) showed the efficiency of Pseudomonas increasing significantly maize plant growth and production when adequate quantity of nitrate has been provided. Previous studies showed that rhizobacteria improve maize productivity increasing the absorption of nitrate, phosphate, potassium, zinc, manganese, copper and iron (Biari et al., 2010). Furthermore, Walker et al. (2011) demonstrated the positive effect of rhizobacteria on 220



the maize plant development via quantitative and qualitative modification of benzoxazinoid compost. Nevertheless, Contesto et al. (2008) showed that rhizobacteria impact positively plants growth inducing vegetal hormone synthesis. In the same tendency, Ahmad et al. (2006) and Estes et al. (2004) evidenced the positive influence of rhizobacteria on plant growth and development through a better seed germination and a greater development of the roots, which induce the increase in the absorption capacity of nutrients and water in plants. These results and observations are in agreement with our finding. Bringing these data together, we showed that rhizobacteria associated to the organic foliar fertilizer can be used to increase or improve the production of maize. However, we are not able to quantify the real impact of rhizobacteria (organic fertilizer) substituting chemical fertilizer. Even if the combination of several potentially nitrogen-fixing rhizobacteria is possible, the selection of plants varieties that favour heterogeneous nitrogen fixation is a new and promising approach.

5. Conclusion The findings of this study have clearly showed that inoculation of maize seeds by rhizobacteria P. fluorescens associated with foliar fertilizer has resulted in obtaining highest dry biomass production, improving maize plant growth and crop yields in the soil of the north of Côte d'Ivoire. These results bode well for the possibility of using these rhizobacteria as organic fertilizer for sustainable production of maize. In the dense region of Korhogo where the pressure on land and the decline of soil fertility constantly threaten agricultural production, the use of bio-fertilizer is a good alternative to increase agricultural yields. However, the experiment should be conducted on a large scale to determine the values of the obtained yields and the contribution of these rhizobaterias in increasing element such as nitrogen, potassium and phosphorus.

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Acknowledgements Many hanks to the Rhizobacter Company in Argentina for providing bio-fertilizer and for their economic support to this work.

Table 1: Pearson correlation between both growth and yield parameters. Ear.Lengt Ear.widt Ear.diamete Seed.Nb Diamete Leaves.Nb Leaves.Lengt h h r r r Height r h Ear.Length

1

Ear.width

0.26

1

Ear.diameter

0.13

0.81*** 1

Seed.Nbr

0.49***

0.35

0.21

1

223



Collar.diamete r -0.21

0.16

0.29

-0.05

1

Height

0.22

0.45***

0.06

0.84*** 1

-0.01

0.82** 0.79*** * 1

-0.06

0.79** 0.96*** * 0.76***

Leaves.Nbr

-0.21

-0.16

Leaves.Length -0.18

0.09

0.13

0.29

0.27

1

***p-value ≤ 0.001; ** 0.001 0.1

Figure 1. Spearman correlation showing the effect of rhizobacteria P. fluorescens biofertilizer treatment on both yield (production) and growth parameters in maize plant (Zea mays. L). Figure 2.

Boxplot analysis assessing the effect of rhizobacteria P. fluorescens on the

variability of both growth and production parameters. Figure 3. Clustering dendrogram of Pearson correlation between growth and yield parameters. Figure 4. Effect of rhizobacteria P. fluorescens bio-fertilizer treatment on both maize ear length and the number of grain per maize ear (ratio of variances 2.20; p-value 0.014). Figure 5. Effect of rhizobacteria P. fluorescens bio-fertilizer treatment on both ear width and diameter parameters (ratio of variance 2.63; p-value=0.003). Figure 6. Effect of rhizobacteria P. fluorescens bio-fertilizer fertilizing treatment on the analysed growth parameters ((A) height of maize plant and collar diameter parameters and (B) leaves length and number parameters) (ratio of variance: 0.57-0.68; p-value: 0.2-0.07). Figure 7. Cluster dendrogram of the Euclidian distance of Pearson correlation between the mean values of the 11 analysed growth and yield parameters (p-value 0.012).

224



Figure 8. (A) Barplot of a comparative analysis of the effect of rhizobacteria P. fluorescens combined with foliar bio-fertilizer on maize dry biomass parameter (B) showing a significant difference between biomass yielded by treatment T2 with respect to the others treatments (t = 6.881, df = 3, p-value = 0.006). Supplementary figure 1. Characteristics of the study and experimental site. Supplementary figure 2. The two varieties of analysed maize (A) DMRESR-Y and (B) EV99-MRP.

225


Figure 1

Figure 2

Figure 3 226



Figure 4

Figure 5

227



Figure 6

228




Figure 7

A

Figure 8

229

B


Supplementary Figure 1

Supplementary Figure 2

230
