Use of Plant Growth Promoting Rhizobacteria in Combination with Chitosan on Maize Crop: Promising Prospects for Sustainable, Environmentally Friendly Agriculture and against Abiotic Stress

Faced with the problems posed by the abusive use of chemical fertilizers and pesticides, it is important to find other alternatives that can guarantee a sustainable and environmentally friendly agriculture. The objective of this study was to evaluate the tolerance of a PGPR (plant growth promoting rhizobacteria) Pseudomonas putida strain to different abiotic stress in in vitro conditions and the synergistic effect of this rhizobacterium in combination with chitosan extracted from crab exoskeletons on the growth of maize in greenhouse conditions. The strain of P. putida was put in culture at different temperatures, pH, and NaCl concentrations to determine its growth. Then, this strain in combination with chitosan extracts were tested for their ability to improve maize growth for 30 days. The results showed that the P. putida strain showed excellent resistance capacities to different salt concentrations, pH, and temperature variations. Moreover, an improvement in plant growth and biomass yield parameters was observed. The highest values of height, diameter, and leaf area were obtained with the plants treated with the combination of chitosan extracted from Cardisoma armatum and P. putida, with increases of 26.8%, 31%, and 55.7%, respectively, compared to the control. This study shows the possibility of using chitosan and rhizobacteria as biostimulants to improve productivity and increase maize yield in a sustainable manner.


Introduction
Agriculture plays a significant role in the economies of countries around the world. However, it is confronted with many problems leading to substantial crop losses. The constant increase in population, rapid urbanization with the decrease in agricultural land, radical change in climatic conditions, and intensive use of agrochemicals in farming practices that have caused environmental disruptions are confronting humanity with growing problems of food security and agricultural sustainability [1,2]. In addition, the excessive use of chemicals for crop fertilization may lead to the accumulation of minerals and nutrients that cannot be easily utilized for plant consumption, ultimately resulting in soil pollution and toxicity [3].
Consequently, the expansion of crop production relies on increasing soil fertility to ensure food for all under the current global food security scenario [4]. It thus becomes essential to develop different environmentally friendly methods of soil fertility improvement such as the use of natural products and/or organisms in order to reduce the effects The chitosans used were chitin derivatives extracted from the exoskeletons of crabs (Callinectes amnicola and Cardisoma armatum) collected in Benin according to the method adapted by [27]. The extracts obtained were stored at the Laboratoire de Biologie et de Typage Moléculaire en Microbioogie (LBTMM). They are solid bioproducts containing natural polymers, biodegradable, and non-toxic for animals and plants.
The PGPR rhizobacterium named Pseudomonas putida was used in this study to evaluate the synergistic effect of PGPR in combination with extracted chitosans. This strain stored at −85 • C in Mueller Hinton nutrient broth with addition of glycerol (10%) in the laboratory was isolated and identified by [28] and characterized by [7] for its PGPR profile. The ability of P. putida to grow under different temperatures (4,16,25,30,37,40,44,48, and 50 • C) was performed by the method adapted by [29] on Mueller Hinton (MH) liquid nutrient broth inoculated with 100 µL of the bacterial culture of the tested strain for 48 h under agitation. The growth of P. putida was evaluated by reading the optical density (OD) with a spectrophotometer (BioMATE 3S, Thermo scientific, Waltham, MA, USA) at the wavelength of 600 nm and the experiments were performed in three r replicates. [29].

Evaluation of the Effects of pH Variation on the Growth of P. putida
The effect of pH was evaluated by the method adapted by [29] on Mueller Hinton (MH) liquid nutrient medium. The medium was adjusted to different pH values, 4, 5, 6, 7, 8, 9, 10, and 12, to evaluate the ability of the strain to grow over a wide range of pH values. The growth of the strain was evaluated by measuring the optical density of the cultures with a spectrophotometer (BioMATE 3S, Thermo scientific) at the wavelength of 600 nm and the experiments were carried out in three replicates.

Evaluation of the Effects of Different Salt Concentrations (NaCl) on the Growth of P. putida
Salt tolerance of P. putida was evaluated by the method adapted by [29] on Mueller Hinton (MH) liquid nutrient broth inoculated with 100 µL of one culture of each rhizobacterium and incubated at 30 • C for 48 h under agitation in the presence of the following increasing concentrations of NaCl: 0, 0.2, 0.4, 0.6, 0.8, and 1 M. The optical density of each culture was read with a spectrophotometer (BioMATE 3S, Thermo scientific) at the wavelength of 600 nm and the experiments were performed in three replicates.  The samples of land crab (Cardisoma armatum) and freshwater crab (Callinectes amnicola) used were collected from anglers and crab sellers, respectively, in the municipalities of Ajohoun and Abomey-Calavi in Benin. After the crab samples were collected, the shells were separated from the meat. These shells were washed with tap water then with distilled water before being dried. The shells were sprayed with acetone and placed in an oven at 50 • C for 24 h to activate the drying process. Drying was continued in a pasteurized oven at 50 • C for 10 days. After drying, all samples were ground and powdered using a Retsch mill type SM 2000/1430/Upm/Smfet [27]. - The powders of the two crab species obtained were used to obtain chitosan in four steps according to the method of [30] adapted by [27], which consists of a demineralization of the powder in acid medium, followed by a deproteinization of the powder in basic medium, then a bleaching of the powder, and finally a deacetylation of the powder by hydrothermochemical process in basic medium.

Refreshing of the Strain and Preparation of P. putida Suspensions
The P. putida strain previously stored at −85 • C was revivified by subculturing on King B agar medium at 30 • C for 24 h at 30 • C. After 24 h, bacterial inocula were obtained by culture in a nutrient medium liquid (Mueller Hinton broth) for 24 h at 30 • C. Then, another culture was carried out from the previous 24 h at the appropriate temperatures. After the 24 h incubation, the cultures were then adjusted to a microbial concentration of about 1 × 10 8 CFU/mL (OD 0.45 at 610 nm) with a spectrophotometer (BioMATE 3S, Thermo scientific) according to the method described by [31].

Filling the Pots
The substrate used was an undegraded ferrallitic-type soil sampled at a depth of 0 to 20 cm using a marked shovel spade. This substrate was collected at the experimental station of the Centre de Recherches Agricoles-Sud du Bénin and dried in the sun. It was then sieved and double sterilized at 120 • C for 20 minutes at an interval of 24 h [32]. Six kilograms of the substrate were weighed and poured into each pot, previously labeled. Each pot was moistened to 2/9th of the Maximum Retention Capacity (MRC) of the substrate water 24 h before sowing [33].

Coating of Seeds with Extracted Chitosans Based on C. amnicola and C. armatum
The coating of the seeds by the chitosans was done on the eve of sowing. Clay was used as a binder to coat the seeds. The amount of chitosan and clay used applied was 10% of the weight of the maize seeds. An amount of water equivalent to 1200 mL/kg of the product was added to obtain the mixture. The seeds were coated in each mixture, thus obtained according to the type of chitosan. They were then air-dried according to the recommendations of [34].

Sowing of Coated Seeds, Inoculation, and Maintenance of Pots
After placing the pots in the greenhouse, two maize seeds previously coated with each chitosan according to the defined treatments were sown after opening the pots about 5 cm deep in the center of each pot. Then, they were immediately inoculated with 10 mL of the P. putida bacterial suspension with a concentration of about 1 × 10 8 CFU/mL according to each treatment, and the pots were immediately closed again.
The pots were watered at 2/9th of their Maximum Water Retention Capacity every 48 h for 30 days. After one week, the plant was de-wedded to one plant per pot. The average temperature values inside the shelter were determined using a thermometer. To obtain the average temperature values inside the greenhouse, a thermometer was used. The average daytime temperature was 35.85 • C and the average night-time temperature was 37.5 • C during the test period.

Chemical Analysis of Soil Used in Greenhouses
The chemical analysis of part of the soil (500 g) used was carried out at the Laboratoire des Sciences du Sol, Eau et Environment (LSSEE) of the Institute National de Recherches Agricoles du Bénin (INRAB). The analyses carried out consisted of pH measurement using an electrode pH meter in a soil/water ratio of 2/5 (g/mL), determination of organic matter [35], determination of assimilable phosphorus using the Bray-1 method [36], and of exchangeable bases (Ca, Mg, K, and Na) by the method of [37].

Collection of Growth and Yield Parameters
Plant growth parameters were assessed by collecting height and crown diameter data on each maize plant at the time of de-mating and every 96 h, i.e., on the 7th, 11th, 15th, 19th, 23rd, 27th, and 30th day after seeding (DAS), while data on leaf length and width were collected on the 30th DAS for leaf area determination. The height of a maize plant is the distance from the crown to the last ligule. It was measured with a tape measure. The diameter at the plant crown was measured using the calliper at the plant crown. The ligulate leaves' surface area was estimated by multiplying the length and width of the leaves by 0.75 [38]. It involved the last two ligulate leaves of each plant. This measurement was carried out only at the 30th DAS. The collected data were recorded in a register. Furthermore, the aerial parts of the plants were cut off, the roots were separated from the culture substrate, and the fresh biomasses were weighed. The dry matter (above and below ground) was determined by weighing after drying in an oven at 70 • C for 72 h.

Statistical Analysis of the Data
Linear mixed-effect models on longitudinal data (using nlme packages for model fitting and lsmeans packages for the calculation of fitted means) were fitted to evaluate the effects of chitosan-based treatments and its combination with P. putida on the growth parameters (height and crown diameter) of maize plants. These effects are graphically represented by the evolution curves per treatment and days after sowing (DAS).
As for the statistical analysis of maize plant yield performance following the different chitosan treatments, a one-factor analysis of variance was performed by yield parameters using the "car" package in the R 4.0.2 software (R Core Team, 2020). This analysis of variance was followed by a post hoc test of pairwise comparisons using Tukey post hoc test [39] was carried out to assess the statistical differences of the means using u multcomp packages. The descriptive statistics calculated required the use of the dplyr and DescTools packages. The packages ggplot2 and ggpur were used to represent the results graphically. A probability (p-value) < 0.05 is considered significant.
A correlation matrix was established in order to assess the dependency between the different parameters measured. The Pearson method was used and using the package corrplot, the coefficients were visualized.

Effect of Temperature Variation on the Growth of P. putida
The growth of P. putida was observed at the different temperatures tested. It was optimal at 30 • C (with an optical density equal to 1.51) but reduced at 40 • C (with an optical density equal to 1.01). At extreme temperatures (4; 16; 48 and 50 • C), P. putida maintained an appreciable but low growth rate ( Figure 1a).

Effect of pH Variation on the Growth of P. putida
Regarding pH tolerance, P. putida grew in a wide range of pH from 4 to 8. At neutrality (pH = 7), P. putida reached its optimal growth with an OD equal to 1.61. On the other hand, inhibition of strain growth was visible from pH 9 (OD = 0.81) until the decline at pH 12 (OD = 0.12). (Figure 1b).

Effect of Salt (NaCl) at Different Concentrations on the Growth of P. putida
The growth of P. putida on nutrient broth was evaluated by measuring the turbidity of the culture at NaCl concentrations ranging from 0 to 1 M. A good tolerance capacity of P. putida to NaCl was noted with a concentration ranging from 0.2 M to 1 M. The P. putida strain subjected to NaCl concentration ranging from 0 to 0.4 M revealed good salt tolerance ability with OD ranging from 1.71 to 1.52. The P. putida strain maintained an appreciable growth rate at the concentration between 0.6 M and 0.8 M and dropped as the amount of NaCl increased (1 M). (Figure 1c).

Effect of pH Variation on the Growth of P. putida
Regarding pH tolerance, P. putida grew in a wide range of pH from 4 to 8. At neutrality (pH = 7), P. putida reached its optimal growth with an OD equal to 1.61. On the other hand, inhibition of strain growth was visible from pH 9 (OD = 0.81) until the decline at pH 12 (OD = 0.12). (Figure 1b).

Effect of Salt (NaCl) at Different Concentrations on the Growth of P. putida
The growth of P. putida on nutrient broth was evaluated by measuring the turbidity of the culture at NaCl concentrations ranging from 0 to 1 M. A good tolerance capacity of P. putida to NaCl was noted with a concentration ranging from 0.2 M to 1 M. The P. putida strain subjected to NaCl concentration ranging from 0 to 0.4 M revealed good salt tolerance ability with OD ranging from 1.71 to 1.52. The P. putida strain maintained an appreciable growth rate at the concentration between 0.6 M and 0.8 M and dropped as the amount of NaCl increased (1 M) (Figure 1c). Table 1 shows the chemical characteristics of the soil used. This substrate is moderately acidic, with a very low percentage of organic matter (1.15%) and organic carbon (0.63%). The content of assimilable phosphorus (8 ppm) is very low and the sum of exchangeable bases of about 4 meq/100 g.

Effects of Chitosan-Based Treatments and Its Combination with P. putida on the Growth Parameters of Maize Plants
The use of chitosan-based bioproducts and PGPR is one of the alternative solutions to increase crop productivity and plant health. The results presented in Table 2 show an improvement in growth parameters at day 30 after sowing following the use of chitosans extracted from C. amnicola and C. armatum and their combination with P. putida on maize growth (Table 2). Moreover, the results show a significant difference between maize plant heights, crown diameters, and leaf areas according to the treatments applied (Figures 2-4).

Effects of Chitosan-Based Treatments and Its Combination with P. putida on t Parameters of Maize Plants
The use of chitosan-based bioproducts and PGPR is one of the alternative solut crop productivity and plant health. The results presented in Table 2 show an im growth parameters at day 30 after sowing following the use of chitosans extracted f and C. armatum and their combination with P. putida on maize growth (Table 2). M sults show a significant difference between maize plant heights, crown diameters according to the treatments applied (Figures 2-4).          Thus, the plants treated with the combination of chitosan extracted from C. armatum and P. putida were clearly more important than the other treatments. The average heights measured on the 30th day after sowing varied between 14.2 ± 0.12 cm and 18 ± 0.71 cm ( Table 2). The lowest values were recorded by control plants while the highest values were recorded by plants treated with the combination of chitosan extracted from C. armatum and P. putida. The treatment based on chitosan extracted from C. armatum alone resulted in a 17% increase in plant height compared to the control. In comparison, its combination with P. putida resulted in a 26.8% improvement in plant performance compared to the control. The same observations were made with the treatment under chitosan extracted from C. amnicola slightly induced an increase of 6%. Furthermore, when combined with P. putida, the increase was 20% compared to the control ( Table 2). In fact, between the 7th and 20th day, plants treated with the combination of chitosan extracted from C. amnicola and P. putida had the best development in diameter, followed by plants treated with the combination of chitosan extracted from C. armatum and P. putida and plants treated with P. putida only which had the same development ( Figure 3). After the 20th day, the development of the plants treated with chitosan extracted from C. armatum and P. putida was significantly higher than the other treatments. The evolution of the plants under the combination of chitosan extracted from C. amnicola and P. putida and chitosan extracted from C. armatum and P. putida was similar (Figure 3). The mean diameters measured on the 30th day after sowing varied between 0.58 ± 0.03 cm and 0.76 ± 0.03 cm ( Table 1). The control plants were those with the low development in diameter. At the same time, those treated with the combination of chitosan extracted from C. armatum and P. putida induced the high development in diameter. In fact, the treatment based on chitosan extracted from C. armatum alone ensured an increase of 24% compared to the control. In comparison, its combination with P. putida induced an improvement in plant performance of 31% compared to the control plants. The combination of chitosan extracted from C. amnicola and P. putida induced an increase of 23% compared to the control. On the other hand, plants treated only with P. putida resulted in a 20% increase compared to the control plants (Table 2).

Leaf Area
In terms of leaf area, combinations between chitosans and P. putida gave the best development ( Figure 4). In addition, the plants treated with the combination of chitosan extracted from C. armatum and P. putida provided the best leaf development at the end of the greenhouse trials (30th day after sowing), and the average value of the leaf area of these plants was 86.7 ± 2.44 cm 2 , an increase of more than 55% of the leaf area of the plants compared to the control plants. The plants treated with chitosan extracted from C. amnicola in combination with P. putida were the second most important with an average of 81.4 ± 3.10 cm 2 and ensured an increase of about 46%. Treatments based solely on chitosan or P. putida gave intermediate results, however, clearly superior to the control treatment. However, it should be noted that the plants treated with P. putida showed a greater development than those treated with chitosan. Thus, treatments with P. putida, chitosan extracted from C. armatum, and chitosan extracted from C. amnicola induced, respectively, an increase in leaf area of 32%, 19%, and 18% (Table 2). Table 3 shows the average biomass obtained from maize plants per treatment. The results indicate that the effect of the different treatments on the performance of the maize plants was significant regardless of the type of biomass measured. In fact, an increase of between 18% and 97% in the fresh aerial biomass of the treated plants was observed compared to the control plants. The average fresh aerial biomass (FAB) observed was respectively 16.7 g, 14.3 g, 13.3 g, 13 g, and 9.99 g for plants treated with chitosan extracted from C. armatum and P. putida, plants treated with P. putida, plants subjected to chitosan extracted from C. amnicola and P. putida, plants subjected to chitosan extracted from C. armatum, and chitosan extracted from C. amnicola.

Effects of Chitosan-Based Treatments and Its Combination with P. putida on Maize Plant Yield Parameters
Statistically, the combination of chitosan extracted from C. armatum and P. putida gave very different results from the other treatments (Table 3). Chitosan extracted from C. armatum and the combination chitosan extracted from C. amnicola and P. putida gave similar results while chitosan extracted from C. amnicola gave similar results to the control. The same trends observed in the fresh aerial biomass (FAB) were observed in the dry above-ground biomass ( Table 3).
The effect of the treatments on the fresh underground biomass (FUB) was greater compared to the results for the fresh above-ground biomass. An increase of between 40% and 110% compared to the control was induced depending on the treatment. Thus, the fresh underground biomass (FUB) was 7.92 g for the combination of chitosan extracted from C. armatum and P. putida; 7.85 g for P. putida; 7.72 g for chitosan extracted from C. armatum; 6.52 g for combination of chitosan extracted from C. amnicola and P. putida; and 5.28 g for chitosan extracted from C. amnicola. The effects induced by these treatments were significant with four groups. Indeed, the treatments C. armatum and P. putida, P. putida, and chitosan extracted from C. armatum formed the first group with similar effects then came groups 2 and 3 constituted, respectively, by the combined treatment of chitosan extracted from C. amnicola and P. putida and chitosan extracted from C. amnicola which give intermediate results relative to the last group constituted by the control (Table 3).
For dry subsurface biomass (Table 3), the results were also broadly similar to the results obtained for fresh subsurface biomass. However, while for the fresh subsurface biomass, the chitosan treatment extracted from C. armatum was in the first group, for the dry subsurface biomass, these effects were similar to those of group 2 (chitosan extracted from C. amnicola and P. putida).

Correlation of the Various Parameters
The measured linear dependence between the growth parameters (height, diameter, and leaf area) and yield (fresh and dry aerial biomass, fresh and dry underground biomass) of the plants is presented in Figure 5. All correlations were positive and important as shown by the size of the circles and the intensity of the colors (rcorr > 0.70). A linear relationship between growth and yield parameters can be deduced from this, the most important of which is the relationship between plant height and diameter (rcorr = 0.99) then the relationship between fresh aerial biomass (FAB) and diameter/height (rcorr = 0.95). The relationship between leaf area and fresh underground biomass (FUB) is the least important (rcorr = 0.72).
Agronomy 2021, 11, 2205 11 of 16 dry subsurface biomass, these effects were similar to those of group 2 (chitosan extracted from C. amnicola and P. putida).

Correlation of the Various Parameters
The measured linear dependence between the growth parameters (height, diameter, and leaf area) and yield (fresh and dry aerial biomass, fresh and dry underground biomass) of the plants is presented in Figure 5. All correlations were positive and important as shown by the size of the circles and the intensity of the colors (rcorr > 0.70). A linear relationship between growth and yield parameters can be deduced from this, the most important of which is the relationship between plant height and diameter (rcorr = 0.99) then the relationship between fresh aerial biomass (FAB) and diameter/height (rcorr = 0.95). The relationship between leaf area and fresh underground biomass (FUB) is the least important (rcorr = 0.72).

Discussion
The deteriorating environmental effects arising from the misuse of pesticides and chemical fertilizers in agriculture has resulted in the pursuit of eco-friendly means of producing agricultural produce without compromising the safety of the environment [40]. These ecological means include the use of PGPR such as P. putida and chitosan, a bioproduct derived from chitin extracted from crustaceans. The knowledge on the tolerant effect of P. putida to abiotic stress under in vitro conditions as well as the synergistic effect of this strain in combination with chitosan on maize cultivation under greenhouse conditions was the subject of this study.

Discussion
The deteriorating environmental effects arising from the misuse of pesticides and chemical fertilizers in agriculture has resulted in the pursuit of eco-friendly means of producing agricultural produce without compromising the safety of the environment [40]. These ecological means include the use of PGPR such as P. putida and chitosan, a bioproduct derived from chitin extracted from crustaceans. The knowledge on the tolerant effect of P. putida to abiotic stress under in vitro conditions as well as the synergistic effect of this strain in combination with chitosan on maize cultivation under greenhouse conditions was the subject of this study.
4.1. Effect of Temperature, pH, and Salinity on the Growth of P. putida Strain A number of abiotic factors, including temperature, salinity, drought, pesticide and fertilizer application, soil pH, and heavy metal contamination, also impede crop productivity [41]. Among these factors, temperature, salinity, and acidity are among the main causes of abiotic stress in plants, causing adverse consequences on the development and quality of harvested products.
The growth of the P. putida strain was observed at the different temperatures tested (Figure 1a). It reached its optimum growth at 30 • C but reduced from 40 • C. At extreme temperatures (4,16,48, and 50 • C), this strain maintained an appreciable but low growth rate. These results are similar to those of [29] with the Pantoea agglomerans lma2 strain that was positive at the different temperatures tested. Indeed, temperature is one of the most important factors that governs the physiology and growth of microorganisms [42].
For the growth of the strain under different pH variations, it was noted that P. putida grew from pH 4 to pH 8 ( Figure 1b). The ability of the strain to grow over a wide range of pH is indicative of its survival ability. This is in agreement with the work of [43] on Pantoea agglomerans CPA-2, able to grow at pH 4.5 to pH 8.6. The same is true for the work of [29], which showed that Pantoea agglomerans lma2 grew in a wide range of pH from pH 4 to pH 8. The use of PGPR will go a long way in supporting the plant to develop both intrinsic and extrinsic ability to tolerate stressful conditions and sustain yield [11].
Concerning salinity, P. putida was found to be salt tolerant at different concentrations (0.2 M to 1 M) (Figure 1c). It retained a strong growth capacity in the presence of NaCl at a concentration of 0.2 to 0.4 M, but from a NaCl concentration equivalent to 0.8 M, the growth of the strains slowed down. Salinity is considered among the abiotic stresses most impacting agriculture for its ability to interfere with crop development and quality. For this reason, practices and innovations that could contain the deleterious effects of such stress are of pivotal importance for maintaining acceptable crop yields [44]. In the last few years, research has shown that the use of salt-tolerant plant growth promoting rhizobacteria (ST-PGPR) in saline agriculture can be harnessed for enhancing productivity and improving soil fertility as well [45]. Authors in [46] also reported that under stress conditions, PGPR can improve the injurious impacts and enhance the yield production under salt conditions.

Effects of Combination Chitosan and P. putida on Growth Parameters
This study shows the positive effect of the combination of chitosan and PGPR on the growth of maize plants. Indeed, inoculation of seeds with chitosan extracted from C. amnicola and C. armatum and their combination with P. putida on day 30 after sowing induced an improvement in growth parameters, which vary from one treatment to another ( Table 2). In addition, the results (Figures 3-5) show a significant difference between maize plant heights, crown diameters, and leaf areas depending on the treatments applied.
Regarding growth parameters, such as height, crown diameter, and leaf area of the plants, the highest values were obtained with plants treated with the combination of chitosan extracted from C. armatum and P. putida, which induced increases of 26.8%, 31%, and 55.7%, respectively, with respect to the control. This rate of improvement in height is higher than the 17% obtained by [47] following the inoculation of maize under greenhouse conditions with chitosan extracted from lobsters combined with strains of Azospirillum lipoferum-P. fluorescens-P. putida. Authors in [48] observed a significant increase in crop yield of 30-50% compared to plants not inoculated with the PGPR strain Azospirillum brasilense (HM053). Various research works have focused mainly on the evaluation of rhizobacteria for their ability to induce improvements in growth and yield parameters in cereals under controlled conditions [12,47,49]. Ref. [50] mentioned that the use of microbial inoculants is a reliable alternative to the use of chemical inputs because these microbial inoculants can act as biofertilizers, bioherbicides, biopesticides, and biocontrol agents. As for the work of [51], it revealed that the application of chitosan increased the percentage of black pepper height and diameter compared to control. Indeed, chitosan has an excellent film-forming property, facilitates the formation of a semi-permeable film on the seed surface that can maintain seed moisture and absorb soil moisture, and thus can promote seed germination [52]. The work of [27] also showed the positive effects of chitosan extracted from the exoskeletons of C. amnicola and C. armatum on in vitro maize seed germination. Several authors have reported the beneficial effects of chitosan application on legume development [52,53]. Authors in [16] reported in their study that the integrated application of PGPR with chitosan significantly reduced symptoms caused by papaya ringspot virus-W (PRSV-W) and tomato chlorotic spot virus (TCSV). The improved effect of chitosan and PGPR has already been reported for other plants and has significantly reduced the severity of tomato leaf curl virus (ToLCV) disease [25] and cucumber mosaic virus [54]. Authors in [20] also reported in their study that foliar application of oligochitosans at different concentrations played a positive role on the growth of tomato and eggplant plants. Chitosan is efficient in agriculture by inducing a signal to synthesize plant hormones such as gibberellins and improve growth and development through a signaling pathway related to auxin biosynthesis [55]. The application of chitosan and PGPR as individual treatments or in combination was effective in improving plant growth and suppressing the severity of disease caused by the Squash Mosaic Virus (SqMV) on cucumber plants [56]. Authors in [57] also showed in their research that the application of chitosan could promote plant germination and growth and reduce harmful salinity. Table 3 shows the average biomass obtained from maize plants per treatment. The results indicate that the effect of the different treatments is very significant on the performance of the maize plants regardless of the type of biomass measured. In fact, an increase of between 18% and 97% in the fresh aerial biomass of the treated plants was observed compared to the control plants, whereas an increase of 40.8% to 111.2% was observed for the underground biomass. This improvement could be explained by the fact that PGPRs readily colonize the root rhizosphere and establish both free life and intimate associations with host plants [5], which could promote plant growth. The stimulating effect of chitosan, on the other hand, can be attributed to increased availability and absorption of water and essential nutrients by adjusting the osmotic pressure of cells, and reduced accumulation of harmful free radicals by increasing antioxidant and enzyme activities [58]. This effect can also be attributed to an increase in the key enzymatic activities of nitrogen metabolism (nitrate reductase, glutamine synthetase, and protease) and to improved nitrogen (N) transport in functional leaves, as well as to an increase in photosynthesis, which promoted plant growth and development [59]. Ref. [60] reported also that PGPRs can help plants absorb nutrients, control the proliferation of plant pathogens, and can have a positive impact on the natural biodiversity of the soil, thus contributing to plant growth and development. These bacteria also serve as plant growth regulators by producing the phytohormones indole-acetic acid (IAA), 1-aminocyclopropane-1-carboxylic acid (ACC), cytokinins, and gibberellins (GA) [61,62].

Effects of Chitosan and P. putida on Biomass Yield Parameters
Furthermore, ref. [23] reported that foliar-applied chitosan, in particular 200 mg/L, increased common bean plant growth, yield, and its quality, as well as physiological constituents in plant shoot under stressed or non-stressed conditions as compared to chitosan untreated plants of common bean. The application of chitosan to rice grains resulted in a significant increase in yield. This increase was due to the induction of phytoalexin accumulation in plant tissues [63]. Given the importance of chitosan and PGPRs in agriculture, their use could therefore reduce dependence on chemical fertilizers and pesticides in the future.

Conclusions
Agricultural practices that reduce or eliminate chemical fertilizers and pesticides through the use of natural products are of paramount importance today for a sustainable and environmentally friendly agriculture. To this end, the application of bioproducts such as PGPR and chitosan in agriculture is one of the promising alternatives to combat abiotic stress in crops and to ensure food and health security of the population. This study revealed through in vitro tests that the strain of PGPR under study, P. putida, showed potentialities of tolerance to abiotic stress such as temperature, pH, and salinity. This study also showed that the application of each of the chitosans extracted from local crabs (C. amnicola and C. armatum) alone or in combination with the rhizobacterium P. putida improved the growth and biomass yield parameters of the maize plants. Plants treated with the combination of chitosan extracted from C. armatum with P. putida induced the best results. This study therefore proves the possibility of using rhizobacteria and chitosan as biostimulants in agricultural production for sustainable and environmentally friendly agriculture in the face of climate change. However, real-world field experiments are needed to better establish the best treatment option for improving maize grain yields.  Data Availability Statement: Main data are contained within the article; further data presented in this study are available on request from the corresponding author.