# Co-Inoculation ofTrichoderma asperellum with Bacillus subtilis to Promote Growth and Nutrient Absorption in Marandu Grass

Authors: Sidney Daniel Araújo da Costa; Aline Figueiredo Cardoso; Gledson Luiz Salgado de Castro; Dalton Dias da Silva Júnior; Thiago Carvalho da Silva; Gisele Barata da Silva
Journal: Applied and Environmental Soil Science
 (2022)

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## Abstract

The authors have investigated the effects of different doses of mineral fertilizers combined with co-inoculation ofBacillus subtilis and Trichoderma asperellum to promote plant growth and use efficient nutrients in a plant Urochloa (Syn. Brachiaria) brizantha cv. Marandu. Individual experiments with doses of the nutrients nitrogen (N), phosphorus (P), and potassium (K) were carried out. The experiment was conducted in a completely randomized design, factorial 3 × 5. With the treatments: control noninoculation, inoculation with Bacillus subtilisUFRA-92, and coinoculation (MIX) with T. asperellum (UFRA-06, UFRA-09, UFRA-12, and UFRA-52) + B. subtilis x nutrient omissions (0%, 25%, 50%, 75%, and 100%). Each treatment had five replications. Biometric parameters, nutrient content, and nutrient use efficiency were evaluated. The results showed that the inoculants promoted growth in Marandu grass. The use of inoculants promoted growth and increased N, P, and K uptake by Marandu grass. Co-inoculation changed leaf area, shoot length, elongation rate, and leaf appearance for N and K and root dry mass for P. In addition, the co-inoculation combined with doses of 75% of N, 50% of P, and 50% of K increased the nutritional content of the leaves by 256% of N, 280% of P, and 29% of K and provided greater agronomic efficiency, with increments of 462% of N, 544% of P, and 177% of K, compared to the control treatment. We present the potential of co-inoculation of B. subtilis and T. asperellum to promote the growth of Urochloa under reduced fertilizer application. There was an improvement in plant growth and nutrient use efficiency.


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## Body

## 1. Introduction

Meeting global food demand requires efficient agricultural management. The projections demand greater land use and an increase in the need for mineral fertilizers, it is estimated that in 2050, 52million tons of fertilizers will be consumed [1]. Currently, Brazil ranks fourth in the world in terms of fertilizer consumption, and this implies a high cost and possibly greater impacts on the environment [2]. Activities such as agriculture have their share of contribution in this scenario. Although historically, fertilizers were used infrequently in the preparation of pastures, currently there is greater use [3]. In the Brazilian Amazon, there are at least 56.6 million hectares of pastures and the main problems are the impoverishment and degradation of soils [4].The intensive use of land demonstrates the need to develop alternative technologies to efficiently use nutrients and do less damage to the environment. Thus, the use of growth-promoting microorganisms can contribute as a potent tool. Studies with the use of microorganisms in different cultures show that they have different modes of action that favor plant growth, mainly due to the better use of nutrients. Growth-promoting microorganisms are responsible for nitrogen mineralization, phosphorus, and potassium solubilization [5]. In addition, they can alter the synthesis of plant hormones, such as auxin and gibberellin [6].Bioinoculation in the grass is a technique used and has shown increases in the growth of the genera (Azospirillum brasilense, Cynodon, and Megathyrsus). Inoculation of A. brasilense associated with nitrogen fertilization in Cynodon dactylon promoted positive effects on shoot dry mass [7]. The use of biopromoter in Megathyrsus (Syn. Panicum) promoted significant increases in the accumulation of shoot and root biomass and higher concentrations of macro and micronutrients [8]. The effect of Trichoderma on grass development needs to be elucidated, to verify if it promotes increases, as shown in other crops of the Poaceae family [9–11]. Inoculation of Trichoderma increased the rate of twinning and growth in Lolium multiflorum compared to the noninoculated treatment [12]. Bacillus inoculation in Lolium multiflorum promoted a significant increase in shoot biomass [13]. Previous results also report that Bacillus sp. and Trichoderma sp. are growth-promoting agents, which contribute to the better development of several crops of the Poaceae family, such as rice, corn, and wheat [14–17].The genera applied in this study, with characteristics that promote growth in other cultures, need to be studied as inoculants in the grass, aiming to increase growth and reduce mineral fertilization. Thus, the objective was to study the effects of different doses of mineral fertilizers combined with the co-inoculation ofBacillus subtilis and Trichoderma asperellum on growth promotion and nutrient use efficiency in Urochloa (Syn. Brachiaria) brizantha cv. Marandu.


## 2. Materials and Methods

### 2.1. Characterization of the Experimental Area

The experiment was carried out in the Seedling Production Unit of the Federal Rural University of Amazonia, Belém, State of Pará, Brazil. The temperature was 32°C and the humidity was 75%, and the regional climate according to the Köppen classification is Afi [18].


### 2.2. Microbial Isolates

The microbial isolates are stored and preserved in the collection of microorganisms owned by the Plant Protection Laboratory (PPL) of the Federal Rural University of Amazonia.Bacillus subtilis UFRA-92 (GenBank MN175193) from açaí palms was isolated and tested for plant growth promotion [19]. In addition, four isolates of Trichoderma asperellum (UFRA-06, UFRA-09, UFRA-12, and UFRA-52) were isolated from rhizosphere soils of reforested and native forest areas in the Amazon and tested for plant growth promotion [20].


### 2.3. Microbial Preparation and Inoculation

TheBacillus subtiliswere grown in 523 solid medium for 24 h at 28°C. This was the culture medium in which the microorganism was grown. Then, bacterial suspensions were prepared with sterilized water. The concentration was adjusted in a spectrophotometer to 550 nm, corresponding to 108 colony-forming units (CFUs) [21, 22]. The isolates of T. asperellum were grown on PDA culture medium at ±28°C, with 12 h photoperiod for seven days. Then, the suspensions were prepared with sterilized water, and the concentration was adjusted to 6 × 108 conidia mL−1 [20]. The irrigation was carried out 14 days after seedling emergence, and each vase of polyethylene received 5 mL of microbial suspension [23]. When co-inoculated, 2.5 mL of B. subtilis and 2.5 mL of T. asperellum were used.


### 2.4. Experimental Design

Simultaneous and independent experiments were carried out to evaluate the effect of fertilization when doses of nitrogen (Exp. 1), phosphorus (Exp. 2), and potassium (Exp. 3) were reduced and associated with the inoculation of microorganisms. The experiment was conducted in a completely randomized design factorial3 × 5. The treatments were control without inoculation, inoculation withBacillus subtilis UFRA-92, and coinoculation (MIX) with T. asperellum (UFRA-06, UFRA-09, UFRA-12, and UFRA-52) + B. subtilis x nutrient omissions (0%, 25%, 50%, 75%, and 100%) and each treatment with five replicates.


### 2.5. Substrate and Sowing

The soil used was collected at the Institute of Agricultural Sciences (UFRA) in 0 to 20 cm layer and classified as a dystrophic yellow latosol with a sandy loam texture [24]. Soil chemical analysis was performed according to [25] and is presented in Table S1. Initially, ten seeds of Urochloa (Syn. Brachiaria) brizantha cv. Marandu in polyethylene pots (20 cm × 30 cm × 0.05 cm), and after 14 days of sowing was performed, leaving one plant per pot.


### 2.6. Mineral Fertilization

Mineral fertilization was carried out seven days before sowing. The doses used for fertilization were 0, 25, 50, 75, and 100% according to the recommendation for the crop [26]. And the sources used were urea (N), simple superphosphate-P2O5 (P), and potassium chloride-K2O (K). In experiment 1, N doses were tested and the recommendation was 50 kg·ha−1, equivalent to 0.208 g/vase. Regardless of the N dose, recommended fertilization of P (80 kg·ha−1) and K (40 kg·ha−1) was performed. In experiment 2, P doses were tested with a recommendation of 80 kg·ha−1, equivalent to 0.834 g/vase. Regardless of the P dose, N (50 kg·ha−1) and K (40 kg·ha−1) fertilization was performed. In experiment 3, K doses were tested and the recommendation was 40 kg·ha−1, equivalent to 0.125 g/vase. Regardless of the K dose, N (50 kg·ha−1) and P (80 kg·ha−1) fertilization was performed.


### 2.7. Morphogenetic and Biometric Analysis

At 35 days after emergence, biometric parameters were evaluated and calculated according to Gomide and Gomide [27]: leaf appearance rate (LAR- leaves. Tiller-1. day-1) was found by the number of leaves that appeared divided by the number of days cycle evaluation, leaf elongation rate (LER- cm. tiller-1. day-1) was found by the difference between the final and initial lengths of leaf blades divided by the number of days of the experiment. Plant height, leaf, and root dry mass were also measured, and leaf area was calculated using equation (Y = 0.7480 × (length × width)) [28] For biomass evaluation, five plants by treatment were collected and separated into shoots and roots. The plant material was dried in an oven (60°C) until reaching constant mass.


### 2.8. Nutritional Analysis

The leaves were collected and placed to dry in an oven with forced air circulation, at an average temperature of 60°C until reaching constant weight. Afterward, milling was carried out in model Willey , ensuring the homogenization of the sample. Subsequently, the decomposition of the plant tissue was carried out, to determine the levels of nutrients (N, P, and K) with perchloric nitric solubilization, using the wet method [25].


### 2.9. Nutrient Use Efficiency (NUE)

The efficiency of nutritional use was calculated from the agronomic efficiency (AE), physiological efficiency (PE), apparent recovery efficiency (ARE), and nutrient response efficiency (NRE) according to Choudhary et al. [29], using the following formulas:(1)AEkgkg:Biomassinoculatedpotskg−BiomassuninoculatedpotskgAppliednutrientcontentkg,PEkgkg:PBVIkg−PBVNIkgNAVA−NAVNAkg,where PBVI = production of biomass from inoculated pots, PBVNI = production of biomass from uninoculated pots, NAVI = nutrient absorbed from inoculated pots, and NAVNI = nutrient absorbed from uninoculated pots.(2)ARE%:NAVIkg−NAVNIkgAppliednutrientcontentkgX100,NREkgkg:ProductionkgAvailablenutrientcontentkg.


### 2.10. Statistical Analysis

Data were evaluated for normality of residuals by the Kolmogorov–Smirnov test. Then, they were subjected to analysis of variance, and whenF values were significant, the SNK test was performed with 5% probability for the isolated effect of microorganisms. When there was an isolated effect of the doses and interaction between the factors, a regression analysis was performed (Table S2). Nutritional analysis data were subjected to the t-test (p<0.05).


## 2.1. Characterization of the Experimental Area

The experiment was carried out in the Seedling Production Unit of the Federal Rural University of Amazonia, Belém, State of Pará, Brazil. The temperature was 32°C and the humidity was 75%, and the regional climate according to the Köppen classification is Afi [18].


## 2.2. Microbial Isolates

The microbial isolates are stored and preserved in the collection of microorganisms owned by the Plant Protection Laboratory (PPL) of the Federal Rural University of Amazonia.Bacillus subtilis UFRA-92 (GenBank MN175193) from açaí palms was isolated and tested for plant growth promotion [19]. In addition, four isolates of Trichoderma asperellum (UFRA-06, UFRA-09, UFRA-12, and UFRA-52) were isolated from rhizosphere soils of reforested and native forest areas in the Amazon and tested for plant growth promotion [20].


## 2.3. Microbial Preparation and Inoculation

TheBacillus subtiliswere grown in 523 solid medium for 24 h at 28°C. This was the culture medium in which the microorganism was grown. Then, bacterial suspensions were prepared with sterilized water. The concentration was adjusted in a spectrophotometer to 550 nm, corresponding to 108 colony-forming units (CFUs) [21, 22]. The isolates of T. asperellum were grown on PDA culture medium at ±28°C, with 12 h photoperiod for seven days. Then, the suspensions were prepared with sterilized water, and the concentration was adjusted to 6 × 108 conidia mL−1 [20]. The irrigation was carried out 14 days after seedling emergence, and each vase of polyethylene received 5 mL of microbial suspension [23]. When co-inoculated, 2.5 mL of B. subtilis and 2.5 mL of T. asperellum were used.


## 2.4. Experimental Design

Simultaneous and independent experiments were carried out to evaluate the effect of fertilization when doses of nitrogen (Exp. 1), phosphorus (Exp. 2), and potassium (Exp. 3) were reduced and associated with the inoculation of microorganisms. The experiment was conducted in a completely randomized design factorial3 × 5. The treatments were control without inoculation, inoculation withBacillus subtilis UFRA-92, and coinoculation (MIX) with T. asperellum (UFRA-06, UFRA-09, UFRA-12, and UFRA-52) + B. subtilis x nutrient omissions (0%, 25%, 50%, 75%, and 100%) and each treatment with five replicates.


## 2.5. Substrate and Sowing

The soil used was collected at the Institute of Agricultural Sciences (UFRA) in 0 to 20 cm layer and classified as a dystrophic yellow latosol with a sandy loam texture [24]. Soil chemical analysis was performed according to [25] and is presented in Table S1. Initially, ten seeds of Urochloa (Syn. Brachiaria) brizantha cv. Marandu in polyethylene pots (20 cm × 30 cm × 0.05 cm), and after 14 days of sowing was performed, leaving one plant per pot.


## 2.6. Mineral Fertilization

Mineral fertilization was carried out seven days before sowing. The doses used for fertilization were 0, 25, 50, 75, and 100% according to the recommendation for the crop [26]. And the sources used were urea (N), simple superphosphate-P2O5 (P), and potassium chloride-K2O (K). In experiment 1, N doses were tested and the recommendation was 50 kg·ha−1, equivalent to 0.208 g/vase. Regardless of the N dose, recommended fertilization of P (80 kg·ha−1) and K (40 kg·ha−1) was performed. In experiment 2, P doses were tested with a recommendation of 80 kg·ha−1, equivalent to 0.834 g/vase. Regardless of the P dose, N (50 kg·ha−1) and K (40 kg·ha−1) fertilization was performed. In experiment 3, K doses were tested and the recommendation was 40 kg·ha−1, equivalent to 0.125 g/vase. Regardless of the K dose, N (50 kg·ha−1) and P (80 kg·ha−1) fertilization was performed.


## 2.7. Morphogenetic and Biometric Analysis

At 35 days after emergence, biometric parameters were evaluated and calculated according to Gomide and Gomide [27]: leaf appearance rate (LAR- leaves. Tiller-1. day-1) was found by the number of leaves that appeared divided by the number of days cycle evaluation, leaf elongation rate (LER- cm. tiller-1. day-1) was found by the difference between the final and initial lengths of leaf blades divided by the number of days of the experiment. Plant height, leaf, and root dry mass were also measured, and leaf area was calculated using equation (Y = 0.7480 × (length × width)) [28] For biomass evaluation, five plants by treatment were collected and separated into shoots and roots. The plant material was dried in an oven (60°C) until reaching constant mass.


## 2.8. Nutritional Analysis

The leaves were collected and placed to dry in an oven with forced air circulation, at an average temperature of 60°C until reaching constant weight. Afterward, milling was carried out in model Willey , ensuring the homogenization of the sample. Subsequently, the decomposition of the plant tissue was carried out, to determine the levels of nutrients (N, P, and K) with perchloric nitric solubilization, using the wet method [25].


## 2.9. Nutrient Use Efficiency (NUE)

The efficiency of nutritional use was calculated from the agronomic efficiency (AE), physiological efficiency (PE), apparent recovery efficiency (ARE), and nutrient response efficiency (NRE) according to Choudhary et al. [29], using the following formulas:(1)AEkgkg:Biomassinoculatedpotskg−BiomassuninoculatedpotskgAppliednutrientcontentkg,PEkgkg:PBVIkg−PBVNIkgNAVA−NAVNAkg,where PBVI = production of biomass from inoculated pots, PBVNI = production of biomass from uninoculated pots, NAVI = nutrient absorbed from inoculated pots, and NAVNI = nutrient absorbed from uninoculated pots.(2)ARE%:NAVIkg−NAVNIkgAppliednutrientcontentkgX100,NREkgkg:ProductionkgAvailablenutrientcontentkg.


## 2.10. Statistical Analysis

Data were evaluated for normality of residuals by the Kolmogorov–Smirnov test. Then, they were subjected to analysis of variance, and whenF values were significant, the SNK test was performed with 5% probability for the isolated effect of microorganisms. When there was an isolated effect of the doses and interaction between the factors, a regression analysis was performed (Table S2). Nutritional analysis data were subjected to the t-test (p<0.05).


## 3. Results

Biometric variables showed an interaction between dose and microorganism factors (p<0.05). For N, the factor doses and microorganisms were significant for the variables height, leaf area, and leaf appearance rate. Plant height presented adjustment to the quadratic regression model with maximum efficiency point at 79%, 53%, and 87% of N using MIX, UFRA-92, and control, respectively (Figure 1(a)). The leaf area as a function of N doses increased linearly for the control and UFRA-92, while it showed a quadratic adjustment for the MIX, with a maximum point at 46% (Figure 1(b)). Leaf appearance rate increased linearly as a function of doses for the three treatments (Figure 1(c)).Figure 1
Effect of inoculation withBacillus subtilis (UFRA-92) and Trichoderma asperellum + UFRA-92 (MIX) as a function of nitrogen doses on height (a), leaf appearance rate (b), and leaf area (c) of Urochloa brizantha cv. Marandu.
(a)(b)(c)There was a significant interaction (p<0.05 for phosphorus doses for root dry mass. It presented fit to the quadratic model for MIX and UFRA-92 with maximum efficiency point at 43% and 64%, respectively, while the control had linear growth (Figure 2).Figure 2
Effect of inoculation withBacillus subtilis (UFRA-92) and Trichoderma asperellum + UFRA-92 (MIX) as a function of phosphorus doses on the dry mass root of Urochloa brizantha cv. Marandu.MIX and UFRA-92 in height presented adjustment to the quadratic model, with maximum efficiency points at 61% and 65%, respectively, while the control showed a positive linear increase for potassium doses (Figure3(a)). The leaf area presented adjustment to the quadratic model in the control and UFRA-92, with maximum efficiency points at 43% and 50%, respectively, while the MIX presented adjustment to the linear model (Figure 3(b)). The leaf elongation rate had a quadratic behavior for the control with a maximum efficiency point at 82%, while the MIX and UFRA-92 had an adjustment to the linear model for the doses (Figure 3(c)).Figure 3
Effect of inoculation withBacillus subtilis (UFRA-92) and Trichoderma asperellum + UFRA-92 (MIX) as a function of potassium doses on height (a), leaf area (b), and leaf elongation rate (c) of Urochloa brizantha cv. Marandu.
(a)(b)(c)The isolated action of the nutrient had a significant effect at (p<0.05) on the biometric variables. For nitrogen, the factor acts in a dependent manner for shoot dry mass, root dry mass, root length, and leaf elongation rate (Figure 4), and the average behavior of the variables had linear growth as a function of nitrogen doses.Figure 4
Isolated effect of reduced nitrogen doses on shoot dry mass (a), root dry mass (b), root length (c), and leaf elongation rate (d) ofUrochloa brizantha cv. Marandu.
(a)(b)(c)(d)For phosphorus, regardless of the microorganisms, all variables had quadratic growth, with maximum efficiency point at 65% for height (Figure5(a)), 53% for shoot dry mass (Figure 5(b)), 67% for the length of root (Figure 5(c)), and 65% for leaf area (Figure 5(d)), while leaf elongation rate (Figure 5(e)) and leaf appearance (Figure 5(f)) had linear behavior.Figure 5
Isolated effect of reduced phosphorus doses on height (a), shoot dry mass (b), root length (c), leaf area (d), leaf elongation rate (e), and leaf appearance rate (f) ofUrochloa brizantha cv. Marandu.
(a)(b)(c)(d)(e)(f)For potassium doses, the isolated action of the doses promoted a quadratic behavior with a maximum efficiency point in 65% in height (Figure6(a)), 66% in root Dry mass (Figure 6(b)), and 62% in root length (Figure 6(c)) and showed a linear increase for leaf appearance rate (Figure 6(d)).Figure 6
Isolated effect of reduced potassium doses on height (a), root dry mass (b), root length (c), and leaf appearance rate (d) ofUrochloa brizantha cv. Marandu.
(a)(b)(c)(d)The effect of inoculation regardless of nutrient doses was significant at (p<0.05) for biometric variables, compared to the control (Table 1). Considering a dose of 75% N, the microorganisms promoted an increase in the growth of U. brizantha, up to 104% in root dry matter, 194% in shoot matter, 22% in root length, and 11% in the rate of leaf elongation, concerning the control.Table 1
Effect of inoculation withBacillus subtilis (UFRA-92) and Trichoderma asperellum + UFRA-92 (MIX), associated with 75% N, 50% P2O5, and 50% K2O, on biometric parameters of Urochloa brizantha cv. Marandu.
TreatmentsRDMSDMRLHeightLALERLApRg/kgg/kgcmcmcm2cm2cm2Control (N)0.50c0.37c34.6b—1.2b—UFRA-920.67b0.53b33.8b——1.4a—MIX1.02a1.09a42.3a——1.3a—Control (P)—0.73b33.70b40.53b15.02b0.7b0.35bUFRA-92—1.87a40.33a61.10a16.99b0.8a0.38aMIX—1.68a36.33b59.40a20.45a0.7b0.35bControl (K)0.66c1.76b35.30c———0.35bUFRA-921.38b2.58a42.83b———0.37bMIX1.83a2.48a47.20a———0.42aInoculation with the microorganisms combined with 50% P increased the growth of Marandu grass. The application of UFRA-92 promoted an increase of 156% in shoot dry mass, 51% in shoot length, 20% in root length, and 9.3% in leaf elongation rate about the control (100% P) (Table1). Only for the variable leaf area (31%), the MIX provided a greater increase concerning UFRA-92 (Table 1).For the dose of 50% K, the MIX provided greater gains than the isolated application of UFRA-92, with an increase of 117% in root dry mass, 34% in root length, and 20% in the rate of leaf appearance, concerning control. The exception was for the variable shoot dry mass (47%) increment in plants that received only UFRA-92 (Table1).


### 3.1. Nutritional Content

From the results found, the best doses were selected as a function of root and aerial biomass to carry out the nutritional analysis. Thus, material collections were carried out at doses of 75% of N, 50% of P, and 50% of K from inoculated plants compared to the 100% control.The nutritional content of shoots increased as a function of microorganisms. The MIX associated with a 75% reduction of N, 50% of P, and 50% of K had an increase of 256%, 280%, and 29%, compared to the control (100%), respectively. Inoculation with UFRA-92 provided increases of 35% in N, 75% in P, and 11% in K when compared to the control (100%) (Figure7).Figure 7
Effect of inoculation withBacillus subtilis (UFRA-92) and Trichoderma asperellum + UFRA-92 on the shoot dry mass nutritional content of Urochloa brizantha cv. Marandu by the t test (p<0.05).
(a)(b)(c)


### 3.2. Nutrient Use Efficiency (NUE)

From the best doses evaluated in the nutritional analysis, 75% of N, 50% of P, and 50% of K, calculations of efficient use were performed, compared to the control (100%). The results showed significant values of (p<0.05) for all indicators of efficient use. Inoculation with MIX and UFRA-92 showed greater agronomic efficiency, generating increments of 462% and 144% for N, 544% and 103% for P, and 177% and 122% for K, respectively, when compared to the control (100%) (Table 2).Table 2
Effect of inoculation withBacillus subtilis (UFRA-92) and Trichoderma asperellum + UFRA-92 (MIX), associated with 75% N, 50% P2O5, and 50% K2O, on agronomic efficiency indices (EA), physiological efficiency (PE), apparent recovery efficiency (ARE), and nutrient response efficiency (NRE) of Urochloa brizantha cv. Marandu.
TreatmentsAEPEARENREkg/kg−1kg/kg−1%kg/kg−1Control (N)1.26c29.87c4.75c6.05cUFRA-923.08b59.89b11.59b13.79bMIX7.08a63.90a36.71a28.37aControl (P)2.68c0.75c1.34c11.86cUFRA-925.45b0.85b3.87b35.62bMIX17.26a3.57a8.98a48.85aControl (K)23.77c48.20b43.19c58.05cUFRA-9252.90b51.75b102.22b121.45bMIX66.00a55.04a136.93a134.55aThe inoculation of MIX and UFRA-92, on physiological efficiency, provided increases of 113% and 100% for N, 376% and 13% for P, and 14% and 7% for K, respectively, concerning the control (100%). The apparent recovery efficiency presented increments of 672% and 144% for N, 570% and 188% for P, and 217% and 136% for K in the MIX and UFRA-92 treatments, respectively. The increments found in the nutrient recovery efficiency were 369% and 128% for N, 312% and 200% for P, and 132% and 109% for K in the MIX and UFRA-92 treatments, respectively, when compared to 100% control (Table2).


## 3.1. Nutritional Content

From the results found, the best doses were selected as a function of root and aerial biomass to carry out the nutritional analysis. Thus, material collections were carried out at doses of 75% of N, 50% of P, and 50% of K from inoculated plants compared to the 100% control.The nutritional content of shoots increased as a function of microorganisms. The MIX associated with a 75% reduction of N, 50% of P, and 50% of K had an increase of 256%, 280%, and 29%, compared to the control (100%), respectively. Inoculation with UFRA-92 provided increases of 35% in N, 75% in P, and 11% in K when compared to the control (100%) (Figure7).Figure 7
Effect of inoculation withBacillus subtilis (UFRA-92) and Trichoderma asperellum + UFRA-92 on the shoot dry mass nutritional content of Urochloa brizantha cv. Marandu by the t test (p<0.05).
(a)(b)(c)


## 3.2. Nutrient Use Efficiency (NUE)

From the best doses evaluated in the nutritional analysis, 75% of N, 50% of P, and 50% of K, calculations of efficient use were performed, compared to the control (100%). The results showed significant values of (p<0.05) for all indicators of efficient use. Inoculation with MIX and UFRA-92 showed greater agronomic efficiency, generating increments of 462% and 144% for N, 544% and 103% for P, and 177% and 122% for K, respectively, when compared to the control (100%) (Table 2).Table 2
Effect of inoculation withBacillus subtilis (UFRA-92) and Trichoderma asperellum + UFRA-92 (MIX), associated with 75% N, 50% P2O5, and 50% K2O, on agronomic efficiency indices (EA), physiological efficiency (PE), apparent recovery efficiency (ARE), and nutrient response efficiency (NRE) of Urochloa brizantha cv. Marandu.
TreatmentsAEPEARENREkg/kg−1kg/kg−1%kg/kg−1Control (N)1.26c29.87c4.75c6.05cUFRA-923.08b59.89b11.59b13.79bMIX7.08a63.90a36.71a28.37aControl (P)2.68c0.75c1.34c11.86cUFRA-925.45b0.85b3.87b35.62bMIX17.26a3.57a8.98a48.85aControl (K)23.77c48.20b43.19c58.05cUFRA-9252.90b51.75b102.22b121.45bMIX66.00a55.04a136.93a134.55aThe inoculation of MIX and UFRA-92, on physiological efficiency, provided increases of 113% and 100% for N, 376% and 13% for P, and 14% and 7% for K, respectively, concerning the control (100%). The apparent recovery efficiency presented increments of 672% and 144% for N, 570% and 188% for P, and 217% and 136% for K in the MIX and UFRA-92 treatments, respectively. The increments found in the nutrient recovery efficiency were 369% and 128% for N, 312% and 200% for P, and 132% and 109% for K in the MIX and UFRA-92 treatments, respectively, when compared to 100% control (Table2).


## 4. Discussion

Grass inoculation with growth-promoting microorganisms has been studied and has shown significant results in the growth and reduction of mineral fertilizer [30–32]. The genera applied in this study, with growth-promoting characteristics in other crops, need to be studied as inoculants in the grass, aiming to increase growth and reduce mineral fertilization.In the present study, from experiments under greenhouse conditions, the synergistic effect of the co-inoculation ofB. subtilis with T. asperellum in improving growth, biomass production, and N, P, and K uptake in Marandu grass was demonstrated, resulting in better nutrient use efficiency. The synergistic interactions that favored plant growth may have occurred from the formation of a biofilm MIX between Bacillus and Trichoderma, due to increased intracellular growth or colonization of the bacteria in the fungal hypha.These mechanisms allow wide colonization of microorganisms along the roots and even penetration into the plant tissue [33], a condition that may contribute to explaining the expressive increase in all variables of the growth of Marandu grass, when inoculated with UFRA-92 and Trichoderma (MIX), independent of the nutrient, concerning the isolated inoculation of UFRA-92.The co-inoculation promoted a greater increase in height, leaf area, and leaf appearance rate (Figure1). The results found to support the hypothesis that plants inoculated with biostimulants favor grass growth in reduced doses of mineral fertilizers. The reduction in N can be attributed to the mineralization of organic N, the acquisition and assimilation of mineral N, and/or the reduction in volatilization and leaching, leading to an increase in grass growth.The greater development of inoculated plants may also be associated with greater production of hormones linked to plant growth (auxin, gibberellin, and cytokinins) and hormones linked to physiological processes that promote greater plant growth. As noted by Paungfoo-Lonhienne et al. [34], Pennisetum clandestinum inoculated with Paraburkholderia sp. and seeded with 50% organic fertilizers and 50% conventional N produced yields similar to those obtained with the conventional 100% N fertilizer. Urochloa sp. inoculated with PGPR at reduced N doses also showed significant increases in the rate of appearance and leaf elongation, possibly due to the greater production of plant hormones [35].Nitrogen is responsible for the vegetative growth of the plant; in this sense, the greater absorption and translocation of N stimulated by the microorganisms MIX and UFRA-92 promoted greater shoot growth and greater leaf expansion. The increase in leaf area from bacterial co-inoculation inU. brizantha was verified by Lopes et al. [23], with an increase of 700% and 108%, when compared to unfertilized plants and fertilized plants, respectively. The increases found in reduced doses of N, associated with microorganisms, can also be related to the greater activity of enzymes responsible for the mineralization of this nutrient, while high doses of inorganic fertilizers can inhibit their action [36].Root development is an important characteristic of the plant, as it allows the establishment and exploitation of water and nutrients, essential for its growth. In the present study, co-inoculation associated with a 50% reduction in the recommended dose of P provided greater root dry mass (Figure2). It is possible to infer that the mechanism of phosphorus solubilization by microorganisms resulted in a high accumulation of P in the plant fertilized with 50% of the dose when compared to fertilization only with mineral fertilizer.The greatest root development found can be related to the participation of phosphorus in plant development, as it acts in physiological processes, root growth, tiller size, and tillering [37]. Inoculated plants showed greater root development, as microorganisms can stimulate root growth by synthesizing or altering hormonal metabolic pathways, such as IAA and cytokinins, which act to elongate hairs and multiply root meristem cells [38]. In previous studies, with Tanzania grass (Panicum maximum) inoculated with rhizobacteria, an increase in root biomass was observed, which was associated with an increase in IAA concentration and a reduction in ethylene [39].The increments found, with 50% of K+, in height, leaf area, and leaf elongation rate can be attributed to differentiated absorption by the inoculated plants (Figure 3). The association with microorganisms causes changes in the architecture of the root system, such as density, length, and amount of root hairs that directly interfere in the exploration and acquisition of the nutrient in the soil, as well as may have provided a lower rate of K+ leaching due to root biomass per soil volume.Results found by Archana et al. [40] showed that the inoculation of Bacillus spp. promoted an increase in corn growth and yield and nutrient accumulation, when compared to plants not inoculated with mineral fertilization. Forage sowed with substrate containing mica (K+ source) and co-inoculated with Bacillus and Azotobacter resulted in biomass accumulation (70%) and higher nutritional content of N (118%) and K (165%), when compared to the uninoculated control [41].The isolated effect of nitrogen provided an increase in shoot and root dry mass, root length, and leaf elongation rate (Figure4). For P, the influenced variables were height, shoot dry mass, root length, leaf area, elongation rate, and leaf appearance (Figure 5), while for K, the variables were height, dry mass, root length, and leaf appearance rate (Figure 6).The increase observed in these variables may be indirectly associated with changes in plant architecture, as root length, influenced by the three experiments, allows for greater exploitation of water and nutrients. N and K in the soil have a high rate of leaching and volatilization; in this sense, a greater number of root hairs may have allowed greater absorption and, as a consequence, greater production of root and aerial biomass, as well as the rate of appearance and leaf elongation. For P, we can infer that produced organic acids contributed to its greater availability. Co-inoculation of microorganisms promotes cumulative effects on the host by increasing the availability of nutrients, from solubilization to the production of hormones linked to plant growth [42].The action of microorganisms regardless of N, P, and K doses was positive on all biometric variables compared to the control (100%). Banayo et al. [43], evaluating the effect of biofertilizers at different rates of inorganic fertilizers on corn yield, found that there was no interaction between the factors, but inoculated treatments showed higher yields at reduced doses of inorganic fertilization, while the isolated use of inorganic fertilizers presents a positive linear behavior, depending on the increase in doses, the same behavior can be verified in this study.In the present study, inoculation with MIX increased the absorption rate and accumulation of macronutrients (Figure7). It is reported that the genus of microorganisms used in this work has effective mechanisms that contribute to nutrient availability [44, 45]. The bacterial isolate is one of the genera reported in the literature with the ability to fix atmospheric nitrogen [46]. The Trichoderma used presents reports of improvements in the nutritional quality of other cultures of the Poaceae family [47].Nitrogen fixation by this microorganism happens because this genus has a group of Nif genes, which is responsible for encoding nitrogenase, the enzyme responsible for nitrogen fixation [48]. A study carried out by Ding et al. [49] based on the isolation of bacteria from the rhizosphere, in the region of China, found that seven isolates, including the genus Bacillus, had the Nif gene.Phosphate fertilizers, when applied to soil, can be rapidly mobilized into insoluble compounds with Al3+ and Fe3+ in acidic soils and Ca2+ in neutral to alkaline soils [50]. On the other hand, the availability of P can occur through the release of organic acids and the enzymes phytases and phospholipases, produced by microorganisms [51]. Acidification of the rhizosphere through the release of H+ causes this proton to compete for the phosphorus adsorption sites bound to iron or aluminum, causing the transformation into orthophosphate (H2PO4)−2, a soluble formula found in acidic soils [52, 53].The solubilization of rock phosphate was recorded in rice inoculated withTrichoderma, which resulted in greater growth and P content [54]. Another mechanism is the production of siderophores, which are low molecular weight binders with high affinity for Fe3+ and Al3+ ions, causing the solubilization of P bound to these ions [55]. The microorganisms that make up the MIX evaluated in this study are producers of siderophores [56] and may have contributed to the high absorption and accumulation of P in U. brizantha inoculated and fertilized with 50% P.Bacillus inoculation resulted in gains in shoot and root, due to greater solubilization of this nutrient [57]. These results demonstrate that the applied microorganisms contributed to nutrient solubilization, even at low doses, favoring their absorption and reducing fixation losses. Rice and wheat inoculated with PGPR and combined with 75% of N, P, and K showed an increase in productivity compared to just complete chemical fertilization (100%), reaching a reduction of 25% of N [58, 59].The study showed that plants inoculated with MIX and UFRA-92 made it possible to reduce 25% of nitrogen, 50% of phosphorus, and 50% of potassium, providing greater efficiency in the use of nutrients, when compared to the control (100%). These results demonstrate that the applied microorganisms contributed to the fixation and solubilization of nutrients, even at low doses, favoring their absorption and reducing losses due to volatilization, leaching, and fixation, and the results of these benefits were reflected in plant growth.The results presented support the hypothesis that inoculation with microorganisms increases the efficient use of nutrients. The greater efficiency found is due to the reduced use of mineral fertilizer in conjunction with inoculation, considering that excessive use can harm the action of microorganisms [60]. The importance of efficient use of nutrients in modern agriculture is of great relevance due to the economic and environmental impacts that can be controlled over the years of production [61].According to Goldstein [62], rhizosphere colonizing microorganisms can replace 25% to 50% of mineral fertilizer, and studies have shown that it is possible to employ the best combinations between microorganisms to obtain better results [63]. The co-inoculation of microorganisms with synergistic effects was verified by Gohil et al. and Vaid et al. [64, 65] in rice plants, which promoted an increase in the yield and absorption of nutrients when compared to noninoculated.An experiment carried out with co-inoculation in grass [41] found that the use of microorganisms promoted an increase in K+ absorption and N fixation when compared to the control treatment, and this behavior is related to the production of organic acids and phytohormones involved in root growth, which increase the area explored by the roots.The efficient use of N, P, and K was also reported by Duarah et al. [66], and the authors evaluated the benefits of the association of a solubilizing microorganism in rice. It was verified that in reduced doses of NPK (50%), the nutritional content and the evaluated growth parameters had better performance when there was inoculation of biopromoters. Elekhtyar [67] found that the yield efficiency of rice plants co-inoculated under doses of NPK showed greater agronomic efficiency in the treatment inoculated with 75% of inorganic fertilizer when compared to the treatment with the recommended dose.The benefits of inoculation with biopromoters are reported by Zeffa et al. [68] in maize plants inoculated with Azospirillum sp., inferring that inoculated plants have higher NUE and reduced damage caused by the reduction of N content in the soil. Similar results are also reported by Yaghoubi Khanghahi et al. [69], who found higher nutritional content (N, P) in rice plants when there was an association of microorganisms versus reduced doses of K, and the evaluated rates of efficient use were significantly higher when compared to 100% of the dose.The application of phosphate-solubilizing bacteria associated with inorganic NPK increased the content of NPK and NUE and reduced the leaching rate of nutrients in grass [66]. Grass inoculation associated with inorganic fertilization with PK promoted higher NUE, representing an increase of 11% in N, 30% in P, and 17% in K when compared to plants fertilized only with inorganic fertilizer [70].New trials from reduced dose macronutrient formulations combined with co-inoculation withB. subtilis + T. asperellum in Urochloa (Syn. Brachiaria) brizantha cv. Marandu should be carried out to investigate the physiological and biochemical mechanisms altered by biostimulation. The co-inoculation of Bacillus subtilis + Trichoderma asperellum in Urochloa brizantha cv. Marandu induced greater growth by changing the absorption behavior using a lower dose of mineral fertilizer in 25% N, 50% P, and 50% K, resulting in increased nutritional efficiency.


## 5. Conclusion

The results of this study showed that the co-inoculation ofTrichoderma asperellum with Bacillus subtilis promoted the growth of Marandu grass under different doses of mineral fertilizer. In addition, the reduction in mineral fertilization of 25% N, 50% P, and 50% K, when associated with microorganisms, favored efficiency in the use of nutrients by more than 100%. This study is an important contribution to new research, and it contributes to agriculture with less environmental impact and to the sustainable use of nonrenewable resources.



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*Source: 3228594-2022-12-17.xml*