Integrated Effect of Bacillus pumilus and Mineral Fertilizers on Growth Parameters on Potato (Solanum tuberosum L. cv. Citlali)

doi:10.21203/rs.3.rs-3143767/v1

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Integrated Effect of Bacillus pumilus and Mineral Fertilizers on Growth Parameters on Potato (Solanum tuberosum L. cv. Citlali)

https://doi.org/10.21203/rs.3.rs-3143767/v1

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Background and aims Plant Growth Promoting Rhizobacteria can make available phosphorus (P) for crops of economic and food importance such as Solanum tuberosum L. (potato), allowing the application of fertilizers to be reduced. The aim was evaluate the integral effect of Bacillus pumilus (phosphate solubilizer) alone and with 30 kg P/ha, as well as a conventional fertilization of 60 kg P/ha on growth, production and sprouting of potato under greenhouse conditions.

Methods Five treatments were assayed: NPK 100% (T1), NK 100% (T2), B. pumilus, BP (T3), BP + NPK 50% (T4), BP + NK 50% (T5). To measure the efect of mixture bacteria and fertilizer on crop development, we recorded plant height, fresh and dry weights, number of internodes, SPAD values, fresh weight of tubers, total number of tubers, number of tubers with diameters greater than or equal to 15 mm, length and width of sprouts.

Results Potato growth parameters indicated a similarity in: plant height (21.65 cm, 20.95 cm), number of internodes (12.42, 13.11), fresh (9.97 g, 5.53 g) and dry weights (1.87 g, 1.08 g) of the treatments T1 and T4, respectively. In mini-tuber production and sprouting, tuber fresh weight (g), number of total tubers, tuber diameter ≥ 15 mm, and sprout width were statistically significant at T4 respect to treatment with B. pumilus alone.

Conclusion Compared to conventional mineral fertilization B. pumilus with 50% mineral fertilizer, have equal effect on plant growth values of potato, production and sprouting of mini tubers.

Bacillus pumilus

potato

phosphate solubilizer

mini-tuber

fertilizer

Potato (Solanum tuberosum L.) is the third most important food crop globally after rice and wheat. In 2021, the global yield of potato was 376,119,974 tons of tubers. At the same year, in Mexico potato production was 1,947,760 tons harvested in 61, 293 ha (FAO 2023a). This crop has many nutrition characteristics as: health promoting components such as vitamin C, phenolic compounds, and iron and has protein content comparable to that of cereal grains (Burlingame et al. 2009). Even potato yields more food more rapidly on less land than any other major crop (Devaux et al. 2020). For potato production, the fertilizer requirements are relatively high and for an irrigated crop they are 80 to 120 kg/ha N, 50 to 80 kg/ha phosphorus P and 125 to 160 kg/ha potassium K; as well as potato crop requires porous soil with pH of 5 to 6 (FAO 2023b). The application of these nutrients in form of mineral fertilizers is essential to promote optimum foliage and tuber growth.

The greatest consumption of these nutrients by the crop occurs during the filling of tuber with a daily demand of 7 kg N/ha and approximately between 0.4 a 0.9 kg P/ha (Horneck and Rosen 2008; Gauhepe and Gutheim 2013). Nitrogen favors foliar development, increasing the photosynthesis surface, which in turn leads to starch production, directly affects the translocation of starch from the leaves to the tubers, influences yield, plant height and number of tubers per unit area (Ramírez et al. 2004). Potassium is an essential nutrient for plant productivity, it influences photosynthesis, chlorophyll content, chloroplast structure, leaf anatomy; it fulfills an important function in the translocation and storage of assimilates since the mobilization of sugars to the tubers allows obtaining a high production and quality (Zhao et al. 2001; Wibowo et al. 2014). While P favors good root and aerial development in seedling establishment stage, in addition, phosphorus participates in various metabolic processes including energy transfer, synthesis of nucleic acids and starch, respiration, synthesis and stability of membranes, activation and deactivation of enzymes, redox reactions and carbohydrate metabolism (Bertsch 2003; Vance et al. 2003). On the other hand, P has also been linked to tuber starch synthesis, as well as increasing tuber specific gravity/dry matter (Stark et al. 2020).

Because potatoes require large amounts of nutrients to increase yields (tuber filling), proper management of the chemical fertilizers that are applied is necessary (Coraspe-León et al. 2008; Rosen and Bierman 2008). However, chemical fertilizer overuse can contribute to soil acidification and soil crust, thereby reducing the content of organic matter, humus content, altering the pH of the soil, growing pests, and even leading to the release of greenhouse gases. The acidity of the soil reduces crop phosphate intake, raises the concentration of harmful ions in the soil and inhibits crop growth (Chandini et al. 2019). According to FAO, total agricultural use of inorganic fertilizers was 189 million tonnes in 2019. The breakdown was 108 million tonnes of N (57% of the total), 37 million tonnes of K (20%) and 43 million tonnes of P (23%); 40 percent higher than in 2000 (33 percent higher for N, 73 percent higher for K and 34 percent higher for P) (FAO 2021). Of the nutrients applied in crops, phosphorus has a high relative demand in the potato crop compared to other crops. According with Nawara et al. (2017), potato needs a soil P concentration of about 76 mg kg^-1 of Olsen P (four times more than needed for crops like cereals) to achieve 95% of its yield potential.

Phosphorus uptake by the plant is interrupted when this nutrient is accumulated in the soil by adsorption processes of metallic cations, such as Ca, Mg, Fe, and Al, becoming unavailable to the plant growth (Sharma et al. 2018). It is estimated that 70% of the inorganic P (Pi) from the application of agrochemicals is quickly converted into insoluble complexes with metal cations aforesaid (Chuang et al. 2007). Some soil microorganisms can transform insoluble P into assimilable forms for plants, including phosphate solubilizing bacteria (Kaur and Reddy 2014). These beneficial bacteria are usually referred to as plant growth-promoting rhizobacteria (PGPRs), can positively influence plant growth through improved mineral nutrition (nitrogen fixation and phosphate solubilization), phytohormone synthesis, siderophore production, increased plant antioxidant and biocontrol properties (Kloepper et al. 1989; Pii et al. 2015; Sirajuddin et al. 2016; Khan et al. 2016). Numerous strains from various genera, such as Azotobacter, Azospirillum, Pseudomonas, Acetobacter, Burkholderia, Enterobacter, Erwinia and Bacillus possess several traits (like the ones mentioned above) plant growth-promoting (Ratti et al. 2001; Esitkenet al. 2010; Bhattacharyya and Jha 2012; Zahir et al. 2012; Israr et al. 2016).

In this context, previous studies have shown that several Bacillus species are model organisms in associative plant–microbe interactions research (Yu et al. 2011; Lee et al. 2014; Saeid et al. 2018; Saxena et al. 2020). By example, Bacillus pumilus a Gram- positive, spore-forming bacteria, which commonly occurs in various environments like marine water, deep-sea sediments, and soil. After B. subtilis, B. pumilus is the second most studied bacterial strain from the genus Bacillus, in terms of plant growth promotion properties (Dobrzyński et al. 2022).

The research suggest B. pumilus is a good growth promoter of plants, by example inoculation with B. pumilus enhanced tolerance stresses salt in culture rice (Oryza sativa L.) (Khan et al. 2016); together with phosphorus enhanced the uptake of boron by rapeseed (Masood et al. 2019). In addition, B. pumilus strain 15.1 to having an effect as a biocontrol agent against Ceratitis capitata larvae (Molina et al. 2010). Importantly, B. pumilus can have a positive effect on native soil bacterial communities. By remaining for a long period of time in the soil and modify the bacterial community of this (Kang et al. 2013). It has even been observed that B. pumilus enhanced bacterial biodiversity, including Shannon diversity in the rhizosphere and root endosphere of rice (Win et al. 2020. However, other mechanisms such as the solubilization of phosphates by B. pumilus have not been so extensively studied. In this sense, the present study aims to evaluate the effect of inoculation of B. pumilus in combination with a mineral fertilization on the growth, production and sprouting of potato under greenhouse conditions.

Phosphate-solubilizing bacterial strain

The bacterial strain Bacillus pumilus Fo03 (MN100586) belongs to the strain of phosphate solubilizing bacteria of the Laboratorio de Edafología y Ambiente, Universidad Autónoma del Estado de México, Toluca, México. It was selected due to its high solubilization capacity of Ca₃(PO₄)₂ 115 mg P l^-1 * day (Yañez-Ocampo et al. 2020) in Pikovskaya liquid medium (PVK) (Pikovskaya 1948).

Inoculum reparation of Bacillus pumilus

The bacterial inoculum was prepared from bacterial colonies preserved in PVK agar at 4°C. In a seed tube with 7 mL of 0.85% NaCl, bacterial biomass was added until obtaining 0.20 absorbance units, at a wavelength of 600 nm in a UV-VIS spectrophotometer (GENESIS 20), equivalent to an approximate concentration of 1x10 ⁶ CFU mL^-1. Of the previous inoculum, 2 mL were added in 500 mL Erlenmeyer flasks with 200 mL of nutrient broth, in triplicate, incubating for 4 days at 28 ± 2°C. After four days, the biomass was transferred to 12 mL centrifuge tubes and centrifuged at 2500 rpm for 15 minutes. The collected biomass was washed with 0.85% NaCl, collected in a 50 mL Erlenmeyer flask and kept refrigerated at 4°C. To determine the population density of bacteria in the collected biomass, the viable live cell count method was performed by surface seeding (Madigan et al. 2003). This procedure was carried out in triplicate for each dilution and the boxes were incubated at 27°C for 24 h. The colony forming units (CFU) grown on the surface of the Petri dishes were counted, then expressed as CFU mL^-1.

Potato microplants of potato cultivar Citlali and growing conditions

Virus-free microplants of potato cv. Citlali, from the Germplasm Bank of the Programa Nacional de Papa del Instituto Nacional de Investigaciones Forestales Agrícolas y Pecuarias (INIFAP) in Toluca, México, microplants were propagated as nodal cuttings in planting tubes (Mora-Herrera et al. 2005), in Murashige and Skoog medium (Murashige and Skoog 1962) modified (Espinoza et al. 1986). The medium contained per liter: NH₄NO₃, 17.5 g; KNO, 20 g; CaCl₂·2H₂O, 4.5 g; KH₂PO₄, 1.75 g; H₃BO₃, 50 mg; MnSO₄, 200 mg; ZnSO₄· 7H₂O, 100 mg; KI, 10 mg; Na₂MoO₄, 2.5 mg; CuSO₄· 5H₂O, 5.0 mg; CoCl₂· 6H₂O, 0.5 mg, and it was supplemented with Myo-Inositol 0.1 g; Fe, 0.065 g; thiamine, 0.0004 g; calcium pantothenate, 0.002 g; glycine, 0.00005 g; GA3, 0.0001 g; and sucrose, 30%. Microplants were incubated at 20 ± 1°C under a 16 h photoperiod (fluorescent lights: 35 µmol m^–2 s^–1, 400–700 nm) and in sterile conditions.

Physicochemical analysis of soil

Soil samples were evaluated for estimation of organic carbon, organic matter, available phosphorus, electrical conductivity, pH and texture (NOM-021-RECNAT-2000). These physicochemical properties of the collected soil sample are presented in Table 1.

Table 1

Physicochemical characterization of the soil before the application of *B. pumilus* and NPK fertilizer
Soil characteristics	Values
Organic carbon (%)	3.96
Organic matter (%)	6.80
Available phosphorus (mg Kg^-1 of soil)	189.78
Electrical conductivity (µS cm^-1)	1103
pH 1:2 (w/v)	5.76
Texture	Clay loam

Experimental location and climatic conditions

The experiment was conducted during the months of May-August 2022 (spring-summer) with 16 h photoperiod, in non technified greenhouse at the facilities of the Laboratorio de Fisiología-Biotecnología. Programa Nacional de Papa, Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias, located in CONJUNTO SEDAGRO, Metepec, Estado de México, México, with geographic reference to 19° 24' 51" north latitude and 99° 58' 44" west longitude (https://smn.conagua.gob.mx/es/). Temperate sub-humid climate with rainfall in summer, with precipitation between 700–1000 mm; at an altitude of between 2,500 and 2,800 m, according to the Instituto Nacional de Estadística y Geografía (https://docplayer.es/99001648-Prontuario-de-informacion-geografica-municipal-de-los-estados-unidos-mexicanos-metepec-mexico-clave-geoestadistica-15054.html). Air temperature and relative humidity were recorded during the growing season of the potato crop under greenhouse conditions with the help of Elitech GSP-6 Digital Temperature and Humidity Data Logger® (Table 2).

Table 2

Climatic data: air temperature (°C) and relative humidity (%) during the potato growing season May-August 2022 under greenhouse conditions
Date	Maximum temperature¹	Minimum temperature¹	Maximum relative humidity²	Minimum relative humidity²
May 10 - June 10	35.6	9.0	88%	15.7%
June 11 - July 11	40.0	9.1	95.7%	26.4%
July 12 - August 12	33.8	9.8	94.5%	71.9%
¹ Average temperature during the May-August season 2022
² Average humidity during the May-August season 2022

Experimental design and treatments

The experimental design was completely randomized; it consisted of 15 replicates per treatment, 1 plant per pot as a replicate. The treatments were as follows: T1: NPK 17/17/17 (T17) full dose of 100% NKP chemical fertilizer, T2: (NH₄)₂SO₄ (NPK 21/00/00/24S) and KNO₃ (NPK 13/00/46) fertilization without phosphorus with a full dose of 100% NK chemical fertilizer, T3: B. pumilus inoculant, T4: B. pumilus inoculant and 50% NPK chemical fertilization T17, T5: B. pumilus inoculant and 50% NK chemical fertilization (NH₄)₂SO₄ and KNO₃.

The fertilizer doses applied to the crop were as follows: 180 kg/ha of N, distributed in 90 kg of N at sowing and 90 kg of N at 50 days after sowing (DAS); 60 kg of P only at sowing and 350 kg of K, distributed in 70 kg of K at sowing and 280 kg of K at 60 DAS. In the treatments where fertilization was at 50%, the following fertilizer doses were added: 90 kg of N, distributed in 45 kg of N at sowing and 45 kg of N at 50 DAS; 30 kg of P only at sowing and 175 kg of K, distributed in 35 kg of K at sowing and 175 kg of K at 60 DAS. The roots of potato plants were immersed for 20 minutes in solution containing B. pumilus strain at 10⁸ CFU mL^-1.

The plants were then planted in 1 kg pots with unsterilized soil from potato crops, horticultural grade perlite and peat moss in a 1:1:1 (w/v). A re-inoculation with B. pumilus strain at a concentration of 10³ CFU mL^-1 was performed after 50 DAS.

Crop management and harvest consisted on irrigation of the plants manually every third day with potable water, while for biological control of Trialeurodes vaporariorum (whitefly), potassium soap was applied by spraying leaves of the plants once a week for 15 days, 30 DAS. Plants were harvested after 90 DAS. The potato mini tubers were harvested 7 days after cutting potato plants.

Data collection

The study variables were plant height (PH), SPAD values using a Konica Minolta® SPAD-502, number of internodes per plant (NI), fresh weight of leaves and stems (FW), dry weight of leaves and stems (DW). SPAD values were measured in the leaflets of the third and fourth leaf, in the middle portion of the leaflets, at 8:00 am, 50 days after planting the crop, according to the methodology described by De Lima et al. (2014). For DW, whole plant biomass was oven-dried at 70°C for 72 h. During harvest, number of tubers smaller than 15 mm in diameter per plant, number of tubers equal to or larger than 15 mm in diameter per plant, number of total tubers per plant (NTT) and total fresh weight of tubers (FWT) per plant were evaluated (Flores-Gutiérrez et al. 2018). The mini tubers were stored in paper bags (individually for each treatment), taking care to maintain a temperature between (15 to 16°C) and diffuse light on the tubers. Afterwards, sprout length and width of the mini tubers were evaluated.

Statistical Analysis

All results presented were statistically analyzed using IBM SPSS statistics 21. Analysis of variance (one-way ANOVA) was performed for data that were normally distributed, while for data that did not meet this normal distribution a Kruskal-Wallis one-way ANOVA was performed. Statistically significant differences were compared using Duncan's multiple range test (p < 0.05) for parameters with a normal distribution; for parameters that did not meet a normal distribution, Dunn's test was performed for each pair of groups, with adjustment of the degree of significance (p) by Bonferroni.

Effect of Bacillus pumilus and NPK fertilizer on plant growth parameters of potato plants

The Table 3 and 4 show plant growth parameters of potato plants from treatments with B. pumilus strain and two rates of inorganic fertilizer with and without phosphorus. In terms of PH and FW of potato plants, our results report that addition of the B. pumilus strain in combination with a 50% dose of NPK and NK fertilizer (T4 and T5) did not present significant differences (p > 0.05) compared to a NPK 100% and NK 100% fertilization (T1 and T2). In conditions where only B. pumilus strain was added, values obtained were nonsignificant for all growth parameters measured.

Table 3

Growth parameters of potato plants treated with *B. pumilus* strain and/or NPK doses
Treatments	Plant height cm ¹	Number of internodes/plant ¹
NPK 100% (T1)	21.65 ± 5.71^a	12.42 ± 2.93^a
NK 100% (T2)	15.97 ± 2.18^ab	10.85 ± 1.95^a
B. pumilus, BP (T3)	13.42 ± 4.71^b	10.07 ± 3.01^a
BP + NPK 50% (T4)	20.95 ± 8.23^a	13.11 ± 3.75^a
BP + NK 50% (T5)	18.94 ± 8.87^ab	12.00 ± 4.94^a
¹ Data are expressed as mean ± standard deviation. Different letters in each column represent statistically significant differences by Duncan p < 0.05

In the number of internodes, there were no significant differences (p > 0.05) between treatments with B. pumilus strain and NPK and NK fertilization at 50% and 100%. In SPAD values, significant differences (p < 0.05) were obtained when treatments T2 and T5 were compared with T1, showing the best SPAD values in T1 followed by T2 and T5, respectively (Fig. 1).

The dry weight of stems and leaves of plants treated with the B. pumilus strain plus 50% NPK fertilization increased by 54% compared to the dry weight of plants treated with 100% NK fertilization.

Table 4

Fresh and dry weight of potato plants 90 DAS, treated with *B. pumilus* strain and/or NPK doses
Treatments	Fresh weight of stems and leaves (g) /plant ¹	Dry weight of stems and leaves (g) /plant ¹
NPK 100% (T1)	9.97 ± 8.54^a	1.87 ± 0.94^a
NK 100% (T2)	3.68 ± 1.28^ab	0.84 ± 0.37^bc
B. pumilus, BP (T3)	1.30 ± 0.81^b	0.23 ± 0.11^c
BP + NPK 50% (T4)	5.53 ± 4.49^ab	1.08 ± 0.72^ab
BP + NK 50% (T5)	5.23 ± 3.13^ab	1.38 ± 0.52^ab
¹Data are expressed as mean ± standard deviation. Different letters in each column represent statistically significant differences Dunn's test p < 0.05

Production of mini tubers of potato plants

It was found that fresh weight of the tubers of treatments T2 and T4 compared to fresh weight of T1 tubers did not show significant differences (p < 0.05). However, inoculation with B. pumilus under 50% NPK and NK conditions (T4 and T5) improved tuber fresh weight by 75% and 67%, respectively, when compared to T3 treatment. B. pumilus inoculum increased the number of total tubers per plant in combination with 50% NPK fertilizer (T4), compared (p < 0.05) to T3. On the other hand, no significant differences (p > 0.05) were found in NTT of T2 and T5 compared to T1 (Table 5).

Table 5

Tuber fresh weight and number of total tubers per plant 90 DAS
Treatments	Fresh weight of tuber g ¹/plant ¹	Total number of tubers/plant ¹
NPK 100% (T1)	22.12 ± 5.59^a	6.57 ± 2.57^a
NK 100% (T2)	17.82 ± 3.39^ab	6.00 ± 2.44^a
B. pumilus, BP (T3)	4.86 ± 2.49^c	2.93 ± 1.43^b
BP + NPK 50% (T4)	19.42 ± 10.50^ab	5.22 ± 1.85^a
BP + NK 50% (T5)	14.87 ± 4.82^b	4.66 ± 2.87^ab
¹Data are expressed as mean ± standard deviation. Different letters in each column represent statistically significant differences Duncan p < 0.05

The number of tubers with a diameter ≥ 15 mm increased in the treatment with B. pumilus inoculum plus 50% NPK (T4) compared to the treatment with B. pumilus alone (T3). While the number of tubers with diameter ≥ 15 mm of treatments T2 and T5 did not show significant differences (p < 0.05) compared to treatment T1. For the number of tubers with diameter < 15 mm, there were no significant differences (p > 0.05) between treatments (Table 6).

Table 6

Number of tubers of diameter greater than/equal to and less than 15 mm per plant
Treatments	Number of tubers with diameter ≥ 15 mm /plant ¹	Number of tubers with diameter < 15 mm / plant ¹
NPK 100% (T1)	4.00 ± 3.40^ab	3.28 ± 2.92^a
NK 100% (T2)	3.14 ± 2.03^ab	2.85 ± 2.03^a
B. pumilus, BP (T3)	1.45 ± 0.93^b	2.54 ± 2.06^a
BP + NPK 50% (T4)	4.66 ± 2.33^a	3.16 ± 1.32^a
BP + NK 50% (T5)	2.66 ± 1.00^ab	2.37 ± 1.59^a
¹Data are expressed as mean ± standard deviation. Different letters in each column represent statistically significant differences Dunn's test p < 0.05

After 180 days of storage, tuber shoot length of the treatment with only B. pumilus (T3) increased compared (p < 0.05) to treatment with 100% NPK fertilization (T1). Treatments T2, T4 and T5 did not show significant differences (p > 0.05) compared to treatment T1 (Fig. 2). The treatment added only with B. pumilus showed the lowest shoot width (p < 0.05) compared to the other treatments (Table 7).

Table 7

Length and width of sprouts of mini potato tubers
Treatments	Sprout length cm ¹/plant ¹	Sprout width cm ¹/plant ¹
NPK 100% (T1)	3.00 ± 1.88^b	0.14 ± .06^a
NK 100% (T2)	3.53 ± 2.41^ab	0.16 ± .06^a
B. pumilus, BP (T3)	4.72 ± 2.45^a	0.10 ± .02^b
BP + NPK 50% (T4)	3.71 ± 2.08^ab	0.17 ± .09^a
BP + NK 50% (T5)	3.54 ± 1.90^ab	0.14 ± .07^a
¹Data are expressed as mean ± standard deviation. Different letters in each column represent statistically significant differences Dunn's test p < 0.05

Inoculating phosphate solubilizing bacteria improves plant growth

Bacillus species are known to their plant-growth-promoting performance. For example, Bacillus pumilus have been reported to promote tomato plant growth by biological nitrogen fixation contributes to the increased N uptake by tomato (Masood et al. 2020) and salt tolerance in rice (Oryza sativa L.) (Khan et al. 2016). At present, has been report that the use of Plant Growth Promoting Bacteria is also essential for the promotion of plant nutrient circulation and reducing the high demand for chemical fertilizers (Çakmakçı et al. 2006; Javaid and Mahmood 2010). In the present study, the inoculation with B. pumilus and a source of 50% NPK fertilizer in the potato crop improved PH, LSFW, LSDW and SPAD values compared to plants that were only inoculated alone with B. pumillus.

Several studies have highlighted the integrated effect of chemical and bio-fertilizers for sustainable agricultural production. In the experiment by Lucas et al. (2004), application of Bacillus licheniformis and a dose of 110 kg/ha of P₂O₅ significantly increased height of plants (cm) and leaf area (cm²) of Lycopersicon esculentum regarding control without bacteria but not the foliage dry weight (g). In another study Ratti et al. (2001), found that the dual application of Glomus aggregatum and Bacillus polymyxa (phosphate solubilizing rhizobacteria) yielded 21.5 g plant dry weight (biomass) as compared to 14.9 g of control plant dry weight (without inoculum) at the same level of 200 mg of tricalcium phosphate for kilogram of soil. Masood et al. (2020), found that the treatment containing B. pumilus inoculum with a nitrogen source and with 100 mg P kg^-1 had no impact on SPAD (leaf chlorophyll content) values of tomato plants compared to the treatment without B. pumilus, without nitrogen source and with 100 mg P kg^-1, as the results obtained in our experiment when comparing treatments 2 and 5 (Fig. 1).

On the other hand, regarding the fresh weight of the tubers, the results found during this experiment are consistent with those obtained by El-Sayed et al. (2015), where it was found that use of biofertilizers (Azospirillum brasilense, Azotobacter chroococcum, Bacillus megaterium, vesicular-arbuscular mycorrhiza and Bacillus cereus) in combination with a compost-type organic amendment (11.9 t ha^-1) and 50% mineral fertilizer, can produce similar results in yield (fresh weight of the tuber) when compared with the yield in tuber tons per hectare, that received a full rate of mineral fertilizer plus compost (11.9 t ha^-1).

Biofertilizers based on PGPR are an alternative for a sustainable agriculture

Currently, biofertilizers like Plant Growth Promoting Rhizobacteria (PGPR) are great alternatives to chemical fertilizers in sustainable agricultural systems, since these biofertilizers enhance plant growth, yield, and quality (Ajmal et al. 2018). A large part of mineral fertilizers, particularly inorganic phosphorus added to the soil, is immobilized by metal cation complexes (Fe³⁺, Al³⁺ and Ca²⁺) present in the soil shortly after it is applied, limiting its bioavailability for plants. The application of phosphate solubilizing bacteria allows the mineralization and solubilization of phosphorus from inorganic fertilizers, which allows adequate availability of mineral content in soils during cultivation (Vessey 2003; Khan et al. 2007; Esitken et al. 2010). In accordance with Kostenko et al. (2020), the use of biological preparations together with mineral fertilizers makes it possible to halve the dose of their application, since the bacteria increase the absorption of microelements by plants, by increasing the volume of the root system and its adsorbing activity.

Integrated effect of B. pumilus on potato yield

Phosphorus can promote root growth and accelerate tuber formation, making it a critical element in the initial period of plant development and tuber formation (Oyarzún et al. 2002). Many studies report that adding P increased the proportion of large tubers harvested (Freeman et al. 1998; Almamori and Abdul-Ratha 2020) while others observed that the increase in the number of small tubers was offset with a decrease in number of large tubers (Rosen and Bierman 2008).

In other hand, other researchers report no effect of P on total tuber number despite observing yield responses and suggest that number of tubers is influence by other nutrients mostly potassium (Flores-López et al. 2016). Regarding the number of total tubers and with a diameter greater than 15 mm, this yield was maintained (Table 5–6) even when the rate of NPK fertilizer was reduced by half plus the addition of B. pumilus compared to a fertilization NPK 100%. In potato cultivation, the seed tuber is used as planting material, physiological status of seed potato has a great impact on sprouting and may depend nutrient management of the crop in the field (Merino et al. 1997; Frazier et al. 2004).

Phosphorus has an important physiological role in the process of sustaining sprouts, since it is an essential component for synthesis, transport and storage of starches, carbohydrates necessary for growth of new sprouts (Viola et al. 2007; Barona et al. 2015). Potato sprouting during storage, in addition to being influenced by the physiological age of the seed tuber and the nutrition that crop received during its life cycle, is also influenced by temperature and the amount of light. Very low temperatures (less than 2°C) cause internal freezing damage, while high temperatures (above 25°C) cause an acceleration in respiration and a greater oxygen requirement and can discolor the internal tissue of the tuber, on the other hand, diffused light generates the production of good quality sprouts (short and vigorous) (Barona et al. 2015).

It is likely that the growth promotion of potato plants that were inoculated with the B. pumilus strain is mainly due to phosphate solubilization by this bacterium. As mentioned above, B. pumilus can solubilize phosphates in PVK medium. In this sense, phosphate solubilization by bacteria occurs when they secrete organic acids such as citric acid, oxalic acid and succinic acid; phosphatase and phytase enzymes, making phosphorus available to plants (Tomer et al. 2016). This has been evidenced by Chawngthu et al. 2020, bacterial strains Bacillus cereus, Bacillus subtilis, produced oxalic acid, malic acid, formic acid, acetic acid tartaric acid, gluconic acid (Chawngthu et al. 2020). In the study reported by Tomer et al. (2016), results similar to the present work were observed, on the effect of the addition of phosphate solubilizing bacteria of the genera, Pseudomonas, Mycobacterium, Bacillus, Pantoea, Rhizobia, Burkholderia with phosphate fertilizers, showed 22% more in wheat grain yield and 26% more in phosphorus uptake, at the same time the fertilizer input was reduced by 30% from 120 to 90 kg P₂O₅ ha^− 1.

In addition, bacteria of the genus Bacillus, such as B. pumilus can produce indoleacetic acid (IAA). In the trial reported by Cruz-Martin et al. (2015), B. pumilus produced 28.9 µg mL^− 1 of IAA. Besides, it significantly increased stem height and thickness, modified root architecture, improved fresh and dry weight of banana plants. In other investigations, B. pumilus strain TRS-3, isolated from the rhizosphere of Camellia sinensis, showed several characteristics characteristic of the PGPR group such as phosphate solubilization, siderophore production and IAA secretion. In addition, it had a strong antagonism against phytopathogenic fungi (Chakraborty et al. 2013).

There is a close relationship between phosphate solubilization and IAA production on growth promotion in crops. It is known that essential plant nutrients are absorbed from the soil by the roots. In this sense, good root growth is characteristic of the effect of auxins such as IAA. Many PGPR stimulate root growth, sometimes through the production of phytohormones by the plant or by bacteria (Torres-Rubio et al. 2000; Lucy et al. 2004). In the experiment of Rajapaksha and Senanayake (2014) suggest that the ability of R2 (B. pumilus) to secrete IAA may have led to better root growth and thus higher P acquisition (P that was added in the form of triple superphosphate and rock phosphate) in the rice crop, furthermore B. pumilus may also have successfully colonized rice roots.

In this report, it was observed an integrated effect of Bacillus pumilus with the half dose of mineral fertilizers (NPK 50%) on growth parameters on potato. In the experiment, we were able to obtain short and vigorous sprout both in the treatments with 100% NPK and NK fertilization and in combination with B. pumilus and fertilizer at 50%. While the longest and thinnest shoots were observed in the treatment with B. pumilus alone. This finding represents an alternative to potato producers, for reducing use of agrichemicals and in consequence having agricultural practices environmentally acceptables that restore soil fertility, besides reduce insumes costs.

N Nitrogen

P phosphorus

K potassium

DAS days after sowing

SPAD Soil Plant Analysis Development

PH Plant height

NI Number of internodes

FW Fresh weight of leaves and stems

DW Dry weight of leaves and stems

NTT Number of total tubers

FWT Fresh weight of tubers

Funding

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Author Contributions

All authors contributed to the study conception and design. Methodology, material preparation, and analysis was performed by Denisse Monserrat de la Paz-Osorio. Formal analysis, data collection and investigation were performed by Rocío Vaca-Paulín and Pedro del Águila-Juárez. Molecular analysis contributed by Arnoldo Wong-Villarreal. Chemical analysis contributed by Laura Sanchez Paz and Nadia de la Portilla-Lopez, Jorge Lugo-de la Fuente The writing - review and editing, conceptualization and first draft of the manuscript was written by Martha Elena Mora-Herrera and Gustavo Yañez Ocampo and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Declarations

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Integrated Effect of Bacillus pumilus and Mineral Fertilizers on Growth Parameters on Potato (Solanum tuberosum L. cv. Citlali)

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Abstract

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Introduction

Materials and Methods

Phosphate-solubilizing bacterial strain

Inoculum reparation of Bacillus pumilus

Potato microplants of potato cultivar Citlali and growing conditions

Physicochemical analysis of soil

Experimental location and climatic conditions

Experimental design and treatments

Data collection

Statistical Analysis

Results

Effect of Bacillus pumilus and NPK fertilizer on plant growth parameters of potato plants

Production of mini tubers of potato plants

Discussion

Conclusions

Abbreviations

Declarations

References

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