Potential application of Bacillus amyloliquefaciens EB13 inoculant for improving soil fertility and Citrus sinensis growth

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Introduction
Chemical fertilizers have been used widely in agriculture to improve soil fertility hence increase crop yield.However, the long-term application of chemical fertilizers entailed the risk that has led to reducing soil quality and the efficacy of chemical fertilizers has been decreasing over time (Savci, 2012).The excessive use of chemical fertilizers in the long-term causes a number of problems, such as the soil degradation, nutrients leaching, reduction of organic matter, and heavy metal contamination (Han et al., 2016;Lin et al., 2019).Because of these concerns, many studies have been conducted to develop green fertilizers, including the development of biofertilizers that utilize plant growth-promoting bacteria.Soil quality is influenced by many factors such as microbial, biochemical, physical, and chemical of soil.The soil microbial activity directly impacts ecosystem stability and fertility.Therefore, a good level of microbial number is essential to maintain the soil quality.The microorganisms and soil enzymes play a key role in nutrient cycling and essential for mineralization, organic matter transformation, and plant nutrients supply in the soil ecosystem.Soil enzyme is commonly used as a sensitive tool to detect changes in the soil ecosystem including fertility and perturbation (Vázquez et al., 2000).Interaction between plant and bacteria varies and occurs in different ways.The interaction may support plant growth, threatens plant health, or has no effect on the plant.The change of soil condition may alters the way bacteria interact.Several studies reported that Bacillus strains, including Bacillus amyloliquefaciens, Bacillus cereus, and Bacillus subtilis could interact with plants by promoting plant growth and suppressing plant disease (Choudhary and Johri, 2009).Bacillus is a group of bacteria that can be found elsewhere in a large variety of environments (Felske et al., 2004;Pignatelli et al., 2009;Connor et al., 2010).As a plant growth-promoting rhizobacteria (PGPR), Bacillus strains can solubilize fixed soil P and increase the availability of inorganic P to plants thus enhance crop yields, for example B. polymyxa, B. pumilus, and B. subtilis (Kumar and Narula, 1999;Matos et al., 2017).There are two mechanisms of how PGPR increases plant growth, via direct and indirect mechanism.Direct mechanism occurs by supplying growth factors to plants, such as nutrients (phosphate solubilization and iron mobilization) and hormones (indole acetic acid, cytokinin, and gibberellin).Indirect mechanism occurs by suppressing the plant pathogens and harmful microorganisms (Glick, 2012;Akinrinlola et al., 2018).The application of bacteria inoculants in the nonsterile condition in field study still encounter a problem.Most of the researches that have been performed in this condition showed less promising on plant response.The bacteria that was introduced could not perform and survive in the field (non-steril soil) because non-sterile soil contains various microorganism species.Then, the introduced bacteria have to compete with others microorganism species in non-sterile soil in order to survive.Therefore, a study to find the suitable PGPR for non-sterile soil conditions is needed.Thus, the objective of our research was to evaluate the effect of Bacillus amyloliquefaciens EB13 inoculant on soil fertility based on soil chemical properties, microorganism population, soil enzymes and microbial activity in non-sterile soil condition.Moreover, this study also investigated the treatment effect on C. sinensis growth.This study is important for reducing the use of chemical fertilizer, particularly in citrus crops.In our previous research, this strain showed the ability to produce plant growth hormone and suppress plant disease (Yuliar et al., 2015).

Material and Methods
Microorganism culture preparation B. amyloliquefaciens EB13 was isolated in our previous study, and it was stored in a 20% glycerol solution at -70 ℃ for further use.A strain of EB13 was pre-cultured overnight with shaking at 30 ℃ in the 100 mL Erlenmeyer flask containing 20 mL of Luria-Bertani (LB) medium.After that,1% (v/v) of pre-cultured were inoculated into 250 mL of No.3 medium in 2 L Erlenmeyer flasks and incubated for 5 days with agitation at 124 rpm and temperature of 30 ℃ (Yuliar et al., 2015) Pot experiments Two hundred mL of B. amyloliquefaciens EB13 culture in No.3 medium was initially inoculated into polybag (25 cm (d) x 30 cm (h)) containing 10 kg of garden soil and mixed.For the control (without inoculation of strain EB13) 200 mL of sterilized distilled water was used instead of the inoculant.C. sinensis tree two years old were transplanted to a polybag and placed in open ground.The citrus was grown for about six months (April -September 2019), and watering was done daily.The second and the third month after citrus transplanted, strain EB13 was re-inoculated into the rhizosphere soil in the polybags as same as the first inoculation by adding and mixing.This experiment was performed in quintuplicate for each treatment.Three months after the third inoculation of strain EB13, the citrus height, diameter, soil pH were measured, and the rhizosphere soil was collected to determine the soil enzymes activity and to monitor the population of bacteria and fungi.Yuliar Yuliar   3/7  Asian J Agric & Biol. 2022(1).

Soil physicochemical analysis
The soil pH was measured using a pH meter by mixing the soil with distilled water (1: 2.5 (w/v)) prior to the measurement.The soil moisture was determined after drying the soil at 105 ℃ for 24 h.The measurement was conducted in duplicate.Soil organic C, total N, total P, available P, and cation exchange capacity (CEC) were conducted by the Indonesian Soil Research Institute with appropriate standard methods.

Soil bacteria and fungi population determination
Rhizosphere soil was collected from the pots of the citrus tree and 1 gram of soil was put into a test tube with 9 mL of sterile distilled water then was vortexed.A serial dilution was made to obtain the final dilution desired.One hundred µL of the dilution samples were spread using a glass rod onto Nutrient Agar (NA) plates and Potatoes Dextrose Agar (PDA) plates for monitoring the population of bacteria and fungi respectively.The NA plates and PDA plates were incubated at room temperature for 2 -3 days and 3 -5 days, respectively.The population density of bacteria in NA plates and fungi in PDA plates were counted (colony forming units/g fresh soil) afterward.

Soil enzymes activity assay
Soil enzyme activities were analyzed in the fresh soil or soil sample that was stored not longer than one week at 4 ℃.Urease was assayed as described by McGarity and Myers (1967).The urease activity was expressed as μg of ammonium that was released by one g of dry weight soil per hour (μgNH 4 -N/g dry soil h).Phosphatase (acid and alkaline) was measured based on the reduction of p-nitrophenyl phosphate (p-NPP) following the method described by Tabatabai (1994).Phosphatase activity was defined as μg of pnitrophenyl (p-NP) was released by one g soil per hour (μg p-NP/g dry soil h).For total soil microbial activity was measured based on the hydrolysis of fluorescein diacetate (FDA) (Green et al., 2006) with a slight modification (Liu et al., 2019).The soil enzyme assay was analyzed in two replication of samples and one control for each treatment.

C. sinensis growth and fruits yield measurement
The height of the citrus tree was measured using a measuring tape and the diameter of the citrus stem was measured with a caliper.Citrus fruits were counted per plant and reported as the mean of three replications.

Data analysis
One-way analysis of variance (ANOVA) was applied to examine the effect of inoculant addition on soil enzyme activity and citrus growth.The treatment means were compared using Tukey's test.Significantly different of means were considered at P < 0.05.

Soil physicochemical properties
The addition of EB13 did not give a significant increase for the total N, P, and available P, total P, available P as P 2 O 5, Cation Exchange Capacity (CEC) between EB13 treatment and control.The soil physicochemical properties were summarized in Table 1.
Bacteria and fungi population number in the rhizosphere soil EB13 treatment significantly has a positive effect on bacteria and the fungi population in the soil.Both the total population of bacteria and fungi in the rhizosphere soil inoculated by EB13 significantly was increased compared with un-inoculated soil (control) (Fig. 1A and 1B).The number of bacteria in soil treated with EB13 was increased five-fold ( 40 to 210 × 107 CFU/g fresh) and the number of fungal was increased three-fold (7.67 to 20.67× 105 CFU/g fresh soil) than control.The total number of microorganisms in the soil rhizosphere was dominated by bacteria.

Soil enzymes response to B. amyloliquefaciens EB13 inoculation
All of the rhizosphere soil enzymes showed a positive response to the inoculation of EB13.The activity of urease, acid and alkaline phosphatase and hydrolysis of FDA on soil inoculated with EB13 was increased significantly (P<0.05)compare to uninoculated soil (control) as shown in Fig. 2. In this study, the highest positive response on EB13 inoculation among soil enzymes was given by urease activity (Fig 2A).Urease activity was increased by 100% (147.89 to 306.55 μg NH4-N/g dry soil h), acid phosphatase by 52% (1848.82 to 2450.50 μg p-NP/g dry soil h), and alkaline phosphatase by 34% (475.15 to 640.98 μg p-NP/g dry soil h).The phosphatase activity was dominated by acid phosphatase as we can see in Fig. 2B and 2C.FDA hydrolysis reflects the total microbial activity in the soil.The hydrolysis of the FDA was increased (1.85 to 2.77 μg fluorescein/g dry soil h) along with the increase of the microorganism population in soil (Fig. 1 and 2D).

C. sinensis growth and fruits yield
Inoculation of the strain of EB13 into soil did not give significant effect on citrus height and diameter.However, strain EB13 inoculation resulted in a significant highest (P<0.05) of citrus fruit production compared with control (without EB13 inoculation).The citrus fruit production was increased by EB13 treatment approximately threefold of control (Table 2).

Discussion
Soil microorganisms are well-considered to play a role in soil health and fertility.They may affect plant growth and soil enzymes activity.Our study showed that the inoculation of B. amyloliquefaciens EB13 in soil has a positive effect on the below-ground (soil) and the above-ground (plant).In the below-ground, EB13 inoculation changed biochemical, and biological properties.These properties have a strong relationship to soil health and fertility.Then, in the above-ground, EB13 treatment enhanced citrus productivity.
Our observation in this study showed that there were improvements in soil quality, particularly in biological properties.However, the soil chemical properties such as C, N, and P have not showed significant improvement yet.It was assumed that soil chemical properties was not change rapidly and needed a long term of observation to get a significant change (Arévalo-Gardini et al., 2015).Since the experiment was conducted in short term (6 month), probably it was too early to find a significant change on soil chemical properties.Nevertheless, bacterial inoculation could increase the availability of nutrients in soil.Studies was reported that inoculation of Bacillus M-13 and B. amyloliquefaciens Y1 increased total N and available P in soil (Canbolat et al., 2006;Jamal et al., 2018).Our results showed that EB13 could survive and was suitable for field applications.It was indicated by the increasing of soil bacterial population after the treatment.Furthermore, the addition of EB13 also promotes the fungi population in the soil.It can be assumed that EB13 can promote the increase of soil microorganism population by providing the amount of available nutrients which is important for soil microorganism growth besides plants.Moreover, EB13 able to suppress pathogens as reported in our previous study.Therefore, it could be suggested that EB13 able to create a favorable conditions for beneficial microorganisms, hence increased their population (Soohee and Kim, 2005).The increase of soil microorganism population in line with the increase of soil enzymes activity, particularly urease, acid and alkaline phosphatase.Enzymes in soil mainly were produced by microorganisms.
Therefore, increasing of microorganism population bring to the increasing soil enzymes activity.The increase of those enzymes in soil was suggested by EB13 inoculant contribution.This result was supported by several studies that have been reported.B. amyloliquefaciens was reported to produce urease, acid and alkaline phophatase (Matos et al., 2017;Meng et al., 2019;Tepe et al., 2019).Soil enzymes take place in many reactions in soils, including nutrients cycling, organic residues decomposition, organic matter formation, and soil structure (Egamberdieva et al., 2010).Therefore, it was expected that increase of urease, acid and alkaline phosphatase activity would increase available N and P resulted from nutrient cycling or decomposition process in soil.Based on the result of this study, there was an improvement in soil quality as indicated by the increase of the total microbial activity in soil after EB13 inoculation.Total microbial activity was the sum of the physiological activities of all the microorganisms in a given soil sample.Total microbial activity was determined by the hydrolysis of fluorescein diacetate method.Soil enzymes activity including fluorescein diacetate (FDA) hydrolysis activity could be used as an indicator of the state of soil quality because they give a quick response to environmental changes compare to other soil properties (Aseri and Tarafdar, 2006;Henry, 2012).In the above-ground, C. sinensis plant growth, particularly in fruits yield.Fruits production in plants has a strong relationship with nutrition availability (e.g.phosphorus and carbon) and plant growth regulator (e.g.hormone and siderophore) in the soil (Furtak and Gajda, 2018).Those can be provided by beneficial microorganism activities in soil.The same result has been reported that the application of B. amyloliquefaciens Y1 promotes pepper plant growth as the increase of the total number of flowers compared to the un-inoculated plant (control) (Jamal et al., 2018).Moreover, fruit production in citrus plants related to the total number of soil microorganisms.Ghagare et al. (2018) reported that the highest bacteria and the fungal number was found in the highest yielding citrus rhizosphere soil and conversely.The same results also was found in this study.The inoculation of EB13 in soil showed a higher fruits yield with a higher total number of bacteria and fungi than un-inoculated (control).All of our finding in this study is in agreement with the previous report which showed B. amyloliquefaciens improved plant growth and soil enzymes activity (Soohee and Kim, 2005;Gowtham et al., 2018;Kang et al., 2015).B. amyloliquefaciens was reported as one of the endophytic bacteria that was found in the citrus plant.This bacteria plays a role as a biocontrol agent and also can promote plant growth.B. amyloliquefaciens was characterized by indole acetic acid (IAA) production, secretion of hydrolytic enzymes, phosphate solubilization, and antibiosis against fungi and bacteria of plant pathogens (Yuliar, 2015;Soares et al., 2016).

Conclusion
The EB13 inoculant significantly increased soil enzyme activities, such as phosphatase activity (1848.82 to 2450.50 μg p-NP/g dry soil h), alkaline phosphatase activity (475.15 to 640.98 μg p-NP/g dry soil h), urease activity (147.89 to 306.55 μgNH4-N/g dry soil h), and total soil microbial activity (1.85 to 2.77 μg fluorescein/g h).Furthermore, the EB13 inoculant significantly increased the population of bacteria and fungi in the rhizosphere.These improvements supported citrus growth by increasing fruits yield approximately threefold (5.33 to 18.33 fruits/plant).Our finding indicated that Bacillus amyloliquefaciens EB13 was able to associate well with C. sinensis in the non-sterile condition and considerable to be used for improving citrus fruits production.

Figure- 1 :
Figure-1: The population of soil bacteria (A) and fungi (B) in the rhizosphere soil on 6 month after treatment, control (un-inoculated) and EB13 (inoculated by B. amyloliquefaciens EB13).Bar with different letter indicates significantly difference (P<0.05).

Table - 2: Growth and fruits yield of C. sinensis after 6 month treatment, control (un-innoculated) and EB13 (inoculated by B. amyloliquefaciensEB13). Treatment Plant height (cm) Plant diameter (cm) Total Number of fruits per plant
aIn columns, means followed by the different letter are significantly different (P< 0.05).