Molecular Identification of Microbes from the Soil Rhizosphere of Cocoa as A Potential Biofertilizer

INTRODUCTION

Biofertilizer is a common fertilizer from a collection of beneficial microorganisms in which its mechanism can bind or dissolve nutrients or produce beneficial phytohormones for plants. The exploration of beneficial microorganisms in various ecosystems and plant vegetation is mostly carried out to find consortia and superior microbial species as a biofetilizer or a pathogen anatagonist. Biofertilizer itself has become an alternative of chemical fertilizers, especially for organic plant cultivators.

Beneficial microorganisms (bacteria) that live in the soil are very essential for plant growth in accelerating the nutrient supply nutrient supply and also as a source of soil organic matter.

The decomposition process of plant remains is reorganized into elements that can be used by plants to grow and develop (Firmansyah, Liferdi,

Khaririyatun, & Yufdy, 2015). This organism belongs to the domain of prokaryotes and microscopic, and has a major role in life on earth. Several groups of bacteria are known as agents that cause infection and disease, while other groups can provide benefits in the fields of food, medicine, and industry such as Azospirillum sp., Azotobacter sp., and Pseudomonas sp. Plant Growth Promoting Rhizobacteria (PGPR) improve the availability of certain nutrients (phosphate solubilization and nitrogen fixation) and plant hormones like indole-3-acetic acid (IAA), ethylene (ET), jasmonic acid (JA), gibberellic acid (GA) and cytokinin (CK) can be synthesized (Sham et al., 2019). Azospirillum sp. produces indole acetic acid (IAA) which is able to accelerate plant growth, lateral root development, stimulates the density and length of root hairs, which in turn causes an increase in nutrient uptake in plants thus make these bacteria function as a biofertilizer. The use of indigenous ARTICLE INFO

Keywords:

Bacillus substilis

Bacillus amyloliquefaciens Biosurfactant

Biofertilizer Article History:

Received: June 29, 2022 Accepted: February 8, 2023

*⁾ Corresponding author:

E-mail: nurmayulis@untirta.ac.id

ABSTRACT

There are three microbes that originated from the roots (Rhizosphere) of the cocoa plant (Theobroma cacao L.) as a potential biofertilizer.

This research aimed to specify the ability of microbial isolates in a consortium with the addition of Biosurfactant Diethanolamide. The three microbes were observed using synergism test, molecular and population identification in the consortium. The synergism test showed the three isolates were not mutually antagonistic indicated there was no clear zone around the colony. The molecular identification showed the isolate 1 was as Bacillus substilis (99.88% hegemony), isolate 2 was as Bacillus substilis (99.75% hegemony) and isolate 3 was as Bacillus amyloliquefaciens (100% hegemony). The population consisted of 3.6 x 10⁶ cfu/ml Azotobacter, 0.55 x 10¹ cfu/ml Azospirillium and 2.5 x 10⁵ cfu/ml phosphate solubilizing bacteria. The results of the calculations on the microbial consortium with the addition of Biosurfactant Diethanolamide were 3.75 x 10⁶ cfu/ml Azotobacter, 0.1 x 10² cfu/ml Azospirillium and 1.65 x 10⁵ cfu/ml phosphate solubilizing bacteria.

ISSN: 0126-0537

Cite this as: Nurmayulis, Sodiq, A. H., Eris, F. R., Hastuti, D., Denny, Y. R., & Susilowati, N. (2023). Molecular identification of microbes from the soil rhizosphere of cocoa as a potential biofertilizer. AGRIVITA Journal of Agricultural Science, 45(1), 124-130. http://doi.org/10.17503/agrivita.v45i1.3840

Molecular Identification of Microbes from the Soil Rhizosphere of Cocoa as A Potential Biofertilizer

Nurmayulis^1*), Abdul Hasyim Sodiq¹⁾, Fitria Riany Eris¹⁾, Dewi Hastuti¹⁾, Yus Rama Denny¹⁾, and Dwi Ningsih Susilowati²⁾

1) Sultan Ageng Tirtayasa University, Serang, Banten, Indonesia

2) Research Center for Horticulture and Estate Crops, National Research and Innovation Agency (BRIN), Indonesia

microbes is more sustainable than introducing the microbes when used as a biofertilizer (Nuraini, Arfarita, & Siswanto, 2015).

Moreover, fertilization efficiency can be increased by using N₂ fixation microbes, P and K nutrient solvents, and plant growth promoters. The use of soil fertilizing microbes can provide nutrients for plants and plant growth regulator metabolites, also protect roots from pests and diseases. Biofertilizer technology is the use of biologically active products consisting of soil-fertilizing microbes to increase fertilization efficiency, fertility and soil health. The use of biofertilizers in Indonesia is currently being widely used, both by farmers, fertilizer factories and various agricultural ministry programs, however, there are still many biofertilizer products that do not meet quality standards. To increase the impact on increasing farmers’ income, the biofertilizer tehnology must be tested with a high level of efficiency. The previous study indicated that the application of the biofertilizer has a positive impact on plants with various benefits from Bacillus subtilis and Lysinibacillus sp. (Sodiq, Setiawati, Santosa,

& Widayat, 2022). Also, a counseling program is needed to extend for farmer to disseminate that the use of biological fertilizers has an impact on increasing crop yields and fertilization efficiency. In order to achieve sustainable agriculture, biofertilizers are a crucial part of integrated nutrient management (Atieno et al., 2020). Biofertilizers, when applied to seeds, plant surfaces, soil or compost, colonize the rhizosphere or plant interior and promote growth by supplying nutritionally important elements such as nitrogen and phosphorus. Products containing living cells of various types of microorganisms facilitate the Biological processes such as nitrogen fixation, phosphate rock solubilization conversion of nutrients from unavailable into plants available to plants.

These potential biofertilizers play an important role in soil fertility productivity and sustainability, protecting the environment with green fertilizers and low-cost inputs for farmers.

Observing the development of the technology of the biofertilizer consortium, this study is an initial study to determine the potential of microbes originating from the rhizosphere soil of cocoa roots.

Soil sampling on cocoa vegetation is intended to be the first target of the application of the microbial consortium to cocoa plants in both at seedling and reprodcutive phases. Free-living beneficial bacteria that provide health benefits to crops are plant growth promoters consisting of various genera such as Azospirillum, Pseudomonas, Azotobacter, Klebsiella,

Enterobacter, Alkaligen, Arthrobacter, Burkholderia and Bacillus and collectively known as plant growth- promoting rhizobacteria (PGPR) (Bashan, de- Bashan, Prabhu, & Hernandez, 2014). However, one of the shortcomings of biofertilizers is the microbial population is less stable so that it cannot be stored for a long time. This condition is exacerbated by the absence of a supportive environment during storage. Therefore, with the addition of additives, it is expected to be able to overcome these limitation so that the effectiveness of biofertilizers can be maintained during storage and application. In the case case as natural insecticide products, they are generally less effective because their formulations are still very simple. The additive combination formula are needed, such as surfactants that function as dispersing and spreading of active ingredients and adhesives to the targeted plant organ (Yusriah, Hambali, & Dadang, 2017).

Additives that have been reported to be able to increase effectiveness of biofertilizers was the addition of biosurfactant Diethanolamide (DEA) palm olein that also able to control cocoa pod borer pests (Nurmayulis et al., 2019). The study was designated to evaluate the effect of DEA on the microbes consortium effectivity, including the microbial population.

MATERIALS AND METHODS

The research was carried out from June to August 2020. The materials used were soil samples of cocoa rhizosphere and materials of laboratory analysis. The population calculation test for Azotobacter, Azospirillium and Phosphate Solubilizing Bacteria (PSB) was carried out at the Microorganism Conservation Laboratory of National Research and Innovation Agency (BRIN), Cimanggu, Bogor. The addition of DEA Biosurfactant was carried out at the Soil and Agroclimate Laboratory, Department of Agroecotechnology, Faculty of Agriculture, Sultan Ageng Tirtayasa University.

Synergism Test

The three selected isolates were streaked from pure cultures into petridish containing TSA (Trypticase Soy Agar) medium and incubated at 27°C for 24 hours. The consortium synergy test results were negative when the isolates failed to form a zone of inhibition when co-cultivated. Therefore, the isolates used in this consortium are not antagonistic to each other and can be cultured together and used in biofertilizer compositions.

Molecular Identification

Molecular identification of bacterial species is required for field applications, especially for safety in their use. Next, identified the bacteria with the highest ammonium production activity by amplification of the 16S rRNA gene and sequenced them. First, DNA isolate was extracted, measured its concentration, and then amplified the 16S rRNA using PCR. PCR was performed by mixing chromosomal DNA with a master mix (containing all dNTPs, buffers, BSA, primers) and then initiated by the addition of Taq polymerase. After amplification, PCR products were examined by 1% agarose gel electrophoresis and purified. Purified PCR products were sent to the DNA Center facility for sequencing. Sequencing results are submitted online to GenBank.

RESULTS AND DISCUSSION Synergism Test

The isolates obtained from previous studies was tested for synergism, so that when microbes are combined in a consortium suspension they would not decrease the the effectiveness of each microbe.

Based on the results of the synergism test, it showed that the three isolates were not mutually antagonistic, as indicated by no clear zone around the bacterial colony as shown in Fig. 1.

The results of this synergism test showed that microbes can be used as biofertilizer in a consortium.

The use of microbes in the consortium has an optimal effect on applied media. The effectiveness of using microorganisms is determined through ability to adapt to the environment. This obstacle can be overcome by using microbes in the form of consortia.

Microbial application in the form of a consortium can reduce the risk of failed microbial applications in the field (Pas, Sopandie, Trikoesoemaningtyas,

& Santosa, 2015). So far, the formulations used to

produce organic fertilizers are typically enriched with various types of microorganisms within consortiums (Sukmadewi, Anas, Widyastuti, & Citraresmini, 2017). Furthermore, the ability of enzymes to work from a wide variety of microorganisms means that microbial consortia tend to yield more optimal results than using individual isolates. A microbial consortium is expected to provide more optimal results in multi- site applications and multi-cultivar experiments (Sodiq, Setiawati, Santosa, & Widayat, 2021).

Molecular Identification Results

The results of molecular identification by analysis of 16S rRNA with the results of the nucleotide sequence are presented in Table 1.

Table 1 shows the identification of 3 isolates with codes NFB-2, LG-1 and PIKOV-2, namely isolates of Bacillus substilis (99.88% homology), Bacillus substilis (99.75% homology) and Bacillus amyloliquefaciens (100% homology), respectively.

These bacterial isolates have potential as enzyme- producing biofertilizers. Bacteria have a more beneficial effect as a source of enzymes because they grow rapidly on inexpensive substrates and are easier to increase yields by adjusting growth conditions. A number of species of the genus Bacillus like Bacillus megaterium, Bacillus circulans, Bacillus coagulans, Bacillus subtilis, Bacillus azotofixans, Bacillus macerans, Bacillus velezensis, etc. are reported as PGPR (Fan et al., 2018). Elucidation of direct and indirect mechanisms of plant growth promotion by Bacillus sp. Nitrogen fixation, solubilization and mineralization of phosphorus and other nutrients, production of plant hormones, production of siderophores, antibacterial compounds and hydrolases, induction of systemic tolerance (ISR) and resistance to abiotic stress (Saxena, Kumar, Chakdar, Anuroopa, & Bagyaraj, 2020).

Fig. 1. The results of the synergism test of 3 selected isolates, all of which showed results not mutually antagonistic as indicated by no clear zone around the bacterial colony

Table 1. Results of molecular identification isolates No. Isolates

Code Result of identification

Nucleotide sequence and type (% similarity)

1. NFB-2 CTAAGGGGCGGAAACCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAG- GGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGACCAGA- GAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTG- GAATTCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCCCGGTT- GAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGAGCCCTTTACGCCCAATA- ATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTAAGCCGT- GGCTTTCTGGTTAGGTACCGTCAAGGTACCGCCCTATTCGAACGGTACTTGTTCTTC- CCTAACAACAGAGCTTTACGATCCGAAAACCTTCATCACTCACGCGGCGTTGCTCCGT- CAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGT- GTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTTGCCTTG- GTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGGTAGC- CGAAGCCACCTTTTATGTTTGAACCATGCGGTTCAAACAACCATCCGGTATTAGCCCCG- GTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTACTCACCCGTCCGC- CGCTAACATCAGGGAGCAAGCTCCCATCTGTCCGCTCGACTTGCATGTGTTAA

Bacillus subtilis (99.88%)

2. LG-1 CACTAAGGGGCGGAAACCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAG- GGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGACCAGA- GAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTG- GAATTCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCCCGGTT- GAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGAGCCCTTTACGCCCAATA- ATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGT- GGCTTTCTGGTTAGGTACCGTCAAGGTGCCGCCCTATTTGAACGGCACTTGTTCTTC- CCTAACAACAGAGCTTTACGATCCGAAAACCTTCATCACTCACGCGGCGTTGCTCCGT- CAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGT- GTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTCGCCTTG- GTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGGTAGC- CGAAGCCACCTTTTATGTCTGAACCATGCGGTTCAGACAACCATCCGGTATTAGCCCCG- GTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTNACTCACCCGTCCGC- CGCTAACATCAGGGAGCAAGCTCGCATCTGTCCGCTCGACTTGCATGTGTT

Bacillus subtilis (99.75%)

3. PIKOV-2 AACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGGACAGATGGGAGCTTGCTC- CCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGG- GATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTCTGAACCGCATGGTTCAGA- CATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTG- GTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGC- CACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTC- CGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATC- GTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGG- TACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGG- CAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGT- GAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAA- GAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGT- GGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGC- GAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGG- GTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGC

Bacillus amyloliquefaciens (100%)

Results of the Consortium Microb Population Test The results of analysis populations of bacteria Azotobacter, Azospirillum, and Phosphate Solubilizing Bacteria (PSB) that have been consorted into biological fertilizers either with the addition of DEA biosurfactant or without the addition of DEA biosurfactant are presented in Table 2.

Table 2 shows beneficial microbes were detected in biofertilizers with or without the addition of DEA. Only in Azospirillium populations showing populations below 10⁴ cfu/ml, this can be corrected by propagation media that need to be close to specific media for Azospirillium considering the importance of the presence of these microbes in the biofertilizer consortium. The results from Widawati

& Suliasih (2018), showed that the combination of PGPR-mix (Azospirillum, Azotobacter, Bacillus) with hydrogels as bio-organic fertilizer and CMC as carriers has promoted the growth of the roots, shoots and vigour index of in vitro sorghum germination, as well as increased the root length, shoot length and total dry weight of sorghum seedlings in pots containing sterile sand. These bacteria are able to fix N and dissolve P and increase plant height. The mechanism of N-fixing bacteria in providing N in the soil is due to the nitrogenase activity of these bacteria which reduces N₂ from the air to NH^3- and increases total soil N (Setiawati, Linda, Kamaluddin, Suryatmana, & Simarmata, 2021).

Among alternative approaches, the use of biological nitrogen fixation (BNF) has emerged as a way to reduce agricultural inputs of N fertilizers and reduce their negative environmental impact. In fact, BNF is a natural process that converts atmospheric nitrogen (N₂) into a readily soluble, non-toxic form (mainly NH⁴⁺) that plant cells use to synthesize a variety of biomolecules. Nitrogen fixation is one of the most important nitrogen sources for plants and a key step in the distribution of this nutrient

in ecosystems (Soumare et al., 2020). According to Roper & Gupta (2016), non-symbiotic nitrogen- fixing bacteria can provide approximately 10–15 kg N/ha/year of nitrogen to plants or soil, depending on carbon sources availability. Bacteria with the ability to live freely and fix nitrogen molecules can be classified into obligate aerobes, facultative aerobes, and anaerobic microorganisms. In some of these genera nitrogen fixation occurs in photoautotrophs.

This is indicated by the presence of intracellular photosynthetic pigments such as the well-known genus Rhodopseudomonas. Desulfovibrio, on the other hand, fixes nitrogen through sulfate reduction (Arfarita, Muhibuddin, & Imai, 2019).

The PSB population was detected up to 10⁵ cfu/ml, this indicates that the biofertilizer has at least 2 functions. Roni, Witariadi, Candraasih, & Siti (2014) stated that PSB activity in dissolving P was not available through the mechanism of releasing metabolites of organic acids with low molecular weights such as malic, fumaric, succinic, and oxalic acids. The cations of Fe³⁺, Al³⁺, Mg²⁺, or Ca²⁺ as phosphate binders will react with organic acids to produce stable organic chelates as a result of which the bound phosphate ions will be released making it easier for plants to absorb them. The phosphatase enzyme from PSB plays a role in dissolving organic P from organic ameliorants. The available P element is needed for better plant root development. Element P is also used in the process of photosynthesis in the leaves of plants. Hence, encouraging bacteria that are particularly important for mobilizing phosphorus that is hard to obtain could help to ensure that plants in soils with little P input have access to enough phosphorus (Grafe et al., 2018). According to Kartikawati, Trisilawati,

& Darwati (2017) the advantages of phosphate solubilizing microbes can also be obtained from the production of growth hormones in addition to phosphate solubilization.

Table 2. Results of analysis populations of bacteria of Azotobacter, Azospirillum, and PSB No. Sample Type

Population of

Azotobacter (cfu/ml) Population of

Azospirillum (cfu/ml) Population of PSB (cfu/ml) Sample

test I Sample

test II Sample

test I Sample

test II Sample

test I Sample test II A. Biofertilizer without the

addition of DEA 3.5 x 10⁶ 4.1 x 10⁶ 0.7 x 10¹ 0.4 x 10¹ 2.4 x 10⁵ 2.6 x 10⁵ B. Biofertilizer + DEA 3.5 x 10⁵ 3.6 x 10⁶ 0.1 x 10² 0.1 x 10² 1.5 x 10⁵ 1.8 x 10⁵

The development of surfactants from palm oil aims to increase the added value of palm oil derivative products because Indonesia’s palm oil production is very high so that the potential for development is very large. Besides, diethanolamine surfactants are more environmentally friendly and biodegradable (Rusdiana, Hambali, &

Rahayuningsih, 2020). The results in Table 2 show that the addition of biosurfactant still shows a good microbial population even though there is a trend of decreasing the number of population compared to without the addition of biosurfactant.

For this reason, the addition of biosurfactants to the biofertilizer consortium has the potential to continue to be developed. Biosurfactants can be used as adhesive components, coagulation, wetting, foaming, emulsifiers, penetrating, and dispersants agents (Nurmayulis, Hastuti, Eris, & Mujahidah, 2021).

CONCLUSION

The results of the synergism test showed that the three isolates were not mutually antagonistic, indicated that there was no clear zone so that they could be used as biofertilizers in a composition. The results of molecular identification showed that 3 isolates with codes NFB-2, LG-1 and PIKOV-2 were isolates of Bacillus substilis (99.88% homology), Bacillus substilis (99.75% homology) and Bacillus amyloliquefaciens (100% homology), respectively.

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