Primer Design of Volatile Synthesis Coding Genes in Ralstonia syzygii subsp. celebesensis

Microbes produce various types of volatile organic compounds (VOCs) through metabolism, which can be used for diagnostic purposes. Microbes' types and classes of VOCs are very wide, including fatty acid derivatives (hydrocarbons, alcohols, and ketones), aromatic compounds, nitrogen-containing compounds, and volatile sulfur compounds. Microbial volatile organic compounds (VOCs) can also be divided into several chemical classes: alkenes, alcohols, ketones, benzos, pyrazines, sulfides, acids, esters, and terpenes. This study aimed to design primers for genes encoding volatile synthesis in Ralstonia syzygii subsp. celebesensis , which causes blood disease in the banana plant. Some of the genes involved are adc (acetone synthesis), adh P (ethanol synthesis), ilvA , nirBD (ammonia synthesis), mdcA (propionic acid synthesis), cysI (hydrogen sulfide synthesis), and speBC (putrescine synthesis). Primers were designed and examined for specificity in silico using Primer3Plus, Geneious Prime, and BLAST programs. The numbers of nine pairs designed primers were successfully amplifying the related nine VOC genes of R. syzygii subsp. celebesensis for qPCR.


INTRODUCTION
Ralstonia syzygii subsp.celebesensis is a gramnegative bacterium pathogenic to banana plants that causes the blood disease bacterium (BDB).It belongs to phylotype IV of the Ralstonia solanacearum species complex (Fegan & Prior, 2005;Safni et al., 2014).BDB is one of the important banana wilt diseases, which can cause damage to banana plantations of up to 27-80%, causing significant crop losses in Indonesia and Malaysia (Blomme et al., 2017;Ray et al., 2021).BDB has been known to attack banana plants in Indonesia since 1921 (Remenant et al., 2011).Blood disease in banana plants was originally only found in Indonesia, but now this disease has spread in Selangor, Malaysia, which is associated with Moko and Fusarium wilt disease (Teng et al., 2016).
Diagnostic methods are an important component in disease management on banana plants that are direct and indirect.Direct detection approaches include molecular technologies and serological technologies such as enzyme-linked immunosorbent assay (ELISA).Meanwhile, the typical indirect method detects morphological changes, changes in transpiration rate, and volatile organic compound (VOC) profiles, which are in accordance with fluorescence imaging technology, hyperspectral techniques, and gas chromatography-mass spectrometry (GC-MS) (Cui et al., 2018).
It is well-known that both microbes and plants emit volatile compounds, some of which may have odors that are characteristic of the species (Blasioli, 2014).Bacteria can produce various types of volatile compounds.Bacterial volatile compounds are known to contribute to interkingdom interactions (plants, fungi, and nematodes), and are also involved in antagonism, mutualism, and internal and inter-species regulation of cellular and growth metabolisms (Bitas et al., 2013;Audrain et al., 2015).The volatile compounds produced by the Ralstonia solanacearum species complex were tested on Ralstonia solanacearum in the other study.The volatile compounds produced were chemical compounds such as alcohols, aldehydes, alkanes, alkyls, amides, amines, benzene, ketones, siloxanes, and thiols (Spraker et al., 2014).

Primer Design
This study used the R. syzygii subsp.celebesensis UGMSS_Db01 strain, whose genome was sequenced and submitted to GenBank and assigned accession numbers NZ_CP068285 for chromosomes and NZ_CP068286 for plasmids (Prakoso, 2022).PCR primers were designed to amplify volatile synthesisencoding genes.The selected encoding genes are those involved in the synthesis of acetone, alcohol, hydrogen sulfide, ammonia, propanoic acid, and putrescine.The candidate gene sequences were taken from gene sequences at the National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/).Gene sequences in nucleotide BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to find out their homologs in NCBI.Then the candidate sequences from the BLAST results were selected that had the highest similarity to R. syzygii subsp.celebesensis UGMSS_Db01.Primer candidates were analyzed using NetPrimer-Biosoft software to check primer pairs' quality and Tm values.The sequence is then BLAST the protein to find out the results of the protein as translated by the gene.The resulting sequences of candidate genes were selected for their primer pairs using the Primer3Plus (https://primer3 plus.com/cgi-bin/dev/primer3plus.cgi) and Geneious Prime software.
The primer candidates were then BLAST to confirm that the gene came from R. syzygii subsp.celebesensis UGMSS_Db01.Then the candidate primer pairs were performed in-silico PCR, or primers were attached to R. syzygii subsp.celebesensis sequences using the SnapGene Viewer software to ensure that the primers were suitable for R. syzygii subsp.celebesensis.

DNA extraction and PCR conditions
The strain R. syzygii subsp.celebesensis UGMSS_ Db01 was streaked onto Casein Peptone Glucose (CPG) agar plates 1 g casein, 10 g peptone, and 5 g glucose per liter and incubated at 30°C for 48−72 hours before further preparation.Total DNA extraction was performed using the Presto™ Mini gDNA Bacteria Kit (Geneaid) according to the manufacturer's instructions for Gram-negative bacteria.All gDNA samples were stored at −20°C until used as templates in the PCR assays.All primers used in this study to determine the existence of genes encoding for volatile synthesis are listed in Table 1.R. syzygii subsp.celebesensis were also amplified using specific primers 121F (5'-CGT ATT GGA TGC CGT AAT GGA-3') and 121R (5'-AAG TTC ATT GGT GCC GAA TCA-3').For each gene, PCR amplification was performed in 10 µL of the total volume, consisting of 5 µl of ReadyMix, 1 µl of F primer and R primer (10 pmol), 2 µl of nucleasefree water, and 1µl of gDNA.DNA fragment application was performed using a PCR machine.After an initial denaturation at 95°C for 3 min, samples were thermocycling for 35 cycles in the BioRad T100TM Thermal Cycle at 95°C for 30 s, 60°C for 30 s, and 72°C for 1 min.The amplification results were visualized using gel electrophoresis. 1 µL of the amplification obtained was injected into 1% agarose and electrophoresed using an electrophoresis set (Bio-Rad) at 50 V for 50 minutes.
The visualization results in the formation of thick bands ranging in size from 100 to 150 bp.

Selection of Primers for the Volatile Synthesis-Encoding Gene
According to the initial primer design results from Primer3Plus, 10 forward primers and 10 reverse primers (10 primer sets) were obtained for each gene, as shown in Table 1.The primer pairs were then selected based on the length of the amplicon (100− 150 bp), as the primers would be used for quantitative PCR.Furthermore, primers were chosen based on melting temperature (Tm) (42−65 °C) with a Tm difference of less than 1 °C, guanine or cytosine content (40−60%), the optimal length of primers (usually between 18 and 30 bases), the presence of G or C ends, the absence of repeating sequences with a maximum number of repeated sequences, the absence of run sequences or a maximum of 3 nucleotides, and no self-complementarity (Arenas & Salazar, 2019).
The products of bacterial metabolism with low vapor pressure, high lipophilicity, and a molecular weight of less than 300 Da are likely to be released as volatile compounds.The volatiles produced by bacteria reflects the specific metabolism of the bacteria.Given the enormous diversity of bacterial metabolism, these volatile compounds can originate from a variety of metabolic pathways (Schulz & Dickschat, 2007).Volatile profiles have been widely used in the identification of plant pathogenic bacteria.In this research, the primer design of the genes involved in the synthesis of volatiles was carried out, namely the genes encoding the synthesis of acetone, alcohol, ammonia, putrescine, hydrogen sulfide, and propanoic acid.
The gene involved in acetone synthesis investigated in this study is the adc gene, which has the function of producing the enzyme acetoacetate decarboxylase, which converts acetoacetate to acetone.Acetone is naturally present in the metabolism of plants, animals, and bacteria.The standard dogma has persisted for so long that acetone is the end product of metabolism in animals and humans.Acetone evaporates easily, even from water and soil (Kalapos, 2014).The addition of acetate and glucose to the bacterial growth substrate can increase the production of acetone (Gao et al., 2015).The medium used in this study was CPG, which consisted ISSN 1410-1637 (print), ISSN 2548-4788 (online) Table 1.Ralstonia syzygii subsp.celebesensis UGMSS_Db01 strain primers designed for qPC of 5 g/L glucose.To obtain energy, bacteria consume glucose via the pyruvic acid metabolism pathway; pyruvic acid is then converted into the compound Acetyl CoA.Acetyl CoA is converted into acetoacetate.Acetoacetate decarboxylase releases CO2 from acetoacetate to form acetone (Berzin et al., 2012).The adc gene R. syzygii subsp.celebesensis is located at the JK151_21925 gene locus, which is involved in butanoate or butyric acid metabolism (KEGG, 2021).
The adh gene is a gene coding for this alcohol dehydrogenase enzyme (Atika et al., 2015).On R. syzygii subsp.celebesensis, the alcohol dehydrogenase enzyme is encoded by adhP, which is located at the JK151_03205 gene locus.The enzyme alcohol dehydrogenase converts acetaldehyde to ethanol.Ethanol is produced from glucose through the consumption of pyruvate during fermentation (Kang, 2015).Glycolysis is a metabolic process that converts glucose into the oxidized product, pyruvate, and supplies ATP for the production of bacterial biomass.Furthermore, under anaerobic conditions, pyruvate can be fermented into ethanol through the sequential reactions of pyruvate decarboxylase and alcohol dehydrogenase, which lose one carbon as carbon dioxide (CO 2 ) (Kang, 2015).
The genes encoding for ammonia synthesis investigated in this study were the ilvA, nirB, and nirD genes.The ilvA gene catabolizes the amino acid threonine to -ketobutyrate, 2-isobutyrate, and ammonia.The nirB and nirD genes, which encode the enzyme nitrite reductase, are also involved in the formation of ammonia by converting nitrite compounds into ammonia.On R. syzygii subsp.celebesensis, the ilvA gene is located at the JK151_00610, the nirB gene is located at the JK151_RS06690, and the nirD gene is located at the JK151_RS06685 gene locus.
Hydrogen sulfide gas is produced during the process of sulfur metabolism by bacteria.Microorganisms can involve sulfur in their metabolism, both in oxidized and reduced states (Fauque & Barton, 2012;Kushkevych et al., 2020).Sulfate reductase performs a major role in sulfur metabolism by catalyzing the reduction of sulfite to hydrogen sulfide and water (Erva et al., 2018).In E. coli, the genes involved are encoded by the cysJIH operon, which consists of the gene for the sulfate reductase subunit (Kushkevych et al., 2020).On R. syzygii subsp.celebesensis, sulfate reductase is encoded by the cysI gene (JK151_17495) (KEGG, 2021).
The mdcA gene is involved in the synthesis of propanoic acid.R. syzygii subsp.celebesensis has a propionate-CoA transferase at the JK151_00505 gene locus, which is involved in propanoate metabolism (KEGG, 2021).Putrescine is a putrid-smelling organic chemical compound produced by the breakdown of amino acids in living and dead organisms.Putrescin is largely responsible for the stench of rotting flesh (Boyle et al., 1984).In R. solanacearum, an abundance of putrescine compounds was found as a virulence metabolite produced by the pathogen (Lowe-Power et al., 2018).The putrescine compounds were formed through the action of the enzymes ornithine decarboxylase (speC) from ornithine compounds and agmatinase (speB) from arginine compounds.On R. syzygii subsp.celebesensis, enzymes involved in putrescine synthesis are located at the JK151_18620 agmatinase (speB) and JK151_17220 ornithine decarboxylase (speC) gene loci.

The Existence of Volatile Synthesis Coding Genes in Ralstonia syzygii subsp. celebesensis
The extraction of total genomic DNA was amplified using the PCR method.Primer 121F/R was used to prove that the extraction gene is R. syzygii subsp.celebesensis.The results of polymerization with primer 121FR showed that the DNA extract used was positive for RSC, indicated by the formation of DNA fragments with a size of 317 bp.The primer used for the detection of genes encoding volatile synthesis was also well amplified, as shown by the formation of DNA fragments with a size of 100−150 bp (Figure 1).The DNA base sequence specifications of primers 121F/R are able to recognize R. syzygii subsp.celebesensis in their total genomic DNA.The special characterization belonging to R. syzygii subsp.celebesensis on its DNA fragments will be the main identity factor as well as a distinguishing character from other bacteria (Edy et al., 2011).The annealing temperature used in PCR was 60°C for the 30s, the same annealing temperature used in quantitative PCR (qPCR) for volatile gene expression analysis.The amplicons produced were also adjusted for the use of qPCR, which is 100−150 bp.In qPCR, the amplicon size directly influences the detection of the target: the larger the amplicons, the longer the detection (Debode et al., 2017).

CONCLUSION
Based on this study, the adc, adhP, ilvA, nirBD, cysI, mdcA, and speBC genes are present in the genome of R. syzygii subsp.celebesensis.The pairs of primers designed in this study have the potential to be used as validations for the presence of volatile organic compounds (VOC) produced by R. syzygii subsp.celebesensis.