Identification of bacterial species from healthy wood of Aquilaria crassna using colony PCR

. Agarwood is one of the most expensive, sacred and valuable woods used in the biotechnology industry. This natural raw material is in high demand as a commodity and can be used to manufacture a variety of products . Isolating a microbe from its environment is crucial because several bacterial strains can produce novel compounds for biotechnological applications . Exploration of bacterial strains, such as bacterial endophytes, has been associated with bioactive compounds of the agarwood plant . The purpose of this study was to isolate and identify bacteria from Agarwood trees in the Kaeng Hang Maeo Sub - district of Chanthaburi Province, Thailand by using molecular biology techniques . A colony PCR technique without prior DNA extraction for rapid and simple detection of bacteria was performed . After the DNA sequence analysis, the results showed that most populations in the normal layer are belonged to Pantoea dispersa ( 99 %). Through analysis of phylogenetic tree by neighbor - joining method, the sequences of the 16s rRNA gene were analyzed to understand evolutionary relationships of bacteria found in agarwood . In conclusion, good amplification of the 16s rRNA sequence ( 1 . 5 kb ) was detected with the specificity of the target sequences of the 16s RNA . In addition, the isolated bacterial strains are the most common species in agarwood, however, there has been no previous research on isolating these bacterial strains in Aquilaria crassna .


Introduction
Aquilaria crassna is an agarwood producing tree of the family Thymalaeaceae [1,2]. This plant is found primarily in China, Bangladesh, India, and Thailand [3]. When aquilaria trees are wounded or stressed, their wood turns from white to yellow, brown, black, and eventually greasy black and spreads out. Furthermore, the resinous substances accumulated in the wood surrounding the wound can be secreted by the wood. It must, however, be left for several years until completely turning to black or brown color [4,5]. Many countries, including Egypt, China, Greece, and India, used agarwood essential oil or fragrant resins for medicinal therapy, incense in religious rites, perfumes, and ornamental materials [6,7].
Many bacteria are ubiquitous; that is, they are present everywhere. They are also highly adaptable to a variety of environmental conditions [8]. A previous report presented endophytic bacteria as playing a crucial role in the production of pharmaceutical compounds present in plants [9]. The wood was also used to decorate boxes, belts and clothing articles and its usage has recently expanded to include new products such as detergent and shampoo [10]. There is also evidence that it has tonic and therapeutic properties [11,12]. According to recent research, endophytic bacteria have been discovered to be abundant among the resident microflora of the healthy inner tissues of numerous plant species [13]. Because the function of these bacteria is unknown, the isolation and identification of bacterial strains in these groups may aid in understanding the relationship between microbes and plants, particularly aquilaria species that produce agarwood, as well as the discovery of novel bacterial species.
In 2005, molecular approaches based on DNA gene markers were widely used to identify the genetic variation of plant species in agarwood producing tree species [14,15]. In the present study, we presented the results of the isolation and identification of major bacterial strains associated with the non-resinous part of the agarwood plant. This study provided the results of the isolation and identification of major bacterial strains associated with the non-resinous part of the agarwood plant. Furthermore, this study was the first investigation to identify bacteria strains that could be employed as an alternative bio-resource for the future production of bioactive compounds.

Bacterial isolation from healthy wood sample
The wood samples (Aquilaria crassna Pierre ex Lec) were collected in the Kaeng Hang Maeo Sub-district of Chanthaburi Province (Figure 1). They were pulverized into fine powder. The powder (0.3 g) was soaked in 10 ml distilled water before being incubated at 37°C for 48 h. One milliliter of suitable dilutions of the suspension was used to inoculate a set of three Petri dishes, each containing 15mL Nutrient Agar (NA) medium (HIMEDIA ® , India). Plates were then incubated at 37°C for 24 h.

Colony PCR
The PCR reaction mixtures were carried out in a volume of 25 μl with 2.5U of Pfu DNA polymerase (Biotechrabbit, Germany), 2.5 μl of 10X reaction buffer, 200μM of each dNTP, 1.5mM MgCl2, 25 pmole of each primer and were adjusted with distilled water. Primer forward (5'AGA GTT TGA TCM TGG CTC AG3') and reverse (5'TAC GGY TAC CTT GTT ACG ACT T3') to 16S rRNA PCR amplification were used for bacterial identification. A single colony of cream whitish-colored colonies was selected as the DNA template for PCR. The PCR reaction was carried out using a DNA Engine thermal cycler (BIORAD) with an initial denaturation step of 95qC for 5 min, followed by 40 cycles of denaturation at 95qC for 30 sec, annealing at 52qC for 30 sec, extension at 72qC for 30 sec, and an additional extension step at 72qC for 10 min. This process used distilled water as a negative control to check for contamination. The PCR reaction components and conditions are shown in Table 1 and Table 2.

Detection of PCR products and sequencing
The presence of PCR products was investigated using 2% agarose gel electrophoresis. PCR yields were estimated by comparing them to a low DNA mass ladder ( Promega, USA). The post PCR purification step was then carried out using a QIAquick PCR purification Kit (QIAGEN, USA). The purified PCR product was sequenced with the ABI PRISM TM BigDye Terminator Cycle Sequencing Kit. The amplification was carried out in a DNA Engine thermal cycler ( BIORAD) under the following conditions: 25 cycles of 96qC for 10 sec, 55qC for 5 sec and 70qC for 1 min. The DNA was recovered by ethanol precipitation after amplification and the dye-labeled DNA was analyzed using an Applied Biosystems 377 sequencer (Perkin-Elmer, Norwalk, CT, USA).

Data analysis using Bioinformatics tools
After obtaining the nucleotide sequences of the 16s rRNA gene, they were checked by using the nucleotide blast database (https: / / blast. ncbi. nlm. nih. gov/ Blast. cgi). All alignment illustrations were created using the ClustalW program [16] and the GeneDoc software was used to display the output [17]. MEGA 7 was used to create the phylogenetic trees using the Neighbor-Joining method and Bootstrapping analysis [18]. The data has been bootstrapped 1,000 times.

Characteristics of colonies obtained by isolating bacteria from wood
The selected isolates underwent presumptive phenotypic identification based on bacterial morphology after gram staining, colony morphology and the ability to grow on NA media and culture conditions. The results showed that the isolated microorganism displayed cream whitishcolored colonies, round, raised, smooth on each plate shown in Figure 2. From the gram staining, it was found gram-negative rod (data not shown). This pattern is similar to that seen in previous research [9, 13].

Colony PCR for detection of the 16s rRNA gene
In this study, colony PCR was used as the universal primers. The PCR products were about 1,500 base pairs long, which was the expected size. As shown in Figure 3, the universal primers produced the amplified fragment of the expected size on the gel when compared with the 100 bp DNA marker. The 16S rRNA gene is universal in bacteria commonly due to its conservation across bacteria and is popularly used to establish the evolution of bacteria through the approximately 1,500 base pair (bp) locus of this gene [19]. According to the results, 16S rRNA sequence analysis found isolates that were more accurate than traditional methods [20]. In addition, the 16S rRNA gene sequences were also generated between groups of known bacteria and the novel isolates of related bacteria to study the evolutionary relationships of living organisms [21].

Sequence analysis of the 16S rRNA gene and Phylogenetic analysis
After obtaining the sequences from this study, they were compared with the bacterial species recorded in the NCBI database ( Figure 4). A single query sequence of query ID 31109 found their best match with the species belonging to the Enterobacteriaceae family. It was found that Pantoea dispersa had the highest identity at 99%. In addition, the sequences of all bacterial strains were aligned with species recorded in the database (No.2-No.12). From the result of multiple sequence alignment, the polymorphic nucleotides were examined using the Clustal W program  Black represents 100% identical nucleotide sequences and white has no DNA sequence similarity Morphological identification becomes difficult because of the invisibility of some features followed by not well-developed specimens, which can even make the identification impossible. Therefore, molecular biology studies are necessary to support the morphological studies of bacteria isolated from natural sources. Phylogenetic trees were constructed based on the multiple sequence alignment of the 16s rRNA using neighbor-joining (NJ). In the phylogenetic trees, the laboratory sequences are checked according to the morphologically identified characteristic, while other species name is given for the sequences downloaded from NCBI GenBank. The evolutionary data was inferred using the Neighbor-Joining method. The percentage of replicating trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) was analyzed to predict tree characteristics [18]. The phylogenetic tree (Figure 6) was established with the outgroup of Aedes albopictus (accession number DQ397921.1). Based on the sequence comparison and phylogenic tree construction, the bacterial isolate from the healthy wood of Aquilaria crassna presents 99% homology with Pantoea dispersa, a known Gram negative bacteria of the family Enterobacteriaceae. Genus Pantoea is isolated from Enterobacteriaceae in 1989 [22].  [22,23]. The genus Pantoea is a gram-negative, flagellated, nonencapsulated, nonspore, and capsule-forming ubiquitous Brevibacterium cruentum [24,25]. This genus is primarily associated to plant pathogens, with a few historical human diseases [25,26]. It was previously reported that this microorganism is useful as a biocontrol agent for black rot in sweet potatoes [27]. In addition, several members of the genus have been potentially found to enhance growth and yield, control plant pathogenic microbes, and increase abiotic tolerance in rice plants [28]. To our knowledge, this is the first report describing the isolation of bacterial species from healthy wood of Aquilaria crassna with rapid detection method.

Conclusion
The shape of the colony and the content in agarwood samples indicated cream whitish-colored colonies and gram-negative rods based on observation and isolation. According to the NCBI database, Pantoea dispersa showed the highest identity at 99%, and was regarded as a candidate bacterial endophyte. Furthermore, we found that species-specific colony PCR can be used to identify P.

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https://doi.org/10.1051/e3sconf/202235502004 2 E3S Web of Conferences 355, 02004 (2022) dispersa without the need for DNA extraction from a natural sample. In comparison to conventional methods, it produces results in less time, requires less effort and resources, and is less expensive, rendering it suitable for biotechnological application.