INTRODUCTION
Rationale of the Study
Potatoes are a short-term agricultural crop known for their starchy tubers and high nutritional value They rank as the fifth most important food crop globally, following rice, maize, wheat, and soybean, making them a valuable export item with significant economic efficiency Potatoes not only provide essential nutrients for humans and animals but also serve as important raw materials for various industries In Vietnam, they are widely cultivated during the winter months in the North, where they play a crucial role in the local diet.
In Vietnam, potato cultivation emphasizes enhancing yield and quality, leading to focused research on improving potato plant growth and development Endophytic bacteria are crucial for the development of potato plants, making the study of endogenous microflora, particularly the endosymbiotic group, essential for advancing effective farming solutions.
The plant endosphere hosts diverse microbial communities that play a crucial role in enhancing plant health Current research on plant-microbe interactions employs both traditional microbiology and advanced genomic technologies Endophytes, which are beneficial microbial symbionts residing within host plants without causing harm, can boost crop yields while reducing agriculture's environmental impact They promote plant growth through mechanisms such as nitrogen fixation, phytohormone production, nutrient absorption, and providing resilience against various stresses The process of endophytic colonization, where these microorganisms enter and proliferate within the host, is vital for these benefits Despite the growing interest in the plant microbiome, the mechanisms by which plants attract endophytes remain poorly understood (Kandel, Joubert, and Doty 2017).
Endophytes have traditionally been extracted from surface-sterilized plant tissue and cultured in nutrient-rich media Nguyen Dac Tien previously isolated several strains from potato roots, evaluating the genetic diversity of 14 isolates, which revealed significant genetic variation However, further identification of these isolates is necessary This research aims to identify and analyze the partial 16S rRNA sequences of endophytic bacteria isolated from potato roots.
Research Objectives
Identification and sequence analysis of partial 16S rRNA of endophytic bacteria isolated from potato root.
Research requirements
Identification of 12 endophytic bacteria isolated from potato root
Sequence diversity analysis by prediction of the secondary structure of endophytic microorganisms by R2DT and Unafold tools.
LITERATURE REVIEW
General introduction of potato
The scientific name of the potato plant is Solanum tuberosum L, belongs to the
Potato plants are herbaceous perennials growing to about 60 cm in height, dying after flowering
Figure 2.1.2 Detailed image of potato
Potato flowers are white, pink, red, blue, or purple, with yellow pistils Potatoes are pollinated mainly by insects, bumblebees carry pollen from one plant to another
After flowering, some potato varieties produce green, cherry-like fruits that can contain up to 300 seeds, but these fruits are inedible due to the presence of toxic alkaloids, specifically solanine New potato varieties are cultivated from seeds rather than tubers When potatoes are cut and soaked in water, the seeds will split and sink to the bottom after a day While any type of potato can be grown from tubers or tuber pieces, some commercial varieties are not grown from seeds due to unfavorable flowering conditions and are instead propagated from tubers, referred to as varietal pieces.
The resting period: usually freshly harvested potatoes are not able to sprout;
The phenomenon of sleep in potato tubers varies in duration based on breed and external factors, including mechanical rubbing and chemical influences Once the dormancy period concludes or treatment occurs, the tubers are capable of sprouting.
The germination period marks the initial stage in the potato plant's growth cycle, characterized by the gradual development of sprouts from the tubers into young plants The optimal planting time occurs when the tubers exhibit multiple vigorous sprouts.
Stem and leaf growth period: after planting, sprouts develop into stems The main stem grows directly from the seed tuber, the secondary stem grows from the main stem
Tuberous rays begin to form 30-40 days after planting, growing horizontally just beneath the soil These white structures have a strong capacity for growth, both in size and quantity, as they accumulate nutrients essential for the later development of tubers.
The tuber development phase includes flowering, fruiting, and ripening, during which tuber rays enlarge and nutrients are transported to promote rapid growth As the tubers develop, inflorescences appear, and following pollination, the fruit matures, leading to the ripening of both fruit and seeds Eventually, the leaves yellow and die.
2.1.4 Factors affecting the growth and development of potato
Temperature: Potatoes prefer a warm and temperate climate, both heat and cold are not tolerated Requires different temperatures at different growth stages
In the period of growth of stem and leaves, suitable temperature: 20-25°C The appropriate temperature for tuber formation and development is 17-20°C, but for starch accumulation, the appropriate temperature is 16-18°C
Potatoes thrive in bright conditions, with optimal light intensity ranging from 40,000 to 60,000 lux for maximum yield Each growth stage of the potato plant necessitates specific light requirements, particularly during tuber formation and development, which benefit from shorter daylight hours of 10 to 12 hours per day.
Water: Potatoes have shallow roots, so they need to be watered regularly According to research, during the growing process, potatoes need about 500- 700mm of water with a humidity of about 60-86%
The ideal soils for potato cultivation are light in texture, including alluvial, loamy, and sandy soils A pH range of 5 to 7 is optimal, with the best conditions found between 6 and 6.5 To ensure robust growth and development, potatoes require a nutrient-rich environment, abundant in macronutrients such as nitrogen (N), phosphorus (P), and potassium (K), as well as essential trace elements like molybdenum (Mo) and calcium (Ca).
Potatoes are a versatile and affordable tuber that is easy to cultivate and maintain, making them a popular choice for many households in Vietnam as a staple in daily meals due to their high nutritional value.
Potatoes cooked in their skinless state are a great source of many essential vitamins and minerals, such as vitamin C or potassium
Potatoes are mostly water, in addition, the main components of potatoes include carbs, protein and a moderate amount of fiber, especially potatoes have almost no fat.
Endophytic bacteria
Research on non-pathogenic bacteria inhabiting plant tissues dates back to 1926 Since 1940, various studies have documented the presence of native endophytic bacteria in the tissues of numerous plants, including seeds, ovules, tubers, roots, stems, and fruits (Quadt-Hallmann, Kloepper, and Benhamou 1997).
Recent studies on endophytic bacteria have primarily concentrated on their beneficial and ecological roles, particularly in understanding their interactions with host plants and parasites (Ryan et al 2008) Scientists are now increasingly screening endogenous bacterial strains to evaluate their effectiveness in promoting plant growth and development while also addressing environmental pollution, ultimately aiming to produce biologically-based medicinal products for agriculture (Kumar 2011).
Endophytic bacteria inhabit nearly all plant species, residing within the endothelium of host plants and establishing various relationships, including mutualistic and vegetative symbiosis While most endoparasitic forms emerge from the root zone or leaf surface, certain types are capable of parasitizing seeds.
Endophytic bacteria, as defined by Kado (1992), are "bacteria that colonize living plant tissue without significant harm or gain other than assuring colonization." However, this definition is viewed as overly restrictive because it overlooks the potential for endophytic bacteria to establish symbiotic relationships with their host plants.
Bacon and White (2000) provided a widely accepted definition of endosymbiotic bacteria, describing plant endophytes as bacteria that colonize living plant tissues without causing immediate negative effects This definition helps to distinguish these beneficial bacteria from plant pathogenic bacteria.
Endophytic bacteria are defined as bacteria that inhabit the endothelium of plants, remaining undetected externally and typically not causing any harm to their host plants (Rathod and Dhale 2016).
Endophytic bacteria are found in nearly all plant species, colonizing plant tissues either latently or actively, both locally and systematically These bacteria primarily originate from the rhizosphere, leaves, seeds, or asexual propagation materials Research indicates that the rhizosphere serves as the main source of endophytic bacteria, leading to their high density in roots during the early growth stages.
Roots serve as the primary entry point for bacteria into the plant body Endophytic bacteria colonize endothelial cells from various locations, including the surface, cilia, root apex, and lateral root origins (Verma et al., 2001) Additionally, bacteria can infiltrate plants through natural openings like stomata and small pores in the plant tissue.
Endophytic bacteria, after entering the host plant, inhabit the endophytic niche, which shields them from environmental stressors and facilitates their colonization within plant cells and tissues These bacteria typically occupy intercellular spaces and can be found in various plant parts, including roots, stems, leaves, fruits, and seeds (Oliveira et al., 2013).
2.2.4 The role of plant endophytes
Endophytic bacteria enhance plant growth similarly to rhizobia, as they are often isolated from the healthy tissues of plants These facultative endophytes can thrive both within plant tissues and in the surrounding rhizosphere, demonstrating their versatility and beneficial role in promoting plant health.
Endophytic bacteria enhance host plant growth by directly promoting the synthesis of the plant growth hormone auxin (IAA), increasing mineral content, aiding in biological nitrogen fixation, and dissolving insoluble phosphorus.
Climate change adversely affects plants, leading to increased resistance to pathogens and reduced turnover (Ryan et al., 2008) Additionally, plants play a crucial role in removing contaminants from the environment (Rosenbkueth and Martínez-Romero, 2006; Ryan et al.).
Endophytic bacteria can enhance seed germination and promote seedling development even in challenging conditions They play a crucial role in preventing pathogen growth by synthesizing endogenous intermediates and producing new metabolites Additionally, these bacteria contribute to the host plant's iron needs through siderophore production, which also aids in resisting infections from pathogens.
Methods for identifying microorganisms
2.3 Molecular indentigication of endophytic bacteria
In recent plant taxonomy studies, the Internal Transcribed Spacer (ITS) region is frequently analyzed at the species level due to its high diversity, approximately 13.6% among closely related species The ITS gene region comprises two distinct loci, ITS1 and ITS2, linked by the 5.8S locus Additionally, research indicates that the ITS region exhibits a low degree of intraspecies variability (Baldwin et al., 1995).
ITS gene cloning by PCR
PCR-ITS product check electrophoresis
16S rRNA gene sequencing is a vital tool for identifying, classifying, and quantifying microbes in complex biological samples, including environmental and gut microbiomes This gene, being a highly conserved element of the transcriptional machinery in all DNA-based life forms, serves as an ideal target for sequencing diverse microbial species Universal PCR primers can effectively amplify the 16S rRNA gene across various microorganisms, thanks to its combination of conserved and variable regions The conserved regions facilitate universal amplification, while the variable regions enable differentiation among specific microorganisms, such as bacteria, archaea, and microbial eukarya However, identifying viruses necessitates metagenomic sequencing, as they do not possess the 16S phylogenetic marker.
Molecular taxon identification relies on marker gene sequencing, where the choice of marker gene and sequencing platform influences sensitivity, resolution, and throughput Sanger sequencing is the standard for single taxon identification, while Illumina MiSeq and third-generation sequencing are preferred for community surveys Fungal marker genes vary in length, resolution, phylogenetic power, and availability of sequences and primers Accurate single taxon identification often requires multiple markers, starting with a phylogeny-based classification using a pre-marker gene, followed by fine-tuning with a group-specific marker The 28S and 18S rRNA gene sequences are key fungal phylogenetic markers, with the 28S rRNA gene providing lower taxonomic resolution, despite the majority of publicly available sequences being 18S rRNA gene sequences Recent decades have seen the development of fungi-specific 18S rRNA gene primers, but their characteristics, overall coverage, and potential co-amplification with non-fungal sequences remain underexplored.
PCR amplification of the 5.8S-ITS rDNA region
PCR amplification of D1/D2 domain of 26S rDNA region
D1/D2 26S rDNA gene sequencing and phylogenetic analysis
Some studies using 16s rRNA
2.4.1 Cultivation-independent population analysis of bacterial endophytes in three potato varieties based on eubacterial and Actinomycetes-specific PCR of 16S rRNA genes
Endophytic bacteria are commonly found in most plants, colonizing them without causing disease Research on the diversity of these bacterial endophytes has primarily focused on characterizing isolates from internal plant tissues Although culture-independent methods for analyzing microbial communities are widely used in various natural environments, there is limited information regarding the species diversity of endophytes This study analyzed the microbial communities present in the stems, roots, and tubers of three potato varieties using 16S rRNA-based techniques, including terminal restriction fragment length polymorphism analysis, denaturing gradient gel electrophoresis, and 16S rDNA cloning and sequencing Two individual plant experiments were conducted to gather comprehensive data.
In the first experiment, plants experienced light deficiency, while the second experiment yielded healthy and robust plants Although both experiments produced comparable endophytic populations, the healthy potato plants exhibited a significantly higher diversity of endophytes compared to the stressed ones Additionally, plant tissue and variety-specific endophytes were identified Sequence analysis of 16S rRNA genes revealed a wide phylogenetic spectrum of bacteria capable of colonizing plants internally, including α-, β-, and γ-Proteobacteria, high-GC Gram-positives, and members of the Flexibacter/Cytophaga/Bacteroides group and Planctomycetales Notably, group-specific analysis of Actinomycetes showed a greater abundance and diversity of Streptomyces scabiei-related species in the Mehlige Mühlviertler variety, which is recognized for its resistance to potato common scab caused by S scabiei.
2.4.2 Analysis of endophytic bacterial communities of potato by plating and denaturing gradient gel electrophoresis (DGGE) of 16S rDNA based PCR fragments
The study evaluated the diversity of endophytic bacterial populations in potato (Solanum tuberosum cv Desirée) using dilution plating of plant macerates and direct PCR-DGGE analysis of extracted DNA Culturable endophytic bacterial communities in potato stem bases and roots were found to range from 10³ to 10⁵ CFU g⁻¹ of fresh plant tissue The dilution plating identified a variety of dominant bacterial types, primarily within the α and γ subgroups of Proteobacteria, as well as the Flavobacterium/Cytophaga group, with various Firmicutes also present Notably, different Pseudomonas spp were the most frequently isolated strains, constituting over 5% of the total isolates.
The study identified several bacterial species, including Pseudomonas aureofaciens, Pseudomonas corrugata, Pseudomonas putida, Agrobacterium radiobacter, Stenotrophomonas maltophilia, and Flavobacterium resinovorans, through fatty acid methyl ester (FAME) analysis and partial 16S ribosomal RNA gene sequencing Additionally, other Proteobacteria and Firmicutes were detected, though less frequently, primarily in potato stem tissue The research also monitored the behavior of three potential potato endophytes—Stenotrophomonas maltophilia, Bacillus sp., and Sphingomonas paucimobilis—after their introduction into potato plants via injection, root dipping, or soil application.
Following stem injection, S maltophilia and Bacillus inoculants were tracked for 22 and 1 day(s) respectively using dilution plating and PCR-DGGE Only S maltophilia successfully colonized and persisted in plant tissue, while S paucimobilis was not recovered regardless of the introduction method The indigenous bacterial flora associated with potato was monitored through PCR-DGGE, revealing limited complexity in bacterial communities, with distinct differences between those from potato stems, stem peel, and roots Evidence indicated the endophytic presence of various organisms within the α, β, and γ subgroups of Proteobacteria and Firmicutes Some sequences matched those from culturable isolates, while others did not, indicating the detection of non-culturable or yet-uncultured endophytic organisms.
MATERIALS AND METHODS
Materials
12 bacterial isolates isolated by Nguyen Dac Tien K62CNSHC.
Study place and time
Place: Department of Plant Biotechnology, Department of Biotechnology, Vietnam National University of Agriculture
Equipment
Technical balance, analysis, petri dish, culture flask, all kinds of test tubes, thermostatic tanks, medium dispensers, autoclaves, incubators, inoculating rods, balls, tools attached to the cabinet refrigerator, incubator, centrifuge
Mortar, sterile porcelain pestle, ice tray, pipettes, tips of all kinds
The article highlights essential laboratory equipment, including the MM 400 RETSCH cell disruptor, Labtech thermostatic bath, and Hettich MIKRO 200R cold centrifuge It also features the EMCLAB vortex, BioPhotometer plus spectrophotometer, and TAKARA PCR Thermal Cycler Additionally, the SCIE-PLAS electrophoresis machine and UVP BioDoc-it gel electrophoresis camera are mentioned, along with a refrigerator and microwave, all crucial for various scientific applications.
Chemicals
Table 1: List of chemicals used in the study
Using the 2x PCR mix Solution kit (i-taq)
Contents and methods
Identification of 12 endophytic bacteria isolated from potato root by sequencing partial 16S sequence
Identify phylogenetic tree by MEGA7
Identified the polymorphism of 16S sequence of 12 accessions by secondary structure
Identify sequence differences in specific nucleotides located in the sequence
3.5.2.1 Quantification of DNA by using spectrophotometer
The concentration of DNA may be evaluated using a spectrophotometer The monochromator optical system in a cuvette spectrophotometer provides light at
The absorbance peak at 260 nm is crucial for quantifying DNA and RNA, making microplate spectrophotometers increasingly popular for nucleic acid analysis The light absorbed at this wavelength correlates with the nucleic acid quantity in the sample Both DNA and RNA absorb light at 260 nm, providing a total nucleic acid measurement Additionally, the absorbance peak for proteins occurs at 280 nm, which is also relevant for assessing nucleic acid samples The purity of nucleic acids is evaluated using the ratio of absorbance at 260 nm to that at 280 nm, with a ratio close to 2 indicating a highly pure RNA sample, while a ratio of approximately 1.8 signifies purified DNA.
Step 2: Selected DNA, select dsDNA Then pressed enter
Step 3: Placed the Hellma traycell cuvette in place Choose mirrored cap 1mm, factor 10
Step 4: Took 2μl of TE buffer (1:10) and placed it onto Hellma traycell cuvette and pressed blank
Step 5: Took 2μl of TE buffer (1:10) and place it in Hellma traycell cuvette and pressed sample Repeated until 3 consecutive measurements gave absorbance value of zero
Step 6: Took 2μl of DNA sample and placed in Hellma traycell cuvette, measured the samples and recorded the OD and DNA measurement results
Table 2: Components of each PCR reaction
PCR reactions were then put into the PCR thermal cycler The following cycle information were applied:
Step Temperature (˚C) Time Number of cycles
Gel electrophoresis is a laboratory method used to separate DNA, RNA, and proteins by their molecular size In this technique, an electrical field drives negatively charged molecules, such as DNA and RNA, toward the positively charged end of a gel with small pores The movement of these molecules is inversely related to their lengths, meaning smaller DNA molecules travel further across the gel compared to larger ones.
Step 1: Use tape to secure the two ends of the casting tray, put in the comb Place the casting tray on a flat surface
Step 2: Weigh 1g of agarose into 100 ml of 1X TAE, microwave until agarose is completely dissolved Fill with water to a capacity of 100 ml Cool it down to about 60°C, add 5 μl Ethidium Bromide and then carefully pour the melted agarose solution into the casting tray Avoid air bubbles
Step 3: After 30 minutes, the gel is completely solidified, gently remove the combs and tape
Step 4: Place the gel into the electrophoresis chamber Add 1X TAE into electrophoresis chamber until the buffer completely cover the gel
Step 5: Add 2 μl 6X loading dye into each sample and load all into the well Repeat for all accessions Add ladders and blank to their own wells
Step 6: Run at 80V for 45 minutes and image with UVP Biodoc-it machine
The data sequence were analyzed by MEGA7
Secondary structure of 16S sequence were predicted by Unafold (http://www.unafold.org/) and R2DT (http://rnacentral.org/r2dt).
RESULTS AND DISCUSSION
PCR reactions were conducted using the 27F and 1492R primer pairs, following the outlined research methodology This process yielded specific products of the anticipated size (see Figure 4.1) Consequently, the PCR products were utilized for sequencing the 16S region.
Figure 4.1 PCR results 4.2 Sequencing results
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Isolate Description Scientific Name Max
E value Per Ident Acc Len Accession isolate_1 Bacillus thuringiensis strain ERP1
Bacillus thuringiensis 756 756 99% 0 94.87% 541 MF432972.1 isolate_2 Bacillus subtilis strain
CUJ-41 Bacillus subtilis 887 887 100% 0 100.00% 1386 ON908577.1 isolate_3 Bacillus paralicheniformis strain HR-1
Bacillus paralicheniformis 889 889 100% 0 100.00% 1198 MT645610.1 isolate_4 Bacillus subtilis strain
CUJ-41 Bacillus subtilis 887 887 100% 0 100.00% 1386 ON908577.1 isolate_5 Bacillus subtilis strain
CUJ-41 Bacillus subtilis 887 887 100% 0 100.00% 1386 ON908577.1 isolate_6 Bacillus paralicheniformis strain AJVR1
Bacillus paralicheniformis 881 881 99% 0 99.79% 1486 MT459810.1 isolate_7 Bacillus subtilis strain
CUJ-41 Bacillus subtilis 887 887 100% 0 100.00% 1386 ON908577.1 isolate_8 Bacillus sp (in: Bacteria) Bacillus sp 887 887 100% 0 100.00% 624 MT126493.1 strain RWP_D7 isolate_9 Bacillus paralicheniformis strain HR-1
Bacillus paralicheniformis 883 883 100% 0 99.79% 1198 MT645610.1 isolate_10 Bacillus paralicheniformis strain HR-1
Bacillus paralicheniformis 889 889 100% 0 100.00% 1198 MT645610.1 isolate_11 Bacillus subtilis strain
CUJ-41 Bacillus subtilis 887 887 100% 0 100.00% 1386 ON908577.1 isolate_12 Bacillus subtilis strain
Results showed in Table 4.1 showed that: All the sequences had the E value
= 0, the query covers ranged from 99% - 100%, other values have the same or similar value which proves that the nucleotide sequences (queries) were statistically matched to sequence in a database
The analysis reveals that the predominant endophytic microorganisms in potato plants belong to the genus Bacillus, including species such as Bacillus thuringiensis, Bacillus subtilis, Bacillus paralicheniformis, and others A study by Bahmani et al (2021) identified 236 endophytic bacteria in potatoes, selecting 11 diverse isolates for 16S sequencing The identified strains included Bacillus pumilus (Bp91, Bp1, Bp49), Bacillus licheniformis (Bl17), Paenibacillus peoriae (Pa86), Pseudomonas brassicacearum (Psb101, Ps169, Ps52), Chryseobacterium indologenes (Ch54, Chl98), and Microbacterium phyllosphaerae (Mi41).
The endophytic bacterial populations in potato (Solanum tuberosum cv Desirée) were found in the bases of stems and roots, ranging from 10³ to 10⁵ CFU/g Dominant species identified through dilution plating primarily belonged to the Flavobacterium/Cytophaga group and Proteobacteria subgroups, with several representatives from Firmicutes also present The most frequently isolated strains, accounting for over 5% of the total, were identified using fatty acid methyl ester (FAME) analysis and partial 16S ribosomal RNA gene sequencing.
Pseudomonas spp (including P aureofaciens, P corrugata, and P putida), Agrobacterium radiobacter, Stenotrophomonas maltophilia, and Flavobacterium resino are notable bacteria associated with potato plants While other Proteobacteria or Firmicutes were occasionally found in potato stem tissue, three potential endophytes—Stenotrophomonas maltophilia, Bacillus sp., and Sphingomonas paucimobilis—were introduced into potato plants through various methods Monitoring revealed that S maltophilia and Bacillus could be tracked via dilution plating and PCR-DGGE for 22 and 1 day(s), respectively, after stem injection However, only S maltophilia successfully colonized and survived in plant tissue from root dipping or soil, while S paucimobilis was not detected in the plants The diversity of the local bacterial ecology associated with potatoes was further examined using PCR-DGGE (Garbeva et al 2001).
A study by Lastochkina investigated the resistance and quality characteristics of stored potato tubers infected with Phytophthora infestans, focusing on the effects of the endophytic bacteria Bacillus subtilis (strain 10-4) and its combination with salicylic acid (SA).
To conduct the tests, potato mini-tubers were grown hydroponically, coated with
B subtilis both alone and in combination with SA, and then infected with Ph infestans before being preserved for six months The findings showed that Ph infestans infection greatly increased tuber late blight incidence (up to 90–100%) and oxidative and osmotic damage (i.e., malondialdehyde and proline) in tubers
B subtilis treatments or using it in conjunction with SA reduced the prevalence of
Endophytic bacteria B subtilis can significantly improve potato postharvest resistance to late blight and enhance tuber quality during long-term storage This enhancement is observed through a reduction in Ph infestans-activated tuber late blight by 30–40%, as well as a decrease in oxidative and osmotic damages, including malondialdehyde and proline levels The combination of B subtilis with salicylic acid (SA) further amplifies these beneficial effects (Lastochkina et al 2022).
A phylogenetic tree is a diagram that illustrates the evolutionary relationships among different species, serving as a historical hypothesis about their connections This "tree of life" visually represents how various organisms are linked and indicates their evolutionary development over time.
The 12 species identified above have not shown genetic relationships, so we have built a phylogenetic tree to be able to determine the relationship between 12 accessions based on the obtained sequence
Use MEGA7 to define the phylogenetic tree After sequence alignment, export data, phylogenetic tree built by Contrust/test Maximum Likelihood tree with Boostrap value of 1000.
The phylogenetic tree of 12 isolates, constructed using MEGA7 software, revealed their relationships and displayed three distinct branches The first branch comprised seven isolates, all identified as Bacillus subtilis, which showed significant similarity to Bacillus subtilis CUJ-41, justifying their grouping The second branch included four isolates, all annotated as Bacillus paralicheniformis, while the final branch contained a single isolate.
(isolate 1) which was annotated as Bacillus thuringiensis Although there were three isolated were identified as Bacillus paralicheniformis strain HR-1 (isolate_3,
Figure 4.2 illustrates the varying branch lengths of the isolates, indicating differences in genetic sequences Longer branches signify greater genetic change or divergence among the isolates.
The phylogenetic analysis revealed that the isolated microorganisms were categorized into distinct groups However, we aimed to investigate whether minor sequence variations could influence their secondary structures Additionally, we sought to explore the polymorphic diversity within these secondary structures To achieve this, we constructed secondary structure models for 12 isolates using two online tools: Unafold.
(https://www.unafold.org/) and R2DT (https://rnacentral.org/r2dt)
Table 4.2: Secondary structure of 12 species of microorganisms determined by R2DT and Unafold
B) Group II predicted by R2DT
Group II predicted by Unafold
In group II, the application of R2DT tools for predicting the second-order structure yielded nearly identical results However, the Unafold tool revealed differences at positions 160, 200, and 240, as indicated by the orange arrow in the accompanying image.
Here we still see that the structures predicted by R2DT are completely similar between the isolates in the group
Also the sequence 2, 4, 5, 7, 11, 12 but when using Unafold, you can see that the secondary structure has a huge difference (circled as above).
C) Group III predicted by R2DT and Unafold tools
The R2DT tool's predictions indicate a strong similarity in structure among isolates within the same group, while also revealing notable differences in the secondary structures of various isolates.
While secondary structures predicted by R2DT tool showed high similarity between isolates, Unafold tool showed significantly more differences in the secondary structure of the isolates
After identifying the polymorphisms through secondary structure prediction, we performed a detailed analysis by aligning the sequences using MEGA7 software, setting the toggle at 50%, gap open at -400, and gap extension parameters.
CONCLUSIONS AND RECOMMENDATIONS
Conclusions
Twelve endophytic bacteria isolated from potato roots were identified through partial 16S rRNA sequencing All twelve isolates were classified within the genus Bacillus A phylogenetic tree was constructed to illustrate the relationships among the 12 isolates.
The MEGA7 software analysis revealed three distinct branches among the isolates The first branch contained seven isolates, all identified as Bacillus subtilis, while the second branch included four isolates, all annotated as Bacillus paralicheniformis The final branch consisted of a single isolate, designated as Bacillus thuringiensis Despite some isolates being grouped together, the secondary structure analysis of partial 16S rRNA using the Unafold tool demonstrated significant diversity among the isolates Additionally, a detailed sequence analysis of the partial 16S rRNA was conducted.
Recommendations
Other genetic diversity analysis of twelve isolates such as molecular markers should be applied to evaluate the divergence of the genetic
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TTAGCGGCGGACAAGGTGAACTAACTGCTGCGGTAACCTGTCCCATAAGACTGGTGATAA CTACCGGGAAAACCCGCTTCTAATACAGAGATAACATTTTGAACTGCATGGTTCGAAATT GAAAAGCTGCTTCGGCTGGCACTTATGGATGGACCCGCGTCGCATTAGCTAGTTGGTGAG GTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGG GACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCACTACGGAATCTTCCGCAATGGAC GAAAGTCTGACGGATCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGT TGTTAGGGAAGAACAAGTGCTAGTTGAATAAGCTGGCACCTTGACGGTACCTAACCAGAA AGCCACGGCTAACTACATGCCAACAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGG AATTATTCC
TTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCG GGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAAACATAAAAGGTGG CTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCT CACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGAC ACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTG ACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGA AGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGC TAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGG
TTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCG GGAAACCGGGGCTAATACCGGATGCTTGATTGAACCGCATGGTTCAATTATAAAAGGTGG CTTTTAGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGC TCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGA CACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCT GACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAACTCTGTTGTTAGGG AAGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGG CTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTG
TTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCG GGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAAACATAAAAGGTGG CTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCT CACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGAC ACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTG ACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGA AGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGC TAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGG
TTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCG GGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAAACATAAAAGGTGG CTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCT CACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGAC ACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTG ACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGA AGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGC TAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGG
TTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCG GGAAACCGGGGCTAATACCGGATGCTTGATTGAACCGCATGGTTCAATTATAAAAGGTGG CTTTTAGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGC TCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGA CACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCT GACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAACTCTGTTGTTAGGG AAGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCATAAAGCCACGG CTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTG
TTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCG GGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAAACATAAAAGGTGG CTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCT CACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGAC ACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTG ACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGA AGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGC TAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGG
TTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCG GGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAAACATAAAAGGTGG CTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCT CACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGAC ACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTG ACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGA AGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGC TAACTACGTGCCAGCAGCCGCGGTAATACGTATGTGGCAAGCGTTGTCCGGAATTATTGG
TTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCG GGAAACCGGGGCTAATACCGGATGCTTGATTGAACCGCATGGTTCAATTATAAAAGGTGG CTTTTAGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGC TCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGA CACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCT GACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAACTCTGTTGTTAGGG AAGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCGCGG CTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTG