Journal of Life Science and Biomedicine

J Life Sci Biomed, 10 (4): 51-58, 2020

ISSN 2251-9939

DOI: https://dx.doi.org/10.51145/jlsb.2020.7

Isolation and identification of Enset wilt disease causing

bacteria using 16S rRNA Gene Sequence samples collected

from Gurage zone, Ethiopia

Belay TILAHUN¹^, Degisew YINUR², Dejene ZENABU²and Flagot Mola MENESHA²

¹Institute of Biotechnology, University of Gondar, P. O. Box 196, Gondar, Ethiopia

²Department of Biotechnology, College of Natural and Computational Sciences, Wolkite University, P. O. Box 07, Wolkite, Ethiopia.

Corresponding author’s Email: belayt21@gmail.com

ABSTRACT

Original Article

Introduction. Xanthomonas campestris is an important bacterium responsible for bacterial

wilt disease, which causes predominantly a serious loss in enset production. In some enset-

PII: S225199392000007-10

growing areas of Ethiopia, farmers are enforced to replace perennial enset plants with

annual crops because of this disease devastates enset production. Aim. Therefore, the study

Rec. 23 May 2020

Rev. 29 June 2020

Pub. 25 July 2020

aimed to identify the molecular diversity of bacterial wilt disease causing bacteria from

infected enset plants that were collected from the Gurage Zone, using the 16S rRNA gene

sequence. Methods. 60 infected enset samples were collected from infected enset plants.

Presumptive identification of the bacterium was done through biochemical tests. 16S rRNA

genes of bacterial isolates were amplified using the bacteria universal primers and the

amplified products were sequenced at MRC-Holland, Amsterdam. Sequence analysis and

comparison were conducted to identify the isolated microbes into species and strain levels.

Results. Based on the biochemical tests, 18 bacterial isolates were motile, indole negative as

well as citrate and catalase positive and they were hydrolyzed starch. The sequence analysis

revealed that from 18 bacterial isolates 17 of them were identified as Xanthomonas campestris

of different strains and one isolate was identified as an uncultured bacterium. In this study,

different Xanthomonas campestris strains that have different virulence factors were

identified in the study area. To effectively control and manage bacterial wilt disease of enset

plant, it is important to examine antipathogenic agent or biological control mechanisms for

all Xanthomonas campestris strains. Additionally, determining plant bacterial interaction

using molecular tools and identify the virulence genes are also beneficial.

Keywords

Xanthomas sp.,

Enset,

16srRNA gene,

DNA sequencing,

Wilt disease

INTRODUCTION

Xanthomonas campestris is one of the most common bacteria responsible for bacterial wilt disease which attacks

and kills enset plants at any developmental stage [1 ]. It causes predominantly a serious loss in enset production

in which the farmer has already invested land, labor, and resources for several years [2 ]. Such situations have

caused farmers to replace enset plants with annual crops in the southern region of the country (Ethiopia). Enset

growing regions are densely populated with very small land coverage (average of 0.17 ha). However, such a

replacement of enset by annual crops on such a small plot cannot fulfill the food demand of the region [2; 3].

Among the genus Xanthomonas bacteria Xanthomonas campestris which is rod-shaped, gram-negative

bacteria and creamy and light yellow mucoid circular colonies on YDC agar [1] with growth temperature from

25-30 °C, causes the serious disease of enset and banana wilt in the most Africa countries [3 ]. The disease

appears on any part of enset but mostly appears on youngest leaves that show greyish-brown at the tip the

plant leaves [1 ]. In different agroecology, the disease is disseminated and the production of enset has been

decreased in the areas where enset is growing and used as a staple food [3 ]. Thereof, it causes the problem of

food insecurity in the enset growing regions of Ethiopia [4 ].

There are variations among the Xanthomonas campestris strains regarding to their pathogenicity which

leads to great damage in crop production and makes it more complicated to manage the disease [3-5 ]. The

ddiscrepancy among Xanthomonas campestri strains may have different virulent factors and difficult to screen

resistance enset clones. Selections of resistant clones are non-reproducible when testing disease resistant enset

clones by different Xanthomonas campestri isolates. Thus, to develop an effective controlling mechanism of wilt

disease which causes by Xanthomonas campestri and to screen resistant enset cones, studying the diversity of

Citation: Tilahun B, Yinur D, Zenabu D and Menesha FM. Isolation and identification of Enset wilt disease causing bacteria using 16S rRNA Gene Sequence

samples collected from Gurage zone, Ethiopia. J Life Sci Biomed, 2020; 10(4): 51-58; DOI: https://dx.doi.org/10.51145/jlsb.2020.7

Xanthomonas campestris is needed. In order to overcome the bottle neck, this study was planned to assess the

molecular diversity of available strain from infected enset plants which were collected in the study area.

MATERIAL AND METHODS

Isolation and selection of bacteria

A total of 60 infected enset samples was collected aseptically from infected enset plants. Those samples

were obtained from different enset plant varieties that were found ten kebele of three woredas (Gumere

Woreda, Cheha Woreda, and Ezia woreda). Then the samples were transported to Wolkite University

Biotechnology Department Laboratory. Then after surface sterilization, 10 mg of tissues from each infected

plant samples were transferred into 90 ml sterile 0.1% peptone water containing conical flask and homogenized

by vortex for 10-15 min. From appropriate serial dilutions, 0.1 ml Aliquots were spread plated onYDC agar and

incubated at 28⁰C for 48 h. The pure isolates were maintained on YDC agar slants at 4^oC and sub-culture every

four weeks was done until biochemical and molecular characterization was carried out [6 ].

Biochemical characterization of bacterial isolates

The morphological characterizations of bacterial isolates were determined according to their cultural

characteristics using a microscope [7- 8]. Biochemical characterizations such as gram, oxidase, catalase, citrate,

indole, H₂S, VP, citrate, catalase, and methyl red tests) were conducted for each bacterial isolates [9-11 ].

Starch hydrolysis test

Bacterial isolates were streak plated on nutrient agar plates containing 0.2% soluble starch (w/v) and

incubated at 30°C until heavy growth occurred. Then plates were flooded with IKI solution (iodine, 1g;

potassium iodide, 2g; distilled water, 100 ml) and the clear zone around a colony was recorded as positive

reaction [12 ].

Amplification of 16S rRNA gene

The genomic DNA of bacterial isolates was extracted using DNeasy DNA Extraction kit, Qiagen [13- 14 ]. To

amplify the 16S rRNA gene of each bacterial isolate, PCR reaction mixtures (50 μl) which contained 1 μl of the

extracted DNA, 5 μl dNTPs, 1 μl of each of primers rD1 (5’-AGA GTT TGA TCC TGG CT C AG-3’) and fD1 (5’-AAG

GAG GTG ATC CAG CC-3’) [15-16 ], 1 μl of Taq DNA polymerase (Fermentas, St. Leon- Rot, Germany), 5 μl PCR

buffer and reverse osmosis purified water were used. The PCR reaction was programed as initial denaturation

at 95°C for 60 sec, followed by 35 cycles of denaturation at 94°C for 60 sec, primer annealing at 51°C for 30sec

and primer extension at 72°C for 60 sec with a final extension at 72°C for 60 sec. Then, the PCR products of the

16S rRNA gene were separated by gel electrophoresis using 1% agarose gel and 1µL loading dye with 5µL PCR

products and stained with ethidium bromide for gel documentation [16-17 ].

Nucleotide sequencing and sequence analyses

The 16S rRNA gene PCR product of each bacterial isolate was sequenced by automated DNA sequencer

(ABI model 377; Applied Biosystems), MRC-Holland, Amsterdam [16 ].

Phylogenetic analysis

After the raw DNA sequences were edited using the FinchTV package and consensus sequences were

obtained. The sequences were compared with the NCBI DNA database using the BLAST search [16- 19 ]. Then

sequences were then aligned using Clustalx 2.1 [19 ] and phylogenetic tree was constructed using MEGA 7 [16-

19 ].

RESULT AND DISCUSSION

Bacterial isolation and morphological characteristics

From 60 infected enset samples, 18 bacterial isolates were obtained and purified. They were yellowish on

nutrient agar, circular in shape, shiny, motile and all of them were also Gram-negative. Most of the isolates had

slimy mucoid yellowish, therefore; results were in line with Tsehay [1 ] and Welde-Micheal et al. [2 ]. As indicated

Citation: Tilahun B, Yinur D, Zenabu D and Menesha FM. Isolation and identification of Enset wilt disease causing bacteria using 16S rRNA Gene Sequence

samples collected from Gurage zone, Ethiopia. J Life Sci Biomed, 2020; 10(4): 51-58; DOI: https://dx.doi.org/10.51145/jlsb.2020.7

in Aritua et al. [20 ] the bacterium is described as a motile, gram-negative rod, possessing a single polar

flagellum and producing typical yellow, convex, mucoid colonies on nutrient agar and YDC media. Regarding on

the morphological characteristics or their colony morphology results, 18 bacterial isolates which were

resembled Xanthomonas spp were selected [20 ].

Biochemical characterization of bacterial isolates

Motility is one of an important differentiating tools in bacteria, has long been recognized as a biological

characteristic of microorganisms [4]. All 18 bacterial isolates were motile and indole negative which was unable

to breakdown tryptophan to indole. Likewise, all isolates were citrate positive, which they change the color of

the medium (deep green) to blue indicates the ability of to metabolize citrate. Besides, all isolates were catalase

positive; produced bubbles when the colony of each isolate was dissolved in a few drops of 3% H₂O₂which is

supported by Haile et al . [4 ] (Table 1) report. After the plates were flooded with IKI solution clear zone around a

colony was recorded as a positive reaction. The clear zone indicates the level of starch hydrolysis capacity of

isolates (Figure 1). Potential isolates can produce large clear zone and indicate complete hydrolysis of starch. All

isolates were able to hydrolyze starch and the clear zones were also observed.

Figure 1. Clear zone observed around bacterial isolates

Molecular Characterization of bacterial isolates

Amplification of 16S rRNA gene of bacterial isolates. The PCR product of all bacterial isolates was shown

in Figure 2 and all the isolates were shown to have PCR amplified fragments with around 1500bp size [16].

Figure 2. PCR amplification 16s rRNA using rD1 and fD1 bacterial universal primers, M1; 1kb DNA ladder,

amplicon size; 1500bp.

Citation: Tilahun B, Yinur D, Zenabu D and Menesha FM. Isolation and identification of Enset wilt disease causing bacteria using 16S rRNA Gene Sequence

samples collected from Gurage zone, Ethiopia. J Life Sci Biomed, 2020; 10(4): 51-58; DOI: https://dx.doi.org/10.51145/jlsb.2020.7

16S rRNA gene of bacterial sequence analyses

After all the raw the 16S rRNA gene sequences of 18 bacterial isolates were edited using the FinchTV

package, consensus sequences (edited sequence) were blasted in Gen Bank of NCBI. Samples which showed

from 92-100% percent of homology were identified as shown in Table 2. According to this study, the seventeen

bacterial isolates were identified as Xanthomonas campestris, one isolate was identified as Pseudomonas

protegens, one isolate was also identified as Bacillus subtilis and four isolates were also identified as uncultured

bacteria.

The seventeen bacterial isolates were found to be the genus Xanthomonas, of which WKU1, WKU3, WKU17

and WKU18 were belongs to Xanthomonas campestris pv. campestris XCC-C7 with 100% of sequence homology.

WKU2, WKU9, WKU10, WKU13, WKU14 and WKU15 were Xanthomonas campestris strain ATCC 33913 with 100%

of homology. WKU4 and WKU21 were identified as Xanthomonas campestris strain Xan-E1with 92% and 100% of

homology respectively.

WKU7 and WKU12 were identified as Xanthomonas campestris strain LMG 568; WKU19 and WKU22 were

identified as Xanthomonas campestris strain PgBe189 and WKU23 were identified as Xanthomonas campestris

strain with 100% of homology. As Young et al. [21 ] indicated that the isolates identified as Xanthomonas species

by Biolog were confirmed as Xanthomonas campestris using the GspDm primers, sequence-based phylogenetic

tree and pathogenicity tests. The diversity of Xanthomonas bacteria in banana (similar to a false banana, enset)

which was studied by Studholme et al. [22 ] has concurred with this study. Hence, identified diversity of

Xanthomonas within the enset plant, reveals that there has been an evolution of Xanthomonas species from time

to time.

Maximum likelihood method of Tamura and Nei [19] was used to infer the evolutionary history of identified

bacterial species. The highest log likelihood (-1679.15) is shown in the tree. The tree is drawn to scale, with

branch lengths measured in the number of substitutions per site as shown in Figure 3. There were a total of

994positions in the final dataset. Evolutionary analyses were conducted in MEGA7 [23 ].

Molecular identification of Xanthomonas spp. from infected enset using 16s rRNA sequence was identified

Xanthomonas campestris which is a more precise and accurate technique for identification of bacterial isolates.

This is supported by Adriko et al., [24 ] the use of multiple tools for precise identification and characterization of

Xanthomonas bacteria in bananas and elucidates the benefit of this microbial diversity in the management of

bacterial wilt disease. Owing to the variable phenotypic and genotypic characteristics of bacteria, serving for

their identification, their diagnosis often requires the use of complementary methods [25 ].

Table 1. Biochemical characteristics of the bacterial isolates

Biochemical tests

Sample

code

Bacterial

isolates

No.

Gram

test

KOH

Test

Starch

hydrolysis

Catalase

test

H₂S

test

Indole

test

Citrate

Motility

BB9

WKU04

WKU05

WKU07

WKU12

WKU02

WKU09

WKU10

WKU13

WKU14

WKU15

WKU01

WKU03

WKU17

WKU18

WKU22

WKU19

WKU21

WKU23

Motile

YM18

XX1(A)

BN7

BB7SH

WY1

XY5

AY13

AV13(A)

WY3

BB10

TZ11

YN16

10.

11.

12.

13.

14.

15.

16.

17.

18.

XX2

NM16SH

Citation: Tilahun B, Yinur D, Zenabu D and Menesha FM. Isolation and identification of Enset wilt disease causing bacteria using 16S rRNA Gene Sequence

samples collected from Gurage zone, Ethiopia. J Life Sci Biomed, 2020; 10(4): 51-58; DOI: https://dx.doi.org/10.51145/jlsb.2020.7

Table 2. Identification of bacteria isolated from Enset as revealed by partial 16S rRNA gene sequence BLAST search.

Bacterial

isolates

Infected Enset

type (local name)

NCBI Accession

Number

No.

Sample code

% homology

ID based on NCBI BLAST

value

19.

20.

21.

BB9

WKU04

WKU05

WKU07

Agadi

Ginbio

Agadi

92%

100%

KT156667

GU272305

NR119219

Xanthomonas campestris strain Xan-T1

Uncultured bacterium clone CF12

Xanthomonas campestris strain LMG 568

YM18

XX1(A)

22.

23.

24.

BN7

WKU12

WKU02

WKU09

Ameratye

Agadi

100%

NR119219

NR074936

Xanthomonas campestris strain LMG 568

Xanthomonas campestris strain ATCC 33913

BB7SH

Ankafuye

25.

26.

27.

28.

29.

30.

31.

WY1

WKU10

WKU13

WKU14

WKU15

WKU01

WKU03

WKU17

Ankafuye

Yshrakinki

Astara

100%

NR074936

MN108237

Xanthomonas campestris strain ATCC 33913

Xanthomonas campestris pv. campestris XCC-C7

XY5

AY13

AV13(A)

WY3

BB10

TZ11

Ankafuye

Ginbio

Ameratye

32.

33.

34.

YN16

WKU18

WKU22

WKU19

Astara

100%

MN108237

MH211280

Xanthomonas campestris pv. campestris XCC-C7

Xanthomonas campestris strain PgBe189

Separa

XX2

Ameratye

35.

36.

WKU21

WKU23

Separa

100%

KT156666

EF059753

Xanthomonas campestris strain Xan-E1

Xanthomonas campestris

NM16SH

Citation: Tilahun B, Yinur D, Zenabu D and Menesha FM. Isolation and identification of Enset wilt disease causing bacteria using 16S rRNA Gene Sequence samples collected from Gurage zone, Ethiopia. J Life Sci Biomed, 2020; 10(4):

51-58; DOI: https://dx.doi.org/10.51145/jlsb.2020.7

GU272305.1) Uncultured bacterium clone CF12

WKU21) Xanthomonas campestris strain Xan-E1

NR 074936.1)Xanthomonas campestris strain ATCC 33913

KT156667.1) Xanthomonas campestris strain Xan-E1

WKU01) Xanthomonas campestris pv. campestris XCC-C7

WKU22) Xanthomonas campestris strain PgBe189

WKU04) Xanthomonas campestris strain Xan-T1

WKU07) Xanthomonas campestris strain LMG 568

WKU05) Uncultured bacterium clone CF12

WKU18) Xanthomonas campestris pv. campestris XCC-C7

WKU09) Xanthomonas campestris strain ATCC 33913

WKU10) Xanthomonas campestris strain ATCC 33913

MN108237.1) Xanthomonas campestris pv. campestris XCC-C7

WKU02) Xanthomonas campestris strain ATCC 33913

WKU03) Xanthomonas campestris pv. campestris XCC-C7

WKU19) Xanthomonas campestris strain PgBe189

NR 119219.1) Xanthomonas campestris strain LMG 568

WKU12) Xanthomonas campestris strain LMG 568

EF059753.1) Xanthomonas campestris

MH211280.1) Xanthomonas campestris strain PgBe189

WKU17) Xanthomonas campestris pv. campestris XCC-C7

WKU15) Xanthomonas campestris strain ATCC 33913

WKU23) Xanthomonas campestris

WKU13) Xanthomonas campestris strain ATCC 33913

WKU14) Xanthomonas campestris strain ATCC 33913

Figure 3. Phylogenetic analysis based 16s rRNA gene sequences of bacterial strains isolated from Enset; coded as

WKU followed by numbers and reference bacterial strains using the Maximum Likelihood method.

CONCLUSION

In this study, important strains of Xanthomonas spp which were found in the study area were identified.

Sequencing based phylogenetic highlight relationships and possible interactions among the diverse bacteria

populations in a plant system were also recognized. Therefore, the result of this study indicated that various

Xanthomonas campestris strains are responsible for bacterial wilt disease of enset plant. Identification of these

isolates could contribute valuable information for the understanding of enset plant pathogenic interaction and

to develop controlling mechanisms of the pathogen. Moreover, Understanding the dynamics of Xanthomonas

spp in enset and could provide insight into the effect of microbial interactions on BXW disease development as

well possibly using some of these endophytic bacteria as bio control agents.

DECLARATIONS

Acknowledgments

First of all, I would like to thank the Research and community service of Wolkite University, Ethiopia for

their financial support of this study. I would like also thank Dr. Zewdu Terefwork and Prof. Garry Wessel for

their unlimited support for sequence analysis.

Authors’ contributions

B.T. performed all the laboratory works, collected samples and materials, analyzed the data and wrote the

manuscript. D.Y. performed molecular laboratory works, wrote and edited the manuscript. D.Z. and F.M.

Citation: Tilahun B, Yinur D, Zenabu D and Menesha FM. Isolation and identification of Enset wilt disease causing bacteria using 16S rRNA Gene Sequence

samples collected from Gurage zone, Ethiopia. J Life Sci Biomed, 2020; 10(4): 51-58; DOI: https://dx.doi.org/10.51145/jlsb.2020.7

collected samples, performed some part of laboratory work and involved in data records. All Authors read and

agreed on the first manuscript.

Conflicts of interest

The Authors declare no conflict of interest.

Data availability statement

All the data of study are available from the corresponding author.

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Citation: Tilahun B, Yinur D, Zenabu D and Menesha FM. Isolation and identification of Enset wilt disease causing bacteria using 16S rRNA Gene Sequence

samples collected from Gurage zone, Ethiopia. J Life Sci Biomed, 2020; 10(4): 51-58; DOI: https://dx.doi.org/10.51145/jlsb.2020.7