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Biochemical and Molecular Characterization of Xanthomonas axonopodis Pv Phaseoli of Common Bean

T
Tibebu Belete1,*
S
Sylvans Ochola1
K
Kubilay Kurtulus Bastas2
D
Duncan Cheruiyot1
1Rwanda Institute for Conservation Agriculture (RICA), Bugesera Campus, Bugesera/Rwanda.
2Department of Plant Protection, Faculty of Agriculture, Selcuk University, Konya/Turkey.

ABSTRACT

Background: Common bacterial blight (CBB), caused by Xanthomonas axonopodis pv. phaseoli (Xap) significantly impacts common bean production worldwide. Timely identification and characterization of Xap are critical for disease management and breeding for resistance.

Methods: The pathogen was isolated from symptomatic leaves, stems and pods of common bean cultivars, followed by morphological, physiological, biochemical and molecular characterization. Pathogenicity and induction of hypersensitivity reactions of the isolates were confirmed on susceptible bean cultivar and tobacco, respectively.

Result: A total of 35 Xap isolates were Gram-negative, oxidase-negative, catalase-positive and tested positive for levan and H2S production, esculin hydrolysis, acid production from carbohydrates and starch hydrolysis and were non-fluorescent on King’s B medium. Pathogenicity and hypersensitivity reactions assays on susceptible bean cultivar and tobacco, respectively, confirmed the virulence of the isolates. PCR amplification using Xap-specific primers yielded the expected 730 bp product for all isolates, confirming their identity. Plasmid profiling revealed all isolates carried plasmids of similar size, suggesting limited plasmid size diversity among strains from the same agroecological zone. The combination of classical, biochemical and molecular methods provided a reliable strategy for Xap identification, laying a foundation for further studies on identification of Xap strains.

INTRODUCTION

Plant disease caused by Xanthomonas axonopodis pv. Phaseoli (Xap) has been a persistent challenge for bean farmers globally. The pathogen infects the plant through natural openings or wounds, triggering symptoms such as leaf spots, wilting and necrosis to cause common bacteria blight (CBB) disease (Belete et al., 2022). Early and accurate detection of Xap is crucial for implementing timely control measures and preventing the spread of the disease. Biochemical techniques have long been employed for bacterial identification, relying on the analysis of physiological and metabolic characteristics (Doddaraju et al., 2019; Lamichhane and Varvaro, 2014; Roach et al., 2018). These methods, including the determination of enzymatic activities and substrate utilization patterns, have proven effective in differentiating bacterial species and strains. Moreover, they serve as valuable tools for initial screening and classification. Several studies have successfully utilized biochemical methods for the identification of Xanthomonas species, emphasizing the importance of these techniques in the early stages of pathogen detection (Sarker et al., 2017; Schaad et al., 2001; Singh et al., 2016).
       
While biochemical techniques provide a foundation for bacterial identification, molecular methods offer a higher level of specificity and sensitivity. Polymerase chain reaction (PCR) and DNA sequencing have revolutionized the field of microbiology, allowing for the precise identification of bacterial pathogens based on their genetic makeup (Adachi and Oku, 2000; Strayer et al., 2016). The use of molecular markers, such as specific DNA sequences, facilitates the differentiation of closely related bacterial strains. Previous studies have successfully employed molecular techniques for species-specific identification and genetic diversity analysis of Xap (Belete et al., 2025; Batista et al., 2021; Boureau et al., 2013; Potnis et al., 2011; Zamani et al., 2011).
       
Plasmids, extrachromosomal DNA elements in bacteria, play a crucial role in the adaptability and virulence of many plant pathogens. The study of plasmid DNA profiles offers insights into the genetic variability of bacterial populations, providing information on the acquisition and exchange of virulence factors and antimicrobial resistance genes. While the role of plasmids in Xap pathogenicity is not fully understood, their investigation holds promise for uncovering novel targets for disease control. Few studies have explored the plasmid profiles of Xap isolates, highlighting the need for comprehensive research in this area (Alavi et al., 2008). This study employed a combination of biochemical and molecular techniques to identify and characterize genetic diversity Xanthomonas axonopodis pv. Phaseoli.

MATERIALS AND METHODS

Sample collection and isolation of the pathogen
 
Naturally infected common bean leaves, stems and pods showing characteristics CBB symptoms were collected from different bean-growing areas of Cumra and Karatay in Konya province of Turkey (Fig 1, A-C). Forty (40) samples were collected from different local common bean cultivars.          

Fig 1: Assessment and scouting of common bacterial blight incidence in a bean field.

                

The samples were sealed in polyethylene bags and transported to the Molecular Plant Bacteriology Laboratory located at the Department of Plant Protection, Faculty of Agriculture, Selcuk University for isolation of the causative pathogen.
       
Isolation of the pathogen was done from infected leaves and pods according to Bradbury (Bradbury, 1970) with some modifications. Plates were incubated at 28oC for 48-72 hours and examined for the appearance of bacterial colonies and their colony morphologies and colors were determined. Pure cultures of the colonies were obtained by sub-culturing representative colonies.
 
Morphological, biochemical and physiological characterization of Xap
 
All Xap isolates were compared and characterized based on their physiological, biochemical and metabolic properties using standard biochemical tests, such as 3% potassium hydroxide (KOH) test as a gram reaction test, Oxidase Reaction Test, Presence of Catalase, Test of Levan Production, Hydrogen Sulfide (H2S) Production from cysteine, esculin Hydrolysis, Fluorescent Pigment on King’s B medium under UV light, Acid Production from Erythritol and Sorbitol, Tween 80 Hydrolysis and Starch Hydrolysis (Schaad et al., 2001) (Table 1). Each test was conducted in triplicate for each strain and repeated three times.

Table 1: Biochemical and physiological characteristics of Xap Isolates.



Pathogenicity and hypersensitivity reaction (HR) test
 
Susceptible common bean cultivar (Aras 98) was grown in a controlled greenhouse as a pot experiment. When the bean plants reached 16-20 days old after planting, all Xap isolates were streaked onto plates of NA and incubated for 48 hours at 28oC for inoculum preparation. Bacterial cells were suspended in SDW and adjusted to an optical density at 600 nm = 0.5, which corresponds to ≈ 108 CFU/ml (Mkandawire et al., 2004). The first fully expanded trifoliate leaves of the susceptible bean plants were sprayed using a hand-held sprayer and the pots were covered by polyethylene bags for maximum humidity for bacterial development and later removed after three days. SDW was also spread onto leaves of bean plants serving as a negative control. At the same time, the bean plants were inoculated by the known source of Xap (145-X) isolate as a positive control. The experiment was arranged in a completely randomized design (CRD) and replicated three times. Inoculated plants were examined daily for the development of symptoms and observations were made and recorded at seven days post inoculation. Bacteria with the same characteristics as those inoculated were re-isolated from the leaves showing symptoms and characterized accordingly.
       
For HR test, bacteria colonies were suspended in sterile distilled water and adjusted using a bio-photometer to give a concentration of approximately 108 CFU/ml (0.15 absorbance at 660 nm). A bacterial inoculum of 1.5-2 ml of each culture suspension was injected into the expanded leaves of a two-month-old tobacco plant (Nicotiana tabacum, cv. White Burley) using a sterile hypodermic syringe. Tobacco leaves were similarly injected with sterile distilled water (SDW) used as a negative control. All the tobacco plants were kept in the green-house for approximately 24h at 25-28oC until symptoms developed (Schaad et al., 2001).
 
DNA extraction and PCR assay
 
The DNA of all Xap isolates were isolated using the Qiagen DNA isolation kit and fully Automatic Isolation Robot (Qiacube, Qiagen) according to the manufacturer’s recommendation. The quantity and quality of the extracted DNA were determined using spectrophotometer (NanodropND-1000) and by running 10µL (10µL+ loading dye) on 1% agarose gel, respectively (Sambrook and Rusell, 2001). PCR amplifications were conducted with DNA extracted from pure bacterial cultures. PCR reactions were prepared in 0.2 ml Eppendorf tubes.
               
To identify representative strains of Xap, polymerase chain reaction (PCR) was performed with the Xap-specific primer pair, X4c (5´-GGC AAC ACC CGA TCC CTA AAC AGG -3´) and X4e (5´-CGC CCG GAA GCA CGA TCC TCG AAG -3´) (Audy et al., 1994), which directs the amplification of the 730 bp DNA fragment. The PCR was done under the following conditions: initial denaturation at 94oC for 3 minutes (single-step reaction), followed by 30 repeated cycles of melting, annealing and DNA extension at 94oC for 1 minute, 65oC for 1 minute and 72oC for 2 minutes, followed with a single-step final extension reaction for 5 minutes at 72oC. The amplified DNA fragments were electrophoresed in 1% agarose gel in 1 × TBE buffer and visualized with UV light after ethidium bromide staining. A 1 kb molecular marker (marker, Fermantas 1 kb Plus DNA Ladder SM 1153) was used to determine the molecular weights of the resulting bands. To characterize the different Xap isolates based on their plasmid DNA profiles, plasmid DNA was extracted using Mini-prep method (Carvalho et al., 2005) with minor modifications. The plasmid DNA was analyzed on 0.8% agarose gel and a 1000 bp molecular marker used to determine and characterize the plasmid DNA size.

RESULTS AND DISCUSSION

Biochemical and physiological characterization
 
Initial classification and identification of bacterial isolates was carried out based on colony morphology and color on general and semi-selective media. Incubation of the bacterial isolates for  48 h at 28oC, resulted in round/domed, convex, mucoid and yellow colonies on NA (Fig 2, A and B). The morphological appearances of the colonies were characteristic of Xanthomonads and were similar to those previously described by (Schaad et al., 2001). Based on the characteristic colony morphology, 35 Xap representative isolates subjected to different determinative biochemical test for bacteria according to (Schaad et al., 2001).                

Fig 2: Isolation and biochemical test of Xap.

                    

The isolates were subjected to Gram reaction, oxidase reaction, catalase activity, levan production, H2S production, esculin hydrolysis, fluorescent pigment production on King’s B medium, acid production from carbohydrates and starch hydrolysis tests (Table 1). The levan production test resulted in translucent shining mucoid colonies with a distinctive raised convex appearance on NA medium containing 5% sucrose after incubation at 28oC (Fig 1C). The results of this study were consistent with previous research (Lelliot, 1987) that levan was produced from the fructose moiety of the sucrose molecule and its formation was usually responsible for the production of mucoid colonies by some species of bacteria.
       
The addition of 3% KOH to bacterial isolate colonies in suspension resulted in the formation of viscous, thread-like mucus when lifted with an inoculating loop (Fig 2D). This is the confirmatory test and positive reaction for Gram-negative bacteria (Suslow et al., 1982). The peptidoglycan layer in the cell wall of Gram-negative bacteria is monolayer and does not contain teichoic acid and can easily be broken down with KOH. As a result, the cytoplasmic fluid becomes free and a viscous elongation can be observed. However, in Gram-positive bacteria, no viscous elongation was observed when the mixture of KOH and bacterial colonies were lifted by a loop while remaining as an aqueous liquid (Schaad et al., 2001). Vauterin et al., (1990) reported that genus Xanthomonas pathogens are Gram-negative, aerobic, rod-shaped, with a single polar flagellum, catalase-positive, oxidase negative, levan positive, HR-positive, mucoid, convex and formed yellow colonies on yeast dextrose carbonate agar (YDCA) medium.
       
When the isolates were subjected to oxidase reaction test, no dark-purple reaction was observed on the impregnated filter paper (Fig 2E). Within a few seconds, the disks became dark-purple when the strain was oxidase positive and those which did not change color within 60 sec were evaluated as oxidase negative (Fig 2F). The Xap isolates exhibited a negative oxidase reaction, which is consistent with the typical biochemical profile of Xanthomonas species (Kovacs, 1956). The observed dark/bluish-purple color indicated that the enzyme reacted with the chemical encoded on the disc and that the bacterium contains cytochrome protein. In other words, the bacterial isolates were discolored by bacterial coating on discs containing 1% tetramethyl-p-phenylenediamine dihydrochloride.  The oxidase reaction test was used to distinguish the isolates that produced cytochrome c protein in the bacteria electron transport chain. The enzyme cytochrome c oxidase is used in the electron transport system to reduce substances and facilitate the formation of cellular energy (ATP) during cellular respiration.
       
When the Xap isolates were tested for presence of catalase enzyme through exposure to hydrogen peroxide (H2O2), gas bubble formation was observed immediately upon mixing the bacterial culture with 3% hydrogen peroxide (H2O2), indicating that the bacterial isolates were catalase-positive, as evidenced by the decomposition of H2O2 into water and oxygen gas (Fig 2G). Bubble formation was observed in test tubes, indicating that the Xap isolates were able to breakdown H2O2 to release oxygen gas and water. These results were consistent with the findings reported by (Li et al., 2015; Roach et al., 2018).
       
The H2S production test showed that filter paper changed to a black color indicating that isolates were able to produce H2S from cysteine. In other words, after four days of incubation at 28oC on lead acetate strips that were suspended over the inoculated tubes and held by a screw cap, a black lead sulfide discoloration was observed. However, no black lead sulfide was discoloration and observed on the negative control tube. Likewise, a negative result was observed for the negative reference culture (522-P).
       
Bacterial isolates grown on starch-rich medium (nutrient agar supplemented with starch) were flooded with iodine-potassium iodide (IKI) solution after seven days of incubation. The presence of clear zones around bacterial colonies indicated positive starch hydrolysis, demonstrating the production of amylase enzymes that cleaved starch into smaller sugar molecules, which do not form the characteristic blue-black complex with iodine (Table 1). However, there was no significant bright area observed in the reference culture 522-P- 522-P- isolate of P. s. pv. phaseolicola that was used as negative control.
       
Except for the negative reference culture (522-P), all bacterial isolates inoculated in esculin medium tubes exhibited a dark brown coloration, indicating positive esculin hydrolysis (S2). The color change became evident after five days of incubation at 28oC, suggesting the presence of esculinase enzyme activity in these isolates. Schaad et al., (2001) reported that esculin was cleaved by all X. axonopodis pathovars producing glucose and a dark brown compound (dihydrocoumarin). This showed a positive reaction of bacterial isolates due to esculin hydrolysis. All Xap isolates were grown on KB medium and observed under UV light under dark conditions. The isolates exhibited non-fluorescent pigmentation. In contrast, the reference culture (522-P) produced green fluorescent pigment when grown on KB medium. 
 
Pathogenicity and hypersensitivity reaction (HR)
 
The pathogenic and non-pathogenic nature of bacterial isolates was confirmed by the reaction of the hypersen-sitivity of tobacco. The bacterial suspension was prepared at a concentration of 108 CFU ml-1 in the spectrometer and then injected into the intercellular area of the tobacco leaf with a syringe. After 24-48 h inoculation, bacterial isolates caused a typical susceptibility of necrosis reaction on tobacco leaves and considered as a positive result (Fig 3 A-C). This test can only be induced by pathogenic bacteria, which is manifested by tissue collapse and necrosis within 24-48 h (Benchouikh et al., 2016; Lelliot, 1987).

Fig 3: Hypersensitivity reaction and typical symptoms of Xap on representative plants.


       
Like HR, the Xap isolates that induced hypersensitive reaction (HR) response on tobacco, caused typical CBB symptoms on susceptible bean variety Aras 98 after 7 - 10 days of inoculation (Fig 3D-F). After 7 days, symptoms in the leaves started to form irregular chlorotic spots and later these spots necrotized with a thin narrow lemon-yellow halo and a burnt appearance.
 
Molecular diagnosis tests
 
After DNA isolation using the Qiagen kit and fully Automatic Isolation Robot (Qiacube) the obtained genomic DNA was run on a 1% agarose gel and the bands obtained are indicated in Supplementary Fig (S1, A and B). As a result of PCR tests performed using X4c and X4e specific primers with the genomic DNAs; all tested strains/isolates formed a band of 730 bp that is the approximate amplicon size of X. axonopodis pv. phaseoli (S1, B). Similarly, (Popović et al., 2019) isolated the Xap pathogen from seeds of 23 bean cultivars and made the diagnosis using X4c and X4e primers that amplified a 730 bp target DNA from all the X. axonopodis pv. phaseoli strains tested. Bastas and Sahin (2017) also identified 61 Xap strains from the Central Anatolia region, Turkey. They concluded that an amplicon of 730 bp was obtained for all Xap strains by X4c and X4e primer sets. Also, using the same specific primer, Tebaldi et al., (2010) obtained same findings and reached a similar conclusion.

Supplementary Fig 1: Bacterial DNA on 1% agarose gel.


       
Based on plasmid DNA profile determination, all the identified Xap isolates were found to contain plasmids of the same size (Supplimentary Fig 2A). In other words, there was no difference in plasmid size identified in each Xap isolate. This indicated that Xap isolates collected from the same agroecology zone may not have any difference in their plasmid size. All the tested Xap isolates contained a single plasmid. In contrast, Carvalho et al., (2005) characterized the plasmid profile of X. axonopodis pv. citri and found that plasmids were observed in all the 22 tested strains with sizes between 57.7 and 83.0 kb. In their result, the 72.6 kb plasmid was the most frequent one and it was present in 15 out of 22 strains. The presence of plasmids indicated their importance in the genome of Xap, considering that within each of the strains sampled in the present study at least one extrachromosomal element was detected. In this study, the plasmid size difference found in the Xap strains studied was not great, which was contrary to the findings of Carvalho et al., (2005) who demonstrated that plasmid DNA in X. axonopodis pv. citri was highly conservative. According to Smalla et al., (2015) plasmids play an important role for rapid adaptation of bacterial populations to changing environmental conditions. Different researchers have confirmed that several virulence and avirulence factors are associated with genes present in plasmids such as A. rhizogenes, P. savastanoi and A. tumefaciens (Comai and Kosuge, 1980; Coplin, 1982; Nester and Kosuge, 1981). Factors related to the ecological adaptability of bacteria are thought also to be present in plasmids (Coplin, 1982).

Supplementary Fig 2: Biochemical reaction tests for Xap isolates.


               
The combination of classic and molecular methods is the most reliable way for the detection and identification of phytopathogenic bacteria. In our study, the diagnosis of isolates identified as Xap by biochemical tests was supported by using specific primers suggested by different researchers. Audy et al., (1994) reported that a higher level of sensitivity was achieved by a PCR-based assay with specific primer pair X4c and X4e that was specific for Xap. There is a need for further studies to better understand and evaluate the plasmid DNA relationship of the X. axonopodis pv. phaseoli strains present in Turkey and to compare them with strains that occur in other countries. In addition, further research should be conducted to investigate the pathogenic nature of the different Xap strains with different plasmid DNA profiles by inoculating them on susceptible bean cultivars. 

CONCLUSION

The combination of biochemical methods and modern molecular techniques proved effective in providing a comprehensive understanding of the identity of common bacterial blight of beans. The findings of this study lay the groundwork for future investigations into the pathogenicity and plasmid DNA relationships of Xap strains in different geographical locations and under varying environmental conditions. Further research is needed to develop effective management strategies against common bacterial blight of beans.

CONFLICT OF INTEREST

The authors declare they have no conflict of interest.

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    Full Research Article

    Biochemical and Molecular Characterization of Xanthomonas axonopodis Pv Phaseoli of Common Bean

    T
    Tibebu Belete1,*
    S
    Sylvans Ochola1
    K
    Kubilay Kurtulus Bastas2
    D
    Duncan Cheruiyot1
    1Rwanda Institute for Conservation Agriculture (RICA), Bugesera Campus, Bugesera/Rwanda.
    2Department of Plant Protection, Faculty of Agriculture, Selcuk University, Konya/Turkey.

    ABSTRACT

    Background: Common bacterial blight (CBB), caused by Xanthomonas axonopodis pv. phaseoli (Xap) significantly impacts common bean production worldwide. Timely identification and characterization of Xap are critical for disease management and breeding for resistance.

    Methods: The pathogen was isolated from symptomatic leaves, stems and pods of common bean cultivars, followed by morphological, physiological, biochemical and molecular characterization. Pathogenicity and induction of hypersensitivity reactions of the isolates were confirmed on susceptible bean cultivar and tobacco, respectively.

    Result: A total of 35 Xap isolates were Gram-negative, oxidase-negative, catalase-positive and tested positive for levan and H2S production, esculin hydrolysis, acid production from carbohydrates and starch hydrolysis and were non-fluorescent on King’s B medium. Pathogenicity and hypersensitivity reactions assays on susceptible bean cultivar and tobacco, respectively, confirmed the virulence of the isolates. PCR amplification using Xap-specific primers yielded the expected 730 bp product for all isolates, confirming their identity. Plasmid profiling revealed all isolates carried plasmids of similar size, suggesting limited plasmid size diversity among strains from the same agroecological zone. The combination of classical, biochemical and molecular methods provided a reliable strategy for Xap identification, laying a foundation for further studies on identification of Xap strains.

    INTRODUCTION

    Plant disease caused by Xanthomonas axonopodis pv. Phaseoli (Xap) has been a persistent challenge for bean farmers globally. The pathogen infects the plant through natural openings or wounds, triggering symptoms such as leaf spots, wilting and necrosis to cause common bacteria blight (CBB) disease (Belete et al., 2022). Early and accurate detection of Xap is crucial for implementing timely control measures and preventing the spread of the disease. Biochemical techniques have long been employed for bacterial identification, relying on the analysis of physiological and metabolic characteristics (Doddaraju et al., 2019; Lamichhane and Varvaro, 2014; Roach et al., 2018). These methods, including the determination of enzymatic activities and substrate utilization patterns, have proven effective in differentiating bacterial species and strains. Moreover, they serve as valuable tools for initial screening and classification. Several studies have successfully utilized biochemical methods for the identification of Xanthomonas species, emphasizing the importance of these techniques in the early stages of pathogen detection (Sarker et al., 2017; Schaad et al., 2001; Singh et al., 2016).
           
    While biochemical techniques provide a foundation for bacterial identification, molecular methods offer a higher level of specificity and sensitivity. Polymerase chain reaction (PCR) and DNA sequencing have revolutionized the field of microbiology, allowing for the precise identification of bacterial pathogens based on their genetic makeup (Adachi and Oku, 2000; Strayer et al., 2016). The use of molecular markers, such as specific DNA sequences, facilitates the differentiation of closely related bacterial strains. Previous studies have successfully employed molecular techniques for species-specific identification and genetic diversity analysis of Xap (Belete et al., 2025; Batista et al., 2021; Boureau et al., 2013; Potnis et al., 2011; Zamani et al., 2011).
           
    Plasmids, extrachromosomal DNA elements in bacteria, play a crucial role in the adaptability and virulence of many plant pathogens. The study of plasmid DNA profiles offers insights into the genetic variability of bacterial populations, providing information on the acquisition and exchange of virulence factors and antimicrobial resistance genes. While the role of plasmids in Xap pathogenicity is not fully understood, their investigation holds promise for uncovering novel targets for disease control. Few studies have explored the plasmid profiles of Xap isolates, highlighting the need for comprehensive research in this area (Alavi et al., 2008). This study employed a combination of biochemical and molecular techniques to identify and characterize genetic diversity Xanthomonas axonopodis pv. Phaseoli.

    MATERIALS AND METHODS

    Sample collection and isolation of the pathogen
     
    Naturally infected common bean leaves, stems and pods showing characteristics CBB symptoms were collected from different bean-growing areas of Cumra and Karatay in Konya province of Turkey (Fig 1, A-C). Forty (40) samples were collected from different local common bean cultivars.          

    Fig 1: Assessment and scouting of common bacterial blight incidence in a bean field.

                    

    The samples were sealed in polyethylene bags and transported to the Molecular Plant Bacteriology Laboratory located at the Department of Plant Protection, Faculty of Agriculture, Selcuk University for isolation of the causative pathogen.
           
    Isolation of the pathogen was done from infected leaves and pods according to Bradbury (Bradbury, 1970) with some modifications. Plates were incubated at 28oC for 48-72 hours and examined for the appearance of bacterial colonies and their colony morphologies and colors were determined. Pure cultures of the colonies were obtained by sub-culturing representative colonies.
     
    Morphological, biochemical and physiological characterization of Xap
     
    All Xap isolates were compared and characterized based on their physiological, biochemical and metabolic properties using standard biochemical tests, such as 3% potassium hydroxide (KOH) test as a gram reaction test, Oxidase Reaction Test, Presence of Catalase, Test of Levan Production, Hydrogen Sulfide (H2S) Production from cysteine, esculin Hydrolysis, Fluorescent Pigment on King’s B medium under UV light, Acid Production from Erythritol and Sorbitol, Tween 80 Hydrolysis and Starch Hydrolysis (Schaad et al., 2001) (Table 1). Each test was conducted in triplicate for each strain and repeated three times.

    Table 1: Biochemical and physiological characteristics of Xap Isolates.



    Pathogenicity and hypersensitivity reaction (HR) test
     
    Susceptible common bean cultivar (Aras 98) was grown in a controlled greenhouse as a pot experiment. When the bean plants reached 16-20 days old after planting, all Xap isolates were streaked onto plates of NA and incubated for 48 hours at 28oC for inoculum preparation. Bacterial cells were suspended in SDW and adjusted to an optical density at 600 nm = 0.5, which corresponds to ≈ 108 CFU/ml (Mkandawire et al., 2004). The first fully expanded trifoliate leaves of the susceptible bean plants were sprayed using a hand-held sprayer and the pots were covered by polyethylene bags for maximum humidity for bacterial development and later removed after three days. SDW was also spread onto leaves of bean plants serving as a negative control. At the same time, the bean plants were inoculated by the known source of Xap (145-X) isolate as a positive control. The experiment was arranged in a completely randomized design (CRD) and replicated three times. Inoculated plants were examined daily for the development of symptoms and observations were made and recorded at seven days post inoculation. Bacteria with the same characteristics as those inoculated were re-isolated from the leaves showing symptoms and characterized accordingly.
           
    For HR test, bacteria colonies were suspended in sterile distilled water and adjusted using a bio-photometer to give a concentration of approximately 108 CFU/ml (0.15 absorbance at 660 nm). A bacterial inoculum of 1.5-2 ml of each culture suspension was injected into the expanded leaves of a two-month-old tobacco plant (Nicotiana tabacum, cv. White Burley) using a sterile hypodermic syringe. Tobacco leaves were similarly injected with sterile distilled water (SDW) used as a negative control. All the tobacco plants were kept in the green-house for approximately 24h at 25-28oC until symptoms developed (Schaad et al., 2001).
     
    DNA extraction and PCR assay
     
    The DNA of all Xap isolates were isolated using the Qiagen DNA isolation kit and fully Automatic Isolation Robot (Qiacube, Qiagen) according to the manufacturer’s recommendation. The quantity and quality of the extracted DNA were determined using spectrophotometer (NanodropND-1000) and by running 10µL (10µL+ loading dye) on 1% agarose gel, respectively (Sambrook and Rusell, 2001). PCR amplifications were conducted with DNA extracted from pure bacterial cultures. PCR reactions were prepared in 0.2 ml Eppendorf tubes.
                   
    To identify representative strains of Xap, polymerase chain reaction (PCR) was performed with the Xap-specific primer pair, X4c (5´-GGC AAC ACC CGA TCC CTA AAC AGG -3´) and X4e (5´-CGC CCG GAA GCA CGA TCC TCG AAG -3´) (Audy et al., 1994), which directs the amplification of the 730 bp DNA fragment. The PCR was done under the following conditions: initial denaturation at 94oC for 3 minutes (single-step reaction), followed by 30 repeated cycles of melting, annealing and DNA extension at 94oC for 1 minute, 65oC for 1 minute and 72oC for 2 minutes, followed with a single-step final extension reaction for 5 minutes at 72oC. The amplified DNA fragments were electrophoresed in 1% agarose gel in 1 × TBE buffer and visualized with UV light after ethidium bromide staining. A 1 kb molecular marker (marker, Fermantas 1 kb Plus DNA Ladder SM 1153) was used to determine the molecular weights of the resulting bands. To characterize the different Xap isolates based on their plasmid DNA profiles, plasmid DNA was extracted using Mini-prep method (Carvalho et al., 2005) with minor modifications. The plasmid DNA was analyzed on 0.8% agarose gel and a 1000 bp molecular marker used to determine and characterize the plasmid DNA size.

    RESULTS AND DISCUSSION

    Biochemical and physiological characterization
     
    Initial classification and identification of bacterial isolates was carried out based on colony morphology and color on general and semi-selective media. Incubation of the bacterial isolates for  48 h at 28oC, resulted in round/domed, convex, mucoid and yellow colonies on NA (Fig 2, A and B). The morphological appearances of the colonies were characteristic of Xanthomonads and were similar to those previously described by (Schaad et al., 2001). Based on the characteristic colony morphology, 35 Xap representative isolates subjected to different determinative biochemical test for bacteria according to (Schaad et al., 2001).                

    Fig 2: Isolation and biochemical test of Xap.

                        

    The isolates were subjected to Gram reaction, oxidase reaction, catalase activity, levan production, H2S production, esculin hydrolysis, fluorescent pigment production on King’s B medium, acid production from carbohydrates and starch hydrolysis tests (Table 1). The levan production test resulted in translucent shining mucoid colonies with a distinctive raised convex appearance on NA medium containing 5% sucrose after incubation at 28oC (Fig 1C). The results of this study were consistent with previous research (Lelliot, 1987) that levan was produced from the fructose moiety of the sucrose molecule and its formation was usually responsible for the production of mucoid colonies by some species of bacteria.
           
    The addition of 3% KOH to bacterial isolate colonies in suspension resulted in the formation of viscous, thread-like mucus when lifted with an inoculating loop (Fig 2D). This is the confirmatory test and positive reaction for Gram-negative bacteria (Suslow et al., 1982). The peptidoglycan layer in the cell wall of Gram-negative bacteria is monolayer and does not contain teichoic acid and can easily be broken down with KOH. As a result, the cytoplasmic fluid becomes free and a viscous elongation can be observed. However, in Gram-positive bacteria, no viscous elongation was observed when the mixture of KOH and bacterial colonies were lifted by a loop while remaining as an aqueous liquid (Schaad et al., 2001). Vauterin et al., (1990) reported that genus Xanthomonas pathogens are Gram-negative, aerobic, rod-shaped, with a single polar flagellum, catalase-positive, oxidase negative, levan positive, HR-positive, mucoid, convex and formed yellow colonies on yeast dextrose carbonate agar (YDCA) medium.
           
    When the isolates were subjected to oxidase reaction test, no dark-purple reaction was observed on the impregnated filter paper (Fig 2E). Within a few seconds, the disks became dark-purple when the strain was oxidase positive and those which did not change color within 60 sec were evaluated as oxidase negative (Fig 2F). The Xap isolates exhibited a negative oxidase reaction, which is consistent with the typical biochemical profile of Xanthomonas species (Kovacs, 1956). The observed dark/bluish-purple color indicated that the enzyme reacted with the chemical encoded on the disc and that the bacterium contains cytochrome protein. In other words, the bacterial isolates were discolored by bacterial coating on discs containing 1% tetramethyl-p-phenylenediamine dihydrochloride.  The oxidase reaction test was used to distinguish the isolates that produced cytochrome c protein in the bacteria electron transport chain. The enzyme cytochrome c oxidase is used in the electron transport system to reduce substances and facilitate the formation of cellular energy (ATP) during cellular respiration.
           
    When the Xap isolates were tested for presence of catalase enzyme through exposure to hydrogen peroxide (H2O2), gas bubble formation was observed immediately upon mixing the bacterial culture with 3% hydrogen peroxide (H2O2), indicating that the bacterial isolates were catalase-positive, as evidenced by the decomposition of H2O2 into water and oxygen gas (Fig 2G). Bubble formation was observed in test tubes, indicating that the Xap isolates were able to breakdown H2O2 to release oxygen gas and water. These results were consistent with the findings reported by (Li et al., 2015; Roach et al., 2018).
           
    The H2S production test showed that filter paper changed to a black color indicating that isolates were able to produce H2S from cysteine. In other words, after four days of incubation at 28oC on lead acetate strips that were suspended over the inoculated tubes and held by a screw cap, a black lead sulfide discoloration was observed. However, no black lead sulfide was discoloration and observed on the negative control tube. Likewise, a negative result was observed for the negative reference culture (522-P).
           
    Bacterial isolates grown on starch-rich medium (nutrient agar supplemented with starch) were flooded with iodine-potassium iodide (IKI) solution after seven days of incubation. The presence of clear zones around bacterial colonies indicated positive starch hydrolysis, demonstrating the production of amylase enzymes that cleaved starch into smaller sugar molecules, which do not form the characteristic blue-black complex with iodine (Table 1). However, there was no significant bright area observed in the reference culture 522-P- 522-P- isolate of P. s. pv. phaseolicola that was used as negative control.
           
    Except for the negative reference culture (522-P), all bacterial isolates inoculated in esculin medium tubes exhibited a dark brown coloration, indicating positive esculin hydrolysis (S2). The color change became evident after five days of incubation at 28oC, suggesting the presence of esculinase enzyme activity in these isolates. Schaad et al., (2001) reported that esculin was cleaved by all X. axonopodis pathovars producing glucose and a dark brown compound (dihydrocoumarin). This showed a positive reaction of bacterial isolates due to esculin hydrolysis. All Xap isolates were grown on KB medium and observed under UV light under dark conditions. The isolates exhibited non-fluorescent pigmentation. In contrast, the reference culture (522-P) produced green fluorescent pigment when grown on KB medium. 
     
    Pathogenicity and hypersensitivity reaction (HR)
     
    The pathogenic and non-pathogenic nature of bacterial isolates was confirmed by the reaction of the hypersen-sitivity of tobacco. The bacterial suspension was prepared at a concentration of 108 CFU ml-1 in the spectrometer and then injected into the intercellular area of the tobacco leaf with a syringe. After 24-48 h inoculation, bacterial isolates caused a typical susceptibility of necrosis reaction on tobacco leaves and considered as a positive result (Fig 3 A-C). This test can only be induced by pathogenic bacteria, which is manifested by tissue collapse and necrosis within 24-48 h (Benchouikh et al., 2016; Lelliot, 1987).

    Fig 3: Hypersensitivity reaction and typical symptoms of Xap on representative plants.


           
    Like HR, the Xap isolates that induced hypersensitive reaction (HR) response on tobacco, caused typical CBB symptoms on susceptible bean variety Aras 98 after 7 - 10 days of inoculation (Fig 3D-F). After 7 days, symptoms in the leaves started to form irregular chlorotic spots and later these spots necrotized with a thin narrow lemon-yellow halo and a burnt appearance.
     
    Molecular diagnosis tests
     
    After DNA isolation using the Qiagen kit and fully Automatic Isolation Robot (Qiacube) the obtained genomic DNA was run on a 1% agarose gel and the bands obtained are indicated in Supplementary Fig (S1, A and B). As a result of PCR tests performed using X4c and X4e specific primers with the genomic DNAs; all tested strains/isolates formed a band of 730 bp that is the approximate amplicon size of X. axonopodis pv. phaseoli (S1, B). Similarly, (Popović et al., 2019) isolated the Xap pathogen from seeds of 23 bean cultivars and made the diagnosis using X4c and X4e primers that amplified a 730 bp target DNA from all the X. axonopodis pv. phaseoli strains tested. Bastas and Sahin (2017) also identified 61 Xap strains from the Central Anatolia region, Turkey. They concluded that an amplicon of 730 bp was obtained for all Xap strains by X4c and X4e primer sets. Also, using the same specific primer, Tebaldi et al., (2010) obtained same findings and reached a similar conclusion.

    Supplementary Fig 1: Bacterial DNA on 1% agarose gel.


           
    Based on plasmid DNA profile determination, all the identified Xap isolates were found to contain plasmids of the same size (Supplimentary Fig 2A). In other words, there was no difference in plasmid size identified in each Xap isolate. This indicated that Xap isolates collected from the same agroecology zone may not have any difference in their plasmid size. All the tested Xap isolates contained a single plasmid. In contrast, Carvalho et al., (2005) characterized the plasmid profile of X. axonopodis pv. citri and found that plasmids were observed in all the 22 tested strains with sizes between 57.7 and 83.0 kb. In their result, the 72.6 kb plasmid was the most frequent one and it was present in 15 out of 22 strains. The presence of plasmids indicated their importance in the genome of Xap, considering that within each of the strains sampled in the present study at least one extrachromosomal element was detected. In this study, the plasmid size difference found in the Xap strains studied was not great, which was contrary to the findings of Carvalho et al., (2005) who demonstrated that plasmid DNA in X. axonopodis pv. citri was highly conservative. According to Smalla et al., (2015) plasmids play an important role for rapid adaptation of bacterial populations to changing environmental conditions. Different researchers have confirmed that several virulence and avirulence factors are associated with genes present in plasmids such as A. rhizogenes, P. savastanoi and A. tumefaciens (Comai and Kosuge, 1980; Coplin, 1982; Nester and Kosuge, 1981). Factors related to the ecological adaptability of bacteria are thought also to be present in plasmids (Coplin, 1982).

    Supplementary Fig 2: Biochemical reaction tests for Xap isolates.


                   
    The combination of classic and molecular methods is the most reliable way for the detection and identification of phytopathogenic bacteria. In our study, the diagnosis of isolates identified as Xap by biochemical tests was supported by using specific primers suggested by different researchers. Audy et al., (1994) reported that a higher level of sensitivity was achieved by a PCR-based assay with specific primer pair X4c and X4e that was specific for Xap. There is a need for further studies to better understand and evaluate the plasmid DNA relationship of the X. axonopodis pv. phaseoli strains present in Turkey and to compare them with strains that occur in other countries. In addition, further research should be conducted to investigate the pathogenic nature of the different Xap strains with different plasmid DNA profiles by inoculating them on susceptible bean cultivars. 

    CONCLUSION

    The combination of biochemical methods and modern molecular techniques proved effective in providing a comprehensive understanding of the identity of common bacterial blight of beans. The findings of this study lay the groundwork for future investigations into the pathogenicity and plasmid DNA relationships of Xap strains in different geographical locations and under varying environmental conditions. Further research is needed to develop effective management strategies against common bacterial blight of beans.

    CONFLICT OF INTEREST

    The authors declare they have no conflict of interest.

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