Molecular identification and pathogenicity of Ralstonia solanacearum isolates collected from north west of Pakistan

In the tomato commercial growing districts of Khyber Pakhtunkhwa (KP); a province in the north west of Pakistan, multiple comprehensive surveys were conducted during 2012. The main objectives of the current study were to identify the Ralstonia solanacearum ( R. solanacearum ) isolate through its colony characteristics, molecular tools; and to investigate the ability of this pathogen to cause Bacterial wilt (BW) disease, when being inoculated into tomato plant using different inoculation methods. For this purpose, a total of 74 locations covering all over the KP were visited for the presence of tomato plants with BW disease, caused by R. solanacearum . The bacterial pathogen was isolated from diseased plant tissues by growing it on the selective 2,3,5-triphenyl-tetrazolium chloride (TTC) medium. Based on colony morphology of R. solanacearum on the agar plates; and pathogenicity assays, about 29 isolates were guessed to be R. solanacearum . To further confirm the identity of these isolates, a species-specific primers-mediated Polymerase chain reaction (PCR) was carried out. Two specific primers i.e. forward primer: 5'GTCGCCGTCAACTCACTTTCC3', and reverse primer: 5'GTCGCCGTAGCAATGCGGAATCG3', were used for amplification of the 281bp band. Twenty five isolates out of the 29 were genetically confirmed to be R. solanacearum based on their amplified 281bp band.


Introduction
Tomato (Solanum lycopersicum L.) is one of the most important vegetable crops of the world, and is a member of the Solanaceae family (also known as night shade family). There were several important diseases Novel Research in Microbiology Journal affecting this well-known and valuable crop. Among which; tomato BW caused by R. solanacearum (Smith) was considered to be one of the most destructive diseases, due to its economic impact. As a Gram-negative rod, R. solanacearum; expressed a major threat to the production of tomatoes, potatoes, tobacco and other solanaceous crops, in both tropics and temperate zones (Yuliar et al., 2015;Aloyce et al., 2017). Up to 30% of annual losses in tomatoes and potatoes crops were common in Pakistan (Junaid et al., 2018). This pathogenic bacterium has a very wide host range, as more than 200 plant species were attacked by this pathogen.
R. solanacearum is a species complex consisting of groups; races, biovars, bio-types, sub-races and strains. Detection and identification of this bacterial pathogen was routinely carried out on the basis of; biochemical and physiological assays (Alvarez et al., 2008;Marco-Noales, et al., 2008;Rahman et al., 2010) such as Biolog Automated Microplate System (Gebreel et al;, 2000), serology using species-specific anti-bodies (Tawfik et al., 2008), and molecular techniques (Fegan and Prior., 2005;Abdurahman et al., 2017). Some assays were done to identify the pathogen above or at genus level such as; Gram staining, KOH solubility test, Catalase test, Oxidase test, Production of fluorescent pigment on iron-deficient medium, Lipase activity, and Levan production from sucrose, etc. (Chaudhry and Rashid, 2011;Pawaskar et al., 2014;Shahbaz et al., 2015). This pathogen could be detected in latent infections and identified using serological methods such as; Enzyme-linked immunosorbent assay ELISA, and Double antibody sandwich DAS-ELISA (Priou et al., 2010). However, such methods were limiting due to their lack of specificity and sensitivity. PCR was better than serology in terms of detection and identification of bacterial pathogens including R. solanacearum. Effective management of BW disease could be only possible if the pathogen was detected and then identified accurately. The current study focuses on the pathogenicity and molecular identification of R. solanacearum.
There exists a lot of controversy regarding the identification of R. solanacearum strains in the various parts of the world. In Pakistan; however, scant information is available about the presence of this pathogen. Accurate identification of a local pathogen and knowledge about its ability to cause a specific disease is very important in developing a successful integrated disease management programme. One of the purposes of the present investigation was to identify pathogenic R. solanacerum through different techniques including colony characteristics and molecular tools. Moreover, the ability of the pathogen to cause the BW was verified using two different inoculation assays.

Survey and sampling
Tomato growing districts of the Khyber Pakhtunkhwa (KP) province form north west of Pakistan were surveyed throughout the growing period (i.e. April-August, 2012) of the crop. A total of 74 (representing almost all tomato growing areas of KP, Table 1; Fig. 1.) samples were uprooted, collected in paper bags, kept cool, brought to the Clinical Plant Pathology Laboratory, and then processed as soon as possible to avoid the chances of contamination. Stems were cut in two cross sections, followed by suspending the tissues in water to observe bacterial streaming (Tahir et al., 2014;Ivey and Lunos, 2015). This method was characteristic for BW and helps in the preliminary identification of bacterial pathogens.

Isolation of R. solanacearum from tomato samples
TTC (2,3,5-triphenyltetrazolium chloride) also known as TZC medium (Champoiseau et al., 2009) was used for the isolation of the pathogen. To one liter of semi-cold (55-60 o C) sterile nutrient agar (NA) Novel Research in Microbiology Journal medium, 5 ml of 1% (w/v) TTC solution was added and mixed well before pouring the medium into plates. Infected stem samples were cut into small pieces; kept in 5-10 ml sterile dist. water for a few minutes, and then crushed using sterilized pistil and mortar to obtain bacterial exudates (ooze). Tissue macerate was left undisturbed for 10-15 min. and then serial dilutions were prepared up to 10 -7 dilution. The last two dilutions i.e. 10 -6 and 10 -7 were streaked on TTC plates to obtain single separate colonies. After 2-3 days incubation at 28-30 o C, typical R. solanacearum colonies (as reported by Chaudhry and Rashid, 2011;Sagar et al., 2013) were selected. Well isolated individual colonies were re-streaked on the TTC medium to purify them. Pure cultures were kept at 4°C for future use.

Identification of R. solanacearum isolates
Bacterial isolates were identified based upon colony morphology on TTC; pathogenicity on host plants, and then confirmed by PCR assay using species-specific primers.

Colony morphology
Colony morphology of R. solanacearum isolates on TTC was studied. Color, shape, growth rate, texture and margins of the R. solanacearum isolates were recorded. These characteristics were then used for the identification of the pathogen. Speculation about virulent and avirulent colonies were guessed as reported by Tahir et al., (2014).

Pathogenicity assay
The objective of the pathogenicity assays was to fulfill Koch's postulates i.e., to make sure that the isolated pathogen was capable of producing wilt disease symptoms on healthy plants similar to those symptoms recorded on the infected tomato samples from which the pathogen was isolated. More than one pathogenicity assays with different inoculation techniques were used to produce typical disease symptoms on healthy tomato host.

Pathogenicity assay I (Root inoculation)
Four weeks old tomato seedlings of susceptible variety (Lyrika) were transplanted into large pots (2 plants/pot), and then inoculated with pure suspension of R. solanacearum culture one week later. For preparation of culture suspension; colonies (24 h old) growing on NA medium were washed off the surface with sterile dist. water, centrifuged and then adjusted to a final concentration of 10 7 cells\ ml, using Photospectrometer (OD=600). Tomato plants were inoculated using two different methods: One set of pots were inoculated by pouring 30 ml of R. solanacearum culture suspension containing 10 7 cells\ ml into the pots around the root zones of the plants. In the other set of pots, the roots (just below the soil surface) of transplants were slightly pierced with sterile needle in order to produce artificial wounds (in mimicry of natural wounds) before transplantation, and then inoculated by pouring R. solanacearum suspension around the root zone immediately after transplanting (Marques et al., 2012). Plants exhibiting BW symptoms were recorded.

Pathogenicity assay II (Leaf inoculation)
In this case, pathogenicity was carried out by inoculating leaves of relatively younger tomato seedlings as reported by Seleim et al., (2014). Pure bacterial culture suspension prepared as stated before. 5-10 susceptible tomato plants at the third true leaf stage were used for inoculation. The leaves were pierced with a sterile needle, and then a sterile cotton swab was used to spread the bacterial inoculum suspension on their surfaces. Another set of plants were also inoculated with the same procedure but without piercing. Inoculated plants were kept at 25-28 o C under high relative humidity conditions for 10 days. Symptoms of wilting; chlorosis, or stunting were noticed. The bacterium was re-isolated from infested plants by taking a stem or petiole sections above the inoculation points; placing them in a small volume of sterile dist. water, and then plated on TTC medium. After incubation, plates were observed for typical R. solanacearum colonies. Identification of colonies were confirmed through amplification of bacterial DNA, using PCR.

PCR-based confirmation of R. solanacearum isolates
To confirm identity of the R. solanacearum isolates based upon colony morphology and pathogenicity assays; PCR was performed using species-specific primers i.e. forward primer: 5'GTCGCCGTCAACTCACTTTCC3' and reverse primer: 5'GTCGCCGTAGCAATGCGGAATCG3', which amplified the 281bp fragment in reference to Popoola et al., (2015). DNA was extracted with the procedure described by Junaid et al., (2018); however, for DNA purification, the Easy-DNA-Extraction-kit (Invitrogen, DeSchelp, the Netherlands) was used. The extracted genomic DNA from each isolate of R. solanacearum was amplified using specific primers. PCR master mixture consisted of 1µl of each primer (0.2 µM), 1µl-of Taq-DNA polymerase, 2.25µl buffer (KCl-50 mM+Tris-HCl-2 mM, pH 8.4), 2µl MgCl 2 (1mM), 2.5µl (100µM, of each dNTP), which were added to 3µl template DNA (1ng) and HPLC water, in order to make the volume of 25µl used in each PCR tube for amplification. A 30-cycles of PCR were performed for all 29 isolates. PCR conditions were: Agarose gel (2%) was used to separate the PCRamplified DNA bands. Blue tracking (3µl) was added to each PCR-product sample (25µl), and then each sample was carefully uploaded to the wells of the gel. 1Kb size DNA ladder was also used for comparison. Electrophoresis was performed at 200 volts for 30 min. To easily visualize the different bands, the gel was Novel Research in Microbiology Journal kept in Ethedium bromide solution (0.5 µg\ ml) for 15 min. The bands were observed under UV light.

Survey and sampling
Surveys conducted in commercial tomato-growing districts of KP revealed that; a total of 74 locations from 26 districts (3-10 fields from each location) were visited, and a total of 74 whole uprooted tomato plants samples exhibiting typical symptoms of BW infection were collected.

Isolation of R. solanacearum from tomato samples
Isolations from apparently infected samples were made on TTC medium. After 48-72 h of incubation at 28-30 o C; 29 samples yielded colonies having typical morphology of R. solanacearum such as; white elevated colonies, mucoid with pinkish to red centers due to reduction of TTC into insoluble red formazan.

Colony morphology
All the isolates had large colonies; which were elevated, fluidal and either entirely white or pinkish in color. The virulent colonies were mostly with pinkish to red centers. There were few colonies which were surrounded by bluish border (Table 2).

Pathogenicity assay
The tentative R. solanacearum isolates (29) were checked for their ability to cause wilt disease symptoms on tomato seedling using four different inoculation techniques. All isolates produced disease symptoms on using most of the inoculation methods (Table 3). The type of the inoculation method used was found to be important for the success of this pathogenicity assay. When plants were infested through root inoculation method (without piercing with needle); isolates from Mansehra (MAN1); Buner (BNR1), Lower Dir (DLO2) and Swat (SWT1, SWT2) didn't produce the typical wilt symptoms. Moreover; the pathogen from the tissues of these plants neither recovered on TTC medium, nor confirmed through PCR. However; when pathogenicity assay was performed on leaves of young seedlings (with piercing), all the 29 isolates produced wilting symptoms, and then died. Meanwhile; when seedlings leaves were inoculated without piercing, the only plant that survived was the one inoculated with R. solanacearum isolate from Mansehra (MAN1).

Molecular identification of R. solanacearum isolates
Using species-specific primers, PCR was used to finally confirm the identity of R. solanacearum isolates. The 281bp specific band was amplified from 25 isolates, confirming them to be R. solanacearum (Fig. 2). However, the template DNA of four isolates (i.e. LMT1, MAN1, DUP1 and NSH2) failed to amplify the 281bp band.

Discussion
The selective medium named 2,3,5-triphenyltetrazolium chloride (TTC) (Champoiseau et al., 2009), was used to isolate R. solanacearum from diseased tomato samples. Colonies of the pathogen on this medium were fluidal with pinkish-red centers. Similar results were reported by Rahman et al., (2010);Chaudhry and Rashid (2011);Tahir et al., (2014). Colony characteristics of R. solanacearum were helpful as they differentiated virulent from avirulent isolates. Chaudhry and Rashid, (2011) reported that the colonies of virulent isolates were whitish with pink centers; irregular margin, raised, large in size, and were more fluidal. On the other hand; colonies of avirulent (spontaneous mutant) isolates were deep red with entire margin, and had bluish border.
R. solanacearum generally invades its tomato host through wounds in the roots, colonizes the xylem Novel Research in Microbiology Journal    , 2005). We used two inoculation methods for our pathogenicity assays; root inoculation (pierced verses un-pierced), and leaf inoculation (pierced verses un-pierced), on healthy tomato seedlings. In both assays; the piercing inoculation methods produced better results than the un-pierced one. The reason was that although bacterial pathogens could use natural openings present on plant surfaces for entry into their host tissues; however, they become more successful pathogens if they entered through wounds in the host, as such they will be able to enter the vascular fluid more rapidly. Our previous study on race-determination assay using tobacco plants (Junaid et al., 2018), grouped R. solanacearum isolates into race 1 (92% isolates), and race 3 (8% isolates). Race identification helps in better characterization of R. solanacearum isolates which was a complex species.
Some bacterial strains gave over-lapping results with respect to their colony morphology and pathogenicity tests, which made their identity uncertain. Our all 29 isolates produced typical R. solanacearum like colonies on TTC; however, some isolates (i.e. LMT1, KOH1, MAN1, DUP1 and NSH2) also produced deep red colonies with bluish border which were thus considered as avirulent strains according to Chaudhry and Rashid, (2011). This avirulence might be attributed to spontaneous mutation as reported by Meng et al., (2011);Schroeder et al., (2017). Currently; to confirm the identity of our isolates, we used R. solanacearum specific primers (Popoola et al., 2015), that amplified the specific 281bp fragment of 25 out 29 of our isolates through PCR (Vanitha and Umesha, 2014). The R. solanacearum specific band (i.e, 281bp fragment), could not be amplified when the genomic DNA of the four isolates (i.e., LMT1, MAN1, DUP1 and NSH2) was used as template in the PCR reaction. This indicated that either these isolates might be mutated in their specific gene complementary to the used primers (Meng et al., 2011;Schroeder et al., 2017), or that they were not R. solanacearum. However, it was also possible that they might be other close relatives of R. solanacearum (Coenye et al., 2003).

Conclusion
Our study reported that TTC medium could specifically distinguish virulent isolates of R. solanacearum from avirulent ones. Moreover, the pathogenicity assays could not deceive the researchers as the avirulnt strains of the bacterium did not produce any wilt symptoms. Molecular techniques such as PCR can be considered as the best method for detection or identification of R.