2.1 Plant MaterialsIn the present study, symptomatic HLB diseased leaves were collected from Pabna district of Bangladesh and symptoms were identified by Bangladesh Council of Scientific and Industrial Research (BCSIR), Binodpur, Rajshahi, Bangladesh on the basis of diseased symptoms. Symptomatic leaves were also confirmed by the Bangladesh Fruit Research Institute, Regional Office, Binodpur, Rajshahi. Citrus Huanglongbing disease-infected leaves were used as plant material. 2.2 Isolation of Candidatus Liberibacterasiaticusof Citrus Huanglongbing DiseaseCitrus Huanglongbing disease-infected leaves were washed by distilled water and then disinfected using dilute sodium hypochlorite solution (10%) and rinsed thoroughly. Then the infected area was cut and placed on Luria and Bertani (LB) liquid media [18] which is composed of 1g peptone, 0.5g yeast extract and 1g NaCl per 100 ml. Then incubated at 37°C for 12-16h.After the bacteria had grown into LB liquid medium, bacteria were streaked onto a solid nutrient agar plate and incubated at 37°C for 12-16h. The single colony was picked by wire loop and streaked on another media plate for pure culture.2.3 Biochemical Characterization of Candidatus LiberibacterasiaticusA series of biochemical tests were performed for the proper biochemical characterization of candidates Liberibacterasiaticus.All the morphological, physiological and biochemical tests including gram staining test were done by standard microbiological techniques according to Bergey et al. [19]. Potassium hydroxide (KOH) solubility test is special test for gram negative bacteria. For the KOH solubility test, bacteria were aseptically removed from petridishes with an inoculating wire loop, mixed with 3% KOH solution on a clean slide for 1 min and observed for the formation of a thread-like mass. KOH test was performed according to Halebian et al. SIM medium is a combination of differential medium that tests three different parameters, which are represented by the three letters in the name Sulfur Reduction, Indole production and Motility (SIM). Using a needle, strains were introduced into test tubes containing SIM medium and were incubated at room temperature until the growth was evident according to Kirsop and Doyle. Turbidity away from the line of inoculation was a positive indicator of motility. Indole production from tryptophan was tested using the method of Clarke and Cowan. We used SIM medium manufactured by High Media Lab. Pvt. Ltd. from India. 36.23gm medium were suspended in 1L distilled water following the manufacturer’s instructions. Catalase test determine the ability of bacteria to produce catalase enzyme. The catalase test was done by adding the H202(3% v/v) to a bacterial culture and the presence of catalase was indicated by bubbles of free oxygen gas. The citrate test was done according to Simmons method. It was performed to determine the ability of organisms to utilize carbon as energy sources. For Kovac oxidase test, a loopful inoculum from pure culture was picked up by sterilized loop. The inoculum was smeared over the area of filter paper containing oxidase reagent to develop deep blue or purple color within ten seconds indicating the oxidation of the reagent. MacConkey agar test was performed for isolation of gram-negative enteric bacteria and the differentiation of lactose fermenting from lactose non-fermenting gram negative bacteria. MacConkey agar was inoculated with bacteria using streak plate technique and incubated the test tubes at 37°C for 16h. Bacteria were inoculated into the MR broth medium in test tubes for methyl red test. Then Test tubes were incubated at 37°C for 16h. After incubation 2-3 drops of Methyl red reagent was added into bacterial suspension. KIA medium was prepared by using a usable amount in 1 liter distilled water and sterilized at 121°C for 20 minutes. The tube cooled in a slanted position to obtain a butt of 1.5-2.0 cm. The 24h of old culture of each isolate were stabbing the butt and streaking the surface of the tube. Tubes were incubated aerobically at 37°C for 16h. Production of H2S was observed after 7 day incubation at 28°C in triple iron salts agar.2.4Isolation and Purification of Candidatus LiberibacterasiaticusGenomic DNAFor the isolation of bacterial genomic DNA, a single colony of Candidatus Liberibacter asiaticuswas cultured in LB liquid medium at 37°C for 16h. The culture was then taken in an Eppendorf tube and centrifuged and the liquid was discarded from the upper portion of the tube. The total genomic DNA were isolated from bacterial mass by heat lysis and selective precipitation of cell debris and polysaccharides with CTAB (Cetyltrimethyl Ammonium-Bromide) and the procedure was done according to Ausbel et al. The DNA was then re-suspended in TE buffer and quantified using a spectrophotometer then electrophoresed on 1% agar gel by comparison with DNA samples of known concentration.2.5PCR Analysis of Candidatus Liberibacter asiaticusGenomic DNAThe amplification of 16S rDNA gene from the Candidatus Liberibacterasiaticusgenomic DNA was done by PCR reaction in a thermo cycler (Nyx, Technic, Inc., USA), using the primers 27F (5′-AGAGTTTGATCCTGGCTC-3′) and 1391R (5′-GACGGCGGTGTGTRCA-3′). PCR was done in total volumes of 25μl, containing nuclease free ddH2O 15μl, dNTP mix 1.0μl, forward primer 1.0μl, reverse primer 1.0μl, DNA template 1.5μl, MgCl22.5μl, Taqbuffer B 2.5μl and Taq polymerase (Takara, Japan) 0.5μl. The procedure for PCR analysis of Candidatus Liberibacterasiaticusgenomic DNA was as follows: initial denaturation at 95°C for 5min; 35 cycles of denaturation for 40s at 95°C, annealing for 1min at 65°C, and extension for 2min at 72°C; the final extension at 72°C for 10 min, followed by cooling to 4°C until the sample was recovered. Gel electrophoresis was used to visualize the PCR products lengths. 0.5x TBE buffer was used in agar gel and visualized under a UV transilluminator. DNA was purified by agar gel electrophoresis method using AccuPrep® Gel Purification, Bioneer kits.2.6Sequencing and Phylogenetic AnalysisofCandidatus Liberibacterasiaticus16S rDNAGenomic DNA was isolated from the Candidatus Liberibacterasiaticusand purified. Then the purified products were sequenced in a sequencing service laboratory, Invent Biotechnology Ltd. Dhaka, Bangladesh. Two samples were sequenced. Sequencing was performed using Candidatus Liberibacterasiaticus16S rDNA gene specific primers 27F (5′-AGAGTTTGATCCTGGCTC-3′) and 1391R (5′-GACGGCGGTGTGTRCA-3′). All sequences were compared with their related strains using BLASTN program in the GenBank nucleotide database (http://www.ncbi.nlm.nih.gov/BLAST). Phylogenetic trees were constructed at Professor Joarder DNA and Chromosome Research Lab., Bangladesh based on the Maximum Likelihood method.2.7Antibiotics Sensitivity TestAntibiotics sensitivity test was done according to Bauer et al. [29]. We used fifteen different types of antibiotic against Candidatus Liberibacterasiaticus. Candidatus Liberibacterasiaticus was cultured in LB liquid medium and incubated at 37°C for 12-16h with continuous shaking at 150 rpm. LB agar medium was prepared in sterile conical flasks, cooled down to 40°C and placed in 90mm petridish. 20ml of liquid medium was poured in each petridish and left the airflow cabinet for solidification. Commercially available antibiotics disc were placed centrally on agar plate and incubated at 37°C for 12-16h. After incubation, zone of inhibition was measured with help of mm scale. 2.8 Screening of Antimicrobial Activity of Medicinal Plant Extracts against candidates LiberibacterasiaticusScreening of antimicrobial activity of selected medicinal plant extracts was done by moderate disc diffusion technique according to Hossain et al. [30]. We used four different types of medicinal plants which are more or less available in Bangladesh and have antimicrobial and medicinal value. Cassia fistula, Curcuma longa, Cuscuta reflexa and Aloe barbadensis plants were used for antimicrobial screening based on their medicinal value [31,32,33]. We collected these medicinal plants from the Botanical garden, University of Rajshahi. We used ethanol and methanol as solvent for the preparation of plant extracts. The collected plant materials were washed, air-dried and grinded in a fine powder by a grinding machine. 20g of dried powder from each plant was soaked in 200ml ethanol and methanol, respectively in round bottom flask at room temperature for seven days with occasional shaking. The extracts were filtered by cotton white cloth followed by Whatman No.41 (Whatman, UK) filter paper. The filtrate was evaporated at 45°C to dryness and the dried substance was kept in sterile bottle under refrigerated condition until use. An inoculum suspension was swabbed uniformly to solidify 20mL LB agar media for bacteria and the inoculum was allowed to dry for 5 minutes. 6mm diameter paper discs were used. Extracts from each solvent were used at 20μg/ml, 40μg/ml and 60μg/ml concentrations and added onto each disc on the seeded medium and allowed to stand on the bench for 1 hour for proper diffusion and thereafter incubated at 37°C for 24h. The resulting diameter of zone of inhibition was measured by mm scale.
2.9Statistical AnalysisAll experiments were performed at least three times. The data were expressed as mean and standard error (Mean±SE) and analyzed by one-way analysis of variance (ANOVA). P<0.05 was considered statistically significant. The data were analyzed using Microsoft Excel 2010 software.