L. asiaticus’ strains from China and Florida. check details amplicon profiles on agarose gel were designated as electrophoretic types or E-types. E-type frequencies were summarized and Chi-square test was used to determine the significance of E-type differences at different geographical locations. DNA sequencing and BAY 11-7082 supplier analysis DNA bands were excised from the gel and purified using QIAquick Gel Extraction kit (Qiagen, Valencia, CA). Purified DNAs were cloned with pGEM T-easy vector (Promega Corp. Fitchburg, WI) and sequenced using BigDye

Terminator v3.1 Cycle Sequencing Kit in a 3130 × 1 Genetic Analyzer (Applied Biosystems, Inc.). Multiple sequence alignments were performed using ClustalW (Ver.1.74) program with the default parameters [22]. Manual adjustment was performed when appropriate. Protein secondary structure prediction was performed by the method of Bryson et al. [23] available in PSIPRED server http://​bioinf.​cs.​ucl.​ac.​uk/​psipred/​. The protein 3-D structure model was built based on a fold prediction protocol with the help of Phyre [24]. Nucleotide sequence accession numbers Nine DNA sequences of ‘Ca. L. asiaticus’ representing

different amplicon sizes and collection origins have been deposited in GenBank with accession numbers JF412691 to JF412699 (Additional file 2). Results Detection of DNA mosaicisms by primer set Lap5640f/Lap5650r A total of 262 HLB samples detected positive AZD8931 nmr with primer set OI1/OI2c [4] and ITSAf/ITSAr [19] were analyzed. Among them, 188 samples were from nine provinces in China and 74 samples were from

Florida (Table 1). The geographical origins of HLB samples in China were from locations of both high altitude region (HAR) and low altitude region (LAR) (Figure 1). PCR amplification with primer set Lap5640f/Lap5650r produced eight E-types, designated as E-type A to H. Each E-type was composed of one or more of five DNA amplicons, designated as P1 Cepharanthine to P5 (Figure 2). DNA polymorphisms were not detected with the other 14 primer sets listed in Additional file 1 (data not shown), i.e. each of the 14 primer sets generated a single amplicon. Figure 2 Electrophoretic profiles (E-types) of representative ‘ Candidatus Liberibacter asiaticus’ strains from PCR amplification with primer set Lap5650f/Lap5650r. Lane M on the left is molecular markers. Size unique amplicons are labeled by numbers and designated through P1-P5 with sequence lengths indicated on the right. The 797 bp calculated amplicon in the genome of ‘Ca. L. asiaticus’ strain psy62 placed the strain to E-type C (Figure 2, Table 1). Surprisingly, E-type C was found in 3 out of the 74 Florida HLB samples (4.1%). Other E-types detected in Florida were A, G, and H. E-type G was predominant (82.4%) followed by E-type A (10.4%) and E-type H (4.1%) (Table 1). Six E-types (A, B, C, D, E, and F) were found in the 188 samples from China (Figure 2, Table 1).