As an intracellular bacterium, ‘Candidatus Liberibacter asiaticus’ (Las) lacks known transposons and IS elements but contains at least two prophages/phages. In this study, we revealed the genetic diversity and population dynamics of this bacterium based on two prophage hyper-variable regions (HVRs) using separate libraries constructed from citrus, periwinkle and psyllid. A total of 9 variants were identified, including 4 abundance types A, B, C, D and 5 rare types E, A1, A2, C1 and C2. The two HVRs, Type A and B, share highly conserved sequences and are localize to the two prophages, FP1 and FP2, respectively. The most abundant type in the psyllid library was Type A (36.71%), followed by Type B (25.17%) and Type C (19.72), but there was no Type D. However, the most abundant type in citrus was Type B (64.24%), followed by Type A (20.14%), C (11.11%) and D (1.39%). More interestingly, the Type A sequence was a very rare group (0.36%) in the periwinkle library. The most abundant type in the periwinkle library was Type B (43.73%) followed by Type C (39.07%) and then Type D (9.32%). Sequence analysis of these variants revealed the variations were due to the recombination and reassortment between two prophages. Conventional PCR results using primers specific to the different types indicated that Type A, B, C and D were present in more than 94.6% of higher titer Las-infected plant hosts; however, only 16.7% of tested psyllids contained D variants, which were very low titer, and Las-infected psyllids possessed higher titer Type A, B and C populations . Typing results for Las-infected citrus field samples indicated that only the Type D population was associated with huanglongbing (HLB) symptoms: high titer of D with typical blotchy mottle and extremely low to no type D with vein yellowing or other atypical HLB symptoms. Our finding that Las population dynamics derive from the prophage/phage activities may lead to a better understanding of how these bacteria evolve and adapt in different ecological niches.

Citrus Huanglongbing (HLB) is associated with the fastidious bacterium,Candidatus Liberibacter, (Las) that is transmitted by a phloem-feeding insect (Citrus Psyllid). An ideal solution to combat citrus HLB is to completely eliminate the bacteria after a single course of the chemotherapy, either active directly on the bacteria or indirectly through induction of host defense compounds. Twenty-seven antimicrobial compounds were screened to test for in vivo activities against HLB bacterium while assessing their phytotoxicity to citrus using the optimized graft-based chemotherapy approach (Zhang et al., 2012). The Las bacterial titers were quantified by qPCR from the leaf samples that were taken at 4-months and 6-months after inoculation, respectively. The Las-infected plants were considered as Las positive with threshold cycle (Ct) values less than 32.0. The efficiency against the HLB bacterium of the tested compound was evaluated by Ct values in the inoculated plants (both scions and rootstocks), scion infected percentage and HLB bacterial transmission percentage. The phytotoxicity was determined by the survival and growth of scions treated by antimicrobial compounds. The clustering results indicated that 27 antimicrobial compounds were divided into 3 groups. The first group including 12 compounds, such as Zineb, was not effective in eliminating the HLB bacteria, with high scion infection (67.9% ± 14.4% on average)， Las transmission percentage (83.4% ± 13.85 on average) as well as the high bacterial titers. The second group of only two compounds was also not effective against the HLB bacterium, but had high phytotoxicity to citrus (less than 40% of the scion survival and 15% of the scion growth). The third group including 13 compounds, such as nicotine, was effective in eliminating the Las bacteria and had no phytotoxicity to citrus. The effective compounds will be further tested in the container-potted plants and in the field.

Citrus Huanglongbing (HLB) is one of the most serious diseases of citrus worldwide. The present study was undertaken to screen antibiotics against Candidatus Liberibacter asiaticus (Las) while simultaneously assessing phytotoxicity to citrus. Twenty-eight antibiotics from ten classes of medical-antibiotics and three agricultural-antibiotics were tested for in vivo activities against HLB bacterium using the previously optimized graft-based chemotherapy method (Zhang et al., 2012). First, samples for DNA extraction were taken at 4 months after inoculation; subsequent samplings were taken at 2 month intervals. The Las-infected plants were considered as Las positive by real-time qPCR with threshold cycle (Ct) values less than 32.0. The efficiency against the HLB bacterium of each compound was evaluated by Ct values in the inoculated plants (both scions and rootstocks), scion infected percentage and HLB bacterial transmission percentage. The phytotoxicity was determined by the survival and growth of scions treated by antibiotics. The results showed that beta-lactam antibiotics (Ampicillin, Pennicillin and Carbenicillin) were highly effective in eliminating the HLB bacteria, with undetectable Las titers in the inoculated plants by qPCR, and had no phytotoxicity to citrus, with more than 75% scion survival. Antibiotics sulfonamide and tetracycline suppressed the HLB bacterium with Ct values of 35.7 on average, less than 30% scion infection and 16.9% Las-transmission percentage. The effectiveness of some antibiotics, such as aminoglycoside and quinolone, depended on their absorptions and permeability throughout the citrus tree. Peptide antibiotics were not effective in eliminating or suppressing Las bacterium with less than 28.0 Ct values by qPCR and higher scion infection percentages and Las transmission rates. Three agric-antibiotics, Actidione, Validoxylamine A and Zhongsenmycin, were also effective in eliminating the HLB bacteria. Antibiotic combinations, such as beta-lactam and aminoglycoside, are suggested as future applications.

The updated PhyloChip^TM G3 were used to explore the differences in the relative abundance and phylogenetic diversity of the bacterial communities associated with HLB-affected citrus plants in the field over a growing season and those treated with antibiotic combinations of AG (Ampicillin at 1000 mg/L and Gentamicin at 100 mg/L) and PS (Penicillin at 1000 mg/L and Streptomycin at 100 mg/L). Both antibiotic treatments resulted in significantly lower Las bacterial titers (Pr<0.05) and their hybridization scores. Of the 50,000+ available operational taxonomic units (OTUs) on PhyloChip™ G3, 7,028 known OTUs in 58 phyla were detected from the field plants, and 7,407 OTUs in 53 phyla from the inoculated potted plants. Proteobacteria was the constantly dominant phylum of bacteria (38.7%~44.1%) vying for prevalence based on the season, followed by Firmicutes (23.5%~29.0%), Actinobacteria (12.4%~16.1%), Bacteroidetes (6.2%~6.6%) and Cyanobacteria (2.3%~3.2%). Circular tree comparing the Las-free and the Las-infected samples indicated that only 17 families present in the Las-free plants, such as Cyanobacteriaceae; but more than 137 families detected in the Las-infected plants, such as Staphylococcaceae and Pseudonocardiaceae. Cyanobacteria are believed to be responsible for introducing oxygen into the atmosphere and fixing nitrogen and phosphorus assimilation for plant growth. Both Staphylococcaceae and Pseudonocardiaceae were recognized as an emerging opportunistic pathogen of plant and animals. When compared to the bacterial populations in the leaves of citrus trees receiving the water control treatment, the Bacteroidete population decreased (Pr<0.05) by 59.6% and 51.8% in the plants receiving AG and PS treatments, respectively. The overall diversity of bacteria also decreased with the antibiotic treatments. Bacterial cells in close proximity may be able to modify their microenvironment; thus, making the composition of the microbial community an important factor in the ability of Las to cause HLB progression. A low Las level was seen as a both a seasonal fluctuation, part of the bacterial population dynamics, and as a response to the antibiotic treatments.

As a phloem-limited, intracellular bacterial pathogen, ‘Candidatus Liberibacter asiaticus’ (Las) has a significantly reduced genome and causes huanglongbing (HLB), a devastating disease of citrus worldwide.  In this study, we characterized two novel autotransporter proteins of Las, and redesignated them as LasA_I and LasA_II in lieu of the previous names Hyv_I and Hyv_II. Proteins secreted by the type V secretion system (T5SS), known as autotransporters, are large extracellular virulence proteins localized to the bacterial poles. Bioinformatic analyses revealed that LasA_I and LasA_II share the structural features of an autotransporter family containing large repeats of a passenger domain and a unique C-terminal translocator domain. When fused to the GFP gene and expressed in E. coli, the LasA_I C-terminus and the full length LasA_II were localized to the bacterial poles, similar to other members of autotransporter family. Despite the absence of the signal peptide, LasA_I was found to localize at the cell surface by immuno-dot blot using a monoclonal antibody against the partial LasA_I protein. Its surface localization was also confirmed by the removal of the LasA_I antigen using a proteinase K treatment of the intact bacterial cells. When co-inoculated with a P19 gene silencing suppressor and transiently expressed in tobacco leaves, the GFP-LasA_I translocator targeted to the mitochondria. This is the first report that Las encodes novel autotransporters that target to mitochondria. These findings may lead to a better understanding of the pathogenesis of this intracellular “energy parasitic” bacterium, and to more efficient characterizing new molecular targets for HLB control.

HLB affects all citrus cultivars and causes tree decline resulting in greatly reduced citrus production in Asia, Africa, the Indian subcontinent, the Arabian Peninsula, Brazil, and the United States. To enable integrated pest management strategies for combating HLB, we have developed a sensitive nucleic acid-based HLB diagnostic test based on Mesa Tech International, Inc.’s (MTI's) new point-of-use diagnostic platform, MTIDx, and targeting hyvI/hyvII genes of Candidatus Liberibacter asiaticus (Las) (Zhou et al., 2011). The MTIDx platform integrates sample preparation, rapid nucleic acid amplification and sequence-specific hybridization-based detection. The simplicity of the test may offer end users with little or no specialized training the opportunity to obtain molecular test results comparable to laboratory-based PCR methods without costly instrumentation. Here, we will report on the efficacy of MTI's nucleic acid testing platform for the detection of Candidatus Liberibacter asiaticus bacteria in greenhouse and field collected citrus tissue and in the insect vector of the pathogen (Diaphorina citri Kuwayama). Comparative studies evaluating the MTIDx platform's performance relative to widely accepted laboratory PCR testing will be presented. Results from these studies will be discussed in the context of their implications for HLB management.

Liberibacters have not yet been successfully cultured; their minimal genomes carry multiple copies of several genes. Sequences identical to phage genomes have been found in many Liberibacters. Available evidences suggest that the Liberibacter genomes are adapting rapidly in different hosts and environments. Characterization of genomes of rapidly changing unculturable organisms can be challenging. We have used a model system based on Candidatus Liberibacter psyllaurous associated with tomato “psyllid yellows” (Hansen et al., 2008) to develop methodologies using alternate techniques for sequencing metagenomes. We have constructed a BAC library from infected tomato psyllids (Bactericera cockerelli). The library consists of 57,600 clones arrayed in 150 plates each with 384 wells. DNA from individual clones were pooled for screening purposes. Initial identification of clones with Liberibacter sequences were conducted based on 16s ribosomal sequences, and contiguous clones were characterized by end sequencing and identified as containing Liberibacter genome fragments. Screening of additional clones from the library was based on probes developed on such sequences. A total of 245 clones with Liberibacter genome fragments have been identified. A total of 63 bar-coded BAC clones were sequenced by using Roche 454 technology. BAC clones from this library contain large inserts (average size 70 kb). Similarities and differences with other well characterized genomes of Liberibacters (Duan et al., 2009, Lin et al., 2011) will be presented.

Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, is the vector of huanglongbing (HLB), the most devastating disease of citrus worldwide. Knowledge of ACP dispersal behavior in locating host plants may contribute to our understanding of the spread of HLB within and between citrus trees. We conducted research in laboratory to evaluate ACP host plant finding behavior. In a free-choice situation, ACP adults initially settled at equal rates among seedlings of Rhododendron simsii (non host plant for ACP), Murraya panciculata and “Lugan”Citrus reticulata. However, at 18 and 42 h after ACP were released, the mean number of adults per plant on R. simsii was significantly lower than on C. reticulata and M. paniculata, respectively. Numbers of adults on C. reticulata and M. paniculata remained statistically equivalenty until 90 h after releases. Within a plant, the ratio of ACP adults on new flush shoots did not significantly increased in comparison to other parts of the tree during 1-4 d after the psyllids were released. Even on 7^th day after ACP release, about 30 % adults were observed on plant locations other than flushing shoots. The results indicated that ACP adults differentiated between host and non-host plants faster than they differentiated between different parts of a host plant.

Huanglongbing (HLB) is the most devastating disease of citrus worldwide and vectored by the Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Psyllidae). The pathogen associated with HLB maintains uncultural in vitro, and there are few effective options against HLB-affected plants. Identification and deployment of ACP- resistance traits of citrus and citrus related germplasm to suppress ACP populations may be a potential management strategy for the management of HLB. In the present study, the susceptibilities of 71 Chinese citrus genotypes to ACP infestation were evaluated and analyzed in a free-choice situation under field conditions by using the method of systematic clustering and dynamic clustering. The results showed that there was significant difference in susceptibility to ACP infestation among the genotypes of citrus. These genotypes can be graded according to the number of psyllids on the trees. Grade I: highly susceptible with a total of 8 genotypes. Grade II: moderately susceptible with a total of 18 genotypes. Grade III: lower susceptibility with a total of 45 genotypes. The 71 genotypes of citrus are used to be classified into 8 groups according to the Chinese classification system “Citrus Varieties in China” (Chinese Citrus Association). There existed significant differences among the 8 groups. Lemons and Pummelos were highly susceptible hosts to ACP, with no significant difference from Murraya panciculata L., which was the most suitable host to ACP. Mandarins, Hybrid citrus, Sweet oranges and Tangerines were moderately susceptible hosts. Kumquat and Sour oranges were lower susceptible hosts. Some genotypes of Kumquat, like Citrus medurenisi var. Variegated Calamondin, Fortunella hindsii var. Chintou, and Sour orange, Citrus aurantium var. Variegated sour orange, had the lowest susceptiblility to ACP infection. Further experiment will be done to confirm the low ACP- susceptibility of genotypes from Kumquat and Sour orange.