Endophytes and Plant Extracts as Potential Antimicrobial Agents against Candidatus Liberibacter Asiaticus, Causal Agent of Huanglongbing.

Huanglongbing (HLB), also known as citrus greening, is an insidious disease in citrus and has become a threat to the sustainability of the citrus industry worldwide. In the U.S., Candidatus Liberibacter asiaticus (CLas) is the pathogen that is associated with HLB, an unculturable, phloem-limited bacteria, vectored by the Asian Citrus Psyllid (ACP, Diaphorina citri). There is no known cure nor treatment to effectively control HLB, and current control methods are primarily based on the use of insecticides and antibiotics, where effectiveness is limited and may have negative impacts on beneficial and non-target organisms. Thus, there is an urgent need for the development of effective and sustainable treatment options to reduce or eliminate CLas from infected trees. In the present study, we screened citrus-derived endophytes, their cell-free culture supernatants (CFCS), and crude plant extracts for antimicrobial activity against two culturable surrogates of CLas, Sinorhizobium meliloti and Liberibacter crescens. Candidates considered high-potential antimicrobial agents were assessed directly against CLas in vitro, using a propidium monoazide-based assay. As compared to the negative controls, statistically significant reductions of viable CLas cells were observed for each of the five bacterial CFCS. Subsequent 16S rRNA gene sequencing revealed that each of the five bacterial isolates were most closely related to Bacillus amyloliquefaciens, a species dominating the market of biological control products. As such, the aboveground endosphere of asymptomatic survivor citrus trees, grown in an organic orchard, were found to host bacterial endophytes capable of effectively disrupting CLas cell membranes. These results concur with the theory that native members of the citrus microbiome play a role in the development of HLB. Here, we identify five strains of Bacillus amyloliquefaciens demonstrating notable potential to be used as sources of novel antimicrobials for the sustainable management of HLB.

An in vitro protocol for rapidly assessing the effects of antimicrobial compounds on the unculturable bacterial plant pathogen, Candidatus Liberibacter asiaticus.

BACKGROUND: Most bacteria are not culturable, but can be identified through molecular methods such as metagenomics studies. Due to specific metabolic requirements and symbiotic relationships, these bacteria cannot survive on typical laboratory media. Many economically and medically important bacteria are unculturable; including phloem-limited plant pathogens like Candidatus Liberibacter asiaticus (CLas). CLas is the most impactful pathogen on citrus production, is vectored by the Asian citrus psyllid (ACP, Diaphorina citri), and lacks an effective treatment or resistant cultivars. Research into CLas pathogenicity and therapy has been hindered by the lack of persistent pure cultures. Work to date has been mostly limited to in planta studies that are time and resource intensive. RESULTS: We developed and optimized an in vitro protocol to quickly test the effectiveness of potential therapeutic agents against CLas. The assay uses intact bacterial cells contained in homogenized tissue from CLas-infected ACP and a propidium monoazide (PMA) assay to measure antimicrobial activity. The applicability of PMA was evaluated; with the ability to differentiate between intact and disrupted CLas cells confirmed using multiple bactericidal treatments. We identified light activation conditions to prevent PCR interference and identified a suitable positive control for nearly complete CLas disruption (0.1% Triton-X 100). Isolation buffer components were optimized with 72 mM salt mixture, 1 mM phosphate buffer and 1% glycerol serving to minimize unwanted interactions with treatment and PMA chemistries and to maximize recovery of intact CLas cells. The mature protocol was used to compare a panel of peptides already under study for potential CLas targeting bactericidal activity and identify which were most effective. CONCLUSION: This psyllid homogenate assay allows for a quick assessment of potential CLas-disrupting peptides. Comparison within a uniform isolate largely eliminates experimental error arising from variation in CLas titer between and within individual host organisms. Use of an intact vs. disrupted assay permits direct assessment of potential therapeutic compounds without generating pure cultures or conducting extensive in planta or field studies.

Hexaacetyl-chitohexaose, a chitin-derived oligosaccharide, transiently activates citrus defenses and alters the feeding behavior of Asian citrus psyllid.

Plants have a perception system triggered by pathogen and pest signals to initiate defense. These signals include evolutionarily conserved molecules from microbes and insects termed pathogen/herbivore-associated molecular patterns (PAMPs/HAMPs). Here we showed that hexaacetyl-chitohexaose (HC), an oligosaccharide from chitin, a structural component in insect exoskeletons and fungi cell walls, upregulated defense-associated genes WRKY22, GST1, RAR1, EDS1, PAL1 and NPR2, and downregulated ICS1 at 1 h after HC treatment in Sun Chu Sha mandarin leaves. The effect was transient as defense gene transcriptional changes were not observed at 18 h after the treatment. Electrical penetration graph (EPG) recordings were used to study the feeding behavior of Asian citrus psyllid (ACP) following the HC treatment. ACP is the hemipteran vector of Candidatus Liberibacter asiaticus (CLas), the pathogen associated with huanglongbing (HLB). Adult ACP displayed reduced intercellular probing, reduced xylem feeding count and duration, and increased non-probing activity on HC-treated citrus compared to controls. During an 18-h recording, percentage for total duration of xylem ingestion, phloem ingestion, intercellular probing were lower, and the percentage of non-probing behavior was higher in HC-treated leaves than in controls. In host-selection behavior studies, HC treatment did not alter the attractiveness of citrus leaves under light or dark conditions. In addition, ACP feeding on HC-treated leaves did not show differences in mortality for up to 10 day of exposure. In summary, we report that HC induced a transient defense in citrus and an inhibitory effect on ACP feeding but did not affect host selection or the insect fitness under the tested conditions.

Temporal and spatial detection of Candidatus Liberibacter asiaticus putative effector transcripts during interaction with Huanglongbing-susceptible, -tolerant, and -resistant citrus hosts.

BACKGROUND: Citrus Huanglongbing (HLB) is a bacterial disease with high economic significance. The associated agent Candidatus Liberibacter asiaticus is a fastidious, phloem-limited, intracellular bacterium that is transmitted by an insect vector the Asian citrus psyllid (ACP). The genome of Ca. L. asiaticus contains protein secretion machinery that suggests host cell modulation capacity of this bacterium. RESULTS: A total of 28 candidate effectors, an important class of secreted proteins, were predicted from the Ca. L. asiaticus genome. Sequence specific primers were designed for reverse transcription (RT) and quantitative PCR (qPCR), and expression was validated for 20 of the effector candidates in infected citrus with multiple genetic background. Using detached leaf inoculation, the mRNA of effectors was detected from 6 h to 7 days post ACP exposure. It was observed that higher bacterial titers were associated with a larger number of effectors showing amplification across all samples. The effectors' expression were compared in citrus hosts with various levels of HLB tolerance, including susceptible Duncan grapefruit and Washington navel orange, tolerant citron and Cleopatra mandarin, and resistant Pomeroy trifoliate and Carrizo citrange. Across all genotypes relatively high expression was observed for CLIBASIA_03695, CLIBASIA_00460, CLIBASIA_00420, CLIBASIA_04580, CLIBASIA_05320, CLIBASIA_04425, CLIBASIA_00525 and CLIBASIA_05315 in either a host-specific or -nonspecific manners. The two genotypes in each HLB-response group also show effector-expression profiles that seem to be different. In a companion study, the expression of effectors was compared between leaves and roots of own-rooted citrus that had been Ca. L. asiaticus-infected for more than a year. Results indicated relatively high expression of CLIBASIA_03875, CLIBASIA_04800 and CLIBASIA_05640 in all leaf and some root tissues of citron, Duncan and Cleopatra. CONCLUSION: This temporal and spatial expression analysis of Ca. L. asiaticus effectors identified candidates possibly critical for early bacterial colonization, host tolerance suppression and long-term survival which are all worthy of further investigation.

Global analysis of target genes of 21 members of the ZAD transcription factor family in Drosophila melanogaster.

The zinc-finger associated domain (ZAD) family is the largest transcription factor family in dipteran insects. Still, their functional significance is barely recognized in the literature due in part to their resistance to mutagenesis screens in genetic studies. Therefore, we employed in vitro techniques to identify the DNA-binding characteristics of several members of the Drosophila melanogaster ZAD family in an effort to study their target genes. In this comprehensive investigation, we constructed a panel of GST-Zinc finger (ZnF) array chimera from 21 selected ZAD proteins and used them to select binding sites from an oligonucleotide library by employing electrophoretic mobility shift assays (EMSA). Samples of the binding population were sequenced and used to derive DNA-binding consensus sequence for each member. These consensus sequences were tested for complex formation with their respective protein chimera and the specificity of binding ascertained by competition EMSA. Bioinformatics tools were used to identify potential genetic targets. The identified consensus sequences were distinct for each member and the putative genomic targets were clustered in the regulatory regions of specific genes. This appears to be consistent with a conservation of function between members and also suggests that the overlapping functions of ZAD proteins are the result of positive selection to maintain redundancy and not simply artifacts of recent expansion. Putative target genes suggest a major role of the ZAD family members in the regulation of several early developmental genes including homeobox transcription factors.

An efficient and cost-effective protocol for selecting transcription factor binding sites that reduces isotope usage.

To function, transcription factors must position themselves by binding to DNA in a sequence-specific manner. Knowing the binding sites of these factors is a necessary step in understanding their activity. The standard protocols used for selecting a consensus-binding sequence for a DNA binding domain often require the use of radioisotopes to attain the necessary level of power in the assay. Alternatives are often less sensitive and may require an expensive apparatus for visualizing. We have created a modified binding site selection (BSS) protocol to improve efficiency and decrease the use of radioisotope. A GST affinity-tagged DNA binding domain construct was immobilized on a GSH affinity column and used to select from a randomized oligonucleotide library identical to those typically used in a radiolabeled BSS protocol. This produced a library specifically pre-enriched for use in a standard sequential EMSA selection. Use of a pre-enriched library reduced the total number of labeled rounds required for selection, decreasing the use of radioisotope while maintaining efficacy. The protocol was used to select for the binding sequence for several Drosophila melanogaster transcription factors. The consensus sequence was then shown by competitive binding experiments to associate with the protein in a sequence-dependent manner.