Unculturable Bacterium Research Articles

Hiding in Plain Sight: A Widespread Native Perennial Harbors Diverse Haplotypes of 'Candidatus Liberibacter solanacearum' and Its Potato Psyllid Vector.

The unculturable bacterium 'Candidatus Liberibacter solanacearum' (CLso) is responsible for a growing number of emerging crop diseases. However, we know little about the diversity and ecology of CLso and its psyllid vectors outside of agricultural systems, which limits our ability to manage crop disease and understand the impacts this pathogen may have on wild plants in natural ecosystems. In North America, CLso is transmitted to crops by the native potato psyllid (Bactericera cockerelli). However, the geographic and host plant range of the potato psyllid and CLso beyond the borders of agriculture are not well understood. A recent study of historic herbarium specimens revealed that a unique haplotype of CLso was present infecting populations of the native perennial Solanum umbelliferum in California decades before CLso was first detected in crops. We hypothesized that this haplotype and other potentially novel CLso variants are still present in S. umbelliferum populations. To test this, we surveyed populations of S. umbelliferum in Southern California for CLso and potato psyllid vectors. We found multiple haplotypes of CLso and the potato psyllid associated with these populations, with none of these genetic variants having been previously reported in California crops. These results suggest that CLso and its psyllid vectors are much more widespread and diverse in North American natural plant communities than suggested by data collected solely from crops and weeds in agricultural fields. Further characterization of these apparently asymptomatic haplotypes will facilitate comparison with disease-causing variants and provide insights into the continued emergence and spread of CLso.

Specifically targeting antimicrobial peptides for inhibition of Candidatus Liberibacter asiaticus.

Huanglongbing (citrus greening) is a plant disease putatively caused by the unculturable Gram-negative bacterium Candidatus Liberibacter asiaticus (CLas), and it has caused severe damage to citrus plantations worldwide. There are no definitive treatments for this disease, and conventional disease control techniques have shown limited efficacy. This work presents an in silico evaluation of using specifically targeting anti-microbial peptides (STAMPs) consisting of a targeting segment and an antimicrobial segment to inhibit citrus greening by inhibiting the BamA protein of CLas, which is an outer membrane protein crucial for bacterial viability. Initially, a set of peptides with a high affinity toward BamA protein were screened and evaluated via molecular docking and molecular dynamics simulations and were verified in vitro via bio-layer interferometry (BLI). In silico studies and BLI experiments indicated that two peptides, HASP2 and HASP3, showed stable binding to BamA. Protein structures for STAMPs were created by fusing known anti-microbial peptides (AMPs) with the selected short peptides. The binding of STAMPs to BamA was assessed using molecular docking and binding energy calculations. The attachment of high-affinity short peptides significantly reduced the free energy of binding for AMPs, suggesting that it would make it easier for the STAMPs to bind to BamA. Efficacy testing in vitro using a closely related CLas surrogate bacterium showed that STAMPs had greater inhibitory activity than AMP alone. In silico and in vitro results indicate that the STAMPs can inhibit CLas surrogate Rhizobium grahamii more effectively compared to AMPs, suggesting that STAMPs can achieve better inhibition of CLas, potentially via enhancing the site specificity of AMPs.

Preliminary assessment of bacterial antibiotic resistance and Candidatus Liberibacter asiaticus titer in three Florida commercial citrus groves

Over the last 16 years, the U.S. citrus industry has been devastated by citrus greening or Huanglongbing (HLB) disease. The causal agent, Candidatus Liberibacter asiaticus (CLas), is an unculturable bacterium transmitted by the invasive Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae), and by grafting. In 2017 and 2019, the Environmental Protection Agency approved foliar applications of the antibiotics streptomycin (STR) and oxytetracycline (OTC) in commercial citrus groves in Florida. This study provides a preliminary assessment of antibiotic spray effects on three commercial citrus groves by quantifying CLas titer from leaves and antibiotic resistant bacterial colony count from leaf, root+rhizosphere, and soil samples. Two groves used antibiotic spray treatment and followed conventional management, one used no antibiotic treatment and was accredited as an organic grove. Samples were plated onto TSA medium containing either STR (100 μg/mL), OTC (15 μg/mL), or no antibiotic (control) then colony-forming units (CFU) per gram of sample were estimated. Among leaf samples, there were no significant differences in CFU or CLas titer based on grove. For soil samples, we found a larger number of resistant CFU in the organic grove. Root+rhizosphere samples from all groves (organic and conventional) showed high prevalence of both STR and OTC-resistant bacteria indicating that plant selective processes during root colonization might favor antibiotic resistant acquisition. The lack of significant difference in CLas titer among the three groves is supported by other studies which suggest that antibiotic spray might not be an effective control for CLas. This research further highlights the need for continued monitoring of antibiotic efficacy to control plant pathogens in commercial settings.

A prophage-encoded effector from "Candidatus Liberibacter asiaticus" targets ASCORBATE PEROXIDASE6 in citrus to facilitate bacterial infection.

Citrus huanglongbing (HLB), associated with the unculturable phloem-limited bacterium "Candidatus Liberibacter asiaticus" (CLas), is the most devastating disease in the citrus industry worldwide. However, the pathogenicity of CLas remains poorly understood. In this study, we show that AGH17488, a secreted protein encoded by the prophage region of the CLas genome, suppresses plant immunity via targeting the host ASCORBATE PEROXIDASE6 (APX6) protein in Nicotiana benthamiana and Citrus sinensis. The transient expression of AGH17488 reduced the chloroplast localization of APX6 and its enzyme activity, inhibited the accumulation of reactive oxygen species (H2 O2 and O2 - ) and the lipid oxidation endproduct malondialdehyde in plants, and promoted the proliferation of Pseudomonas syringae pv. tomato DC3000 and Xanthomonas citri subsp. citri. This study reveals a novel mechanism underlying how CLas uses a prophage-encoded effector, AGH17488, to target a reactive oxygen species accumulation-related gene, APX6, in the host to facilitate its infection.

Evaluation of Candidatus Liberibacter Asiaticus Efflux Pump Inhibition by Antimicrobial Peptides.

Citrus greening, also known as Huanglongbing (HLB), is caused by the unculturable bacterium Candidatus Liberibacter spp. (e.g., CLas), and has caused a devastating decline in citrus production in many areas of the world. As of yet, there are no definitive treatments for controlling the disease. Antimicrobial peptides (AMPs) that have the potential to block secretion-dependent effector proteins at the outer-membrane domains were screened in silico. Predictions of drug-receptor interactions were built using multiple in silico techniques, including molecular docking analysis, molecular dynamics, molecular mechanics generalized Born surface area analysis, and principal component analysis. The efflux pump TolC of the Type 1 secretion system interacted with natural bacteriocin plantaricin JLA-9, blocking the β barrel. The trajectory-based principal component analysis revealed the possible binding mechanism of the peptides. Furthermore, in vitro assays using two closely related culturable surrogates of CLas (Liberibacter crescens and Rhizobium spp.) showed that Plantaricin JLA-9 and two other screened AMPs inhibited bacterial growth and caused mortality. The findings contribute to designing effective therapies to manage plant diseases associated with Candidatus Liberibacter spp.

Bacterial diversity and community structure shapes pederin polymorphism but lacks association with host genotype specificity in the rove beetle, Paederus fuscipes.

Paederus beetles are notorious for their irritant haemolymph toxin pederin, which is synthesised by an unculturable bacterial symbiont, causing dermatitis when in contact with human skin. Pederin polymorphism is observed when (+) females carrying the toxin pederin and (-) females lacking this co-occur in natural populations. Despite reports detecting pederin polymorphism in several Paederina beetles, symbiont infection frequencies in their natural populations and the bacterial diversity differences underlying the polymorphism between these female types are understudied. Herein we report a high prevalence (>80%) of female Paederus fuscipes carrying bacterial ped genes in all six study populations. This finding suggests that selection pressure favouring pederin-producing females is crucial for survival in the natural environment. 16S rRNA metabarcoding analysis revealed significant dominance of the unculturable pederin-producing Pseudomonas-like bacterium (Gammaproteobacteria) in (+) females, consistent with previous studies. The microbial diversity of the (-) females revealed a significant abundance of Apibacter (Bacteroidia), previously undocumented, suggesting its importance in the functionality of (-) females. Predicted functions related to metabolisms are enriched in (-) females, suggesting fitness advantage possibilities in sustaining the population in the absence of predation. Further investigations on the possible genetic basis of the host genotype revealed no association of host mtDNA and pederin polymorphism in Pa. fuscipes.

Development on Citrus medica infected with 'Candidatus Liberibacter asiaticus' has sex-specific and -nonspecific impacts on adult Diaphorina citri and its endosymbionts.

Huanglongbing (HLB) is a deadly, incurable citrus disease putatively caused by the unculturable bacterium, ‘Candidatus Liberibacter asiaticus’ (CLas), and transmitted by Diaphorina citri. Prior studies suggest D. citri transmits CLas in a circulative and propagative manner; however, the precise interactions necessary for CLas transmission remain unknown, and the impact of insect sex on D. citri-CLas interactions is poorly understood despite reports of sex-dependent susceptibilities to CLas. We analyzed the transcriptome, proteome, metabolome, and microbiome of male and female adult D. citri reared on healthy or CLas-infected Citrus medica to determine shared and sex-specific responses of D. citri and its endosymbionts to CLas exposure. More sex-specific than shared D. citri responses to CLas were observed, despite there being no difference between males and females in CLas density or relative abundance. CLas exposure altered the abundance of proteins involved in immunity and cellular and oxidative stress in a sex-dependent manner. CLas exposure impacted cuticular proteins and enzymes involved in chitin degradation, as well as energy metabolism and abundance of the endosymbiont ‘Candidatus Profftella armatura’ in both sexes similarly. Notably, diaphorin, a toxic Profftella-derived metabolite, was more abundant in both sexes with CLas exposure. The responses reported here resulted from a combination of CLas colonization of D. citri as well as the effect of CLas infection on C. medica. Elucidating these impacts on D. citri and their endosymbionts contributes to our understanding of the HLB pathosystem and identifies the responses potentially critical to limiting or promoting CLas acquisition and propagation in both sexes.

Genomic Characterization of Mycobacterium leprae to Explore Transmission Patterns Identifies New Subtype in Bangladesh.

Mycobacterium leprae, the causative agent of leprosy, is an unculturable bacterium with a considerably reduced genome (3.27 Mb) compared to homologues mycobacteria from the same ancestry. In 2001, the genome of M. leprae was first described and subsequently four genotypes (1–4) and 16 subtypes (A–P) were identified providing means to study global transmission patterns for leprosy. In order to understand the role of asymptomatic carriers we investigated M. leprae carriage as well as infection in leprosy patients (n = 60) and healthy household contacts (HHC; n = 250) from Bangladesh using molecular detection of the bacterial element RLEP in nasal swabs (NS) and slit skin smears (SSS). In parallel, to study M. leprae genotype distribution in Bangladesh we explored strain diversity by whole genome sequencing (WGS) and Sanger sequencing. In the studied cohort in Bangladesh, M. leprae DNA was detected in 33.3% of NS and 22.2% of SSS of patients with bacillary index of 0 whilst in HHC 18.0% of NS and 12.3% of SSS were positive. The majority of the M. leprae strains detected in this study belonged to genotype 1D (55%), followed by 1A (31%). Importantly, WGS allowed the identification of a new M. leprae genotype, designated 1B-Bangladesh (14%), which clustered separately between the 1A and 1B strains. Moreover, we established that the genotype previously designated 1C, is not an independent subtype but clusters within the 1D genotype. Intraindividual differences were present between the M. leprae strains obtained including mutations in hypermutated genes, suggesting mixed colonization/infection or in-host evolution. In summary, we observed that M. leprae is present in asymptomatic contacts of leprosy patients fueling the concept that these individuals contribute to the current intensity of transmission. Our data therefore emphasize the importance of sensitive and specific tools allowing post-exposure prophylaxis targeted at M. leprae-infected or -colonized individuals.

An In Vitro Pipeline for Screening and Selection of Citrus-Associated Microbiota with Potential Anti-"Candidatus Liberibacter asiaticus" Properties.

Huanglongbing (HLB) is a destructive citrus disease that is lethal to all commercial citrus plants, making it the most serious citrus disease and one of the most serious plant diseases. Because of the severity of HLB and the paucity of effective control measures, we structured this study to encompass the entirety of the citrus microbiome and the chemistries associated with that microbial community. We describe the spatial niche diversity of bacteria and fungi associated with citrus roots, stems, and leaves using traditional microbial culturing integrated with culture-independent methods. Using the culturable sector of the citrus microbiome, we created a microbial repository using a high-throughput bulk culturing and microbial identification pipeline. We integrated an in vitro agar diffusion inhibition bioassay into our culturing pipeline that queried the repository for antimicrobial activity against Liberibacter crescens, a culturable surrogate for the nonculturable "Candidatus Liberibacter asiaticus" bacterium associated with HLB. We identified microbes with robust inhibitory activity against L. crescens that include the fungi Cladosporium cladosporioides and Epicoccum nigrum and bacterial species of Pantoea, Bacillus, and Curtobacterium Purified bioactive natural products with anti-"Ca. Liberibacter asiaticus" activity were identified from the fungus C. cladosporioides Bioassay-guided fractionation of an organic extract of C. cladosporioides yielded the natural products cladosporols A, C, and D as the active agents against L. crescens This work serves as a foundation for unraveling the complex chemistries associated with the citrus microbiome to begin to understand the functional roles of members of the microbiome, with the long-term goal of developing anti-"Ca Liberibacter asiaticus" bioinoculants that thrive in the citrus holosystem.IMPORTANCE Globally, citrus is threatened by huanglongbing (HLB), and the lack of effective control measures is a major concern of farmers, markets, and consumers. There is compelling evidence that plant health is a function of the activities of the plant's associated microbiome. Using Liberibacter crescens, a culturable surrogate for the unculturable HLB-associated bacterium "Candidatus Liberibacter asiaticus," we tested the hypothesis that members of the citrus microbiome produce potential anti-"Ca Liberibacter asiaticus" natural products with potential anti-"Ca Liberibacter asiaticus" activity. A subset of isolates obtained from the microbiome inhibited L. crescens growth in an agar diffusion inhibition assay. Further fractionation experiments linked the inhibitory activity of the fungus Cladosporium cladosporioides to the fungus-produced natural products cladosporols A, C, and D, demonstrating dose-dependent antagonism to L. crescens.

Characterization of the heavy metal binding properties of periplasmic metal uptake protein CLas-ZnuA2.

Candidatus Liberibacter asiaticus (CLas), a phloem-limited unculturable Gram-negative bacterium, causes citrus greening disease. The proteome analysis of CLas showed the presence of a heavy metal permease and Co/Zn/Cd cation exporter system. However, there is no designated metal uptake protein specific for the heavy metal permease in CLas. One of the metal uptake proteins, designated as CLas-ZnuA2, in our previous studies, showed a lower metal-binding affinity for Mn2+ and Zn2+ and was postulated to bind and transport metals rather non-specifically. The present study focused on the characterization of the heavy metal binding properties of CLas-ZnuA2 using SPR, CD, DSC and crystallographic studies. The crystal structure analysis of Cd2+ bound CLas-ZnuA2 showed octahedral geometry for Cd2+ binding as compared to a non-preferred square-pyramidal geometry for Mn2+ and Zn2+ binding in earlier reported crystal structures. In SPR analysis, the binding affinities of 4.7 × 10-6 M, 7.2 × 10-6 M, 5.3 × 10-5 M and 4.3 × 10-5 M for Hg2+, Cd2+, Ba2+ and Co2+ respectively were higher as compared to earlier reported values for Mn2+ and Zn2+. Likewise, CD and DSC analysis showed relatively higher thermal stability for CLas-ZnuA2 on heavy metal binding. Taken together with the expression of the permease and exporter system for heavy metals, our results indicate that CLas-ZnuA2 may be involved in sequestering and transport of various transition divalent metals in environmentally stressed conditions.

Synthesis and structure-activity relationships of teixobactin.

The discovery of antibiotics has led to the effective treatment of bacterial infections that were otherwise fatal and has had a transformative effect on modern medicine. Teixobactin is an unusual depsipeptide natural product that was recently discovered from a previously unculturable soil bacterium and found to possess potent antibacterial activity against several Gram positive pathogens, including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci. One of the key features of teixobactin as an antibiotic lead is that resistance could not be generated in a laboratory setting. This is proposed to be a result of a mechanism of action that involves binding to essential cell wall synthesis building blocks, lipid II and lipid III. Since the initial isolation report in 2015, significant efforts have been made to understand its unique mechanism of action, develop efficient synthetic routes for its production, and thus enable the generation of analogues for structure-activity relationship studies and optimization of its pharmacological properties. Our review provides a comprehensive treatise on the progress in understanding teixobactin chemistry, structure-activity relationships, and mechanisms of antibacterial activity. Teixobactin represents an exciting starting point for the development of new antibiotics that can be used to combat multidrug-resistant bacterial ("superbug") infections.

Raman-Deuterium Isotope Probing for in-situ identification of antimicrobial resistant bacteria in Thames River

The emergence and widespread distribution of antimicrobial resistant (AMR) bacteria has led to an increasing concern with respect to potential environmental and public health risks. Culture-independent and rapid identification of AMR bacteria in-situ in complex environments is important in understanding the role of viable but non-culturable and antibiotic persistent bacteria and in revealing potential pathogens without waiting for colony formation. In this study, a culture-independent and non-destructive phenotyping approach, so called Raman Deuterium Stable Isotope Probing (Raman-DIP), was developed to identify AMR bacteria in the River Thames. It is demonstrated that Raman-DIP was able to accurately identify resistant and susceptible bacteria within 24 hours. The work shows that, in the River Thames, the majority of the bacteria (76 ± 2%) were metabolically active, whilst AMR bacteria to carbenicillin, kanamycin and both two antibiotics were 35 ± 5%, 28 ± 3%, 25 ± 1% of the total bacterial population respectively. Raman activated cell ejection (RACE) was applied to isolate single AMR bacteria for the first time, linking AMR phenotype (reistance to antibiotics) and genotype (DNA sequence). The sequences of the RACE sorted cells indicate that they were potential human pathogens Aeromonas sp., Stenotrophomonas sp. and an unculturable bacterium. This work demonstrates Raman-DIP and RACE are effective culture-independent approach for rapid identification of AMR bacteria at the single cell level in their natural conditions.

Functional characterization of LotP from Liberibacter asiaticus.

Summary Liberibacter asiaticus is an unculturable parasitic bacterium of the alphaproteobacteria group hosted by both citrus plants and a psyllid insect vector (Diaphorina citri). In the citrus tree, the bacteria thrive only inside the phloem, causing a systemically incurable and deadly plant disease named citrus greening or Huanglongbing. Currently, all commercial citrus cultivars in production are susceptible to L. asiaticus, representing a serious threat to the citrus industry worldwide. The technical inability to isolate and culture L. asiaticus has hindered progress in understanding the biology of this bacterium directly. Consequently, a deep understanding of the biological pathways involved in the regulation of host–pathogen interactions becomes critical to rationally design future and necessary strategies of control. In this work, we used surrogate strains to evaluate the biochemical characteristics and biological significance of CLIBASIA_03135. This gene, highly induced during early stages of plant infection, encodes a 23 kDa protein and was renamed in this work as LotP. This protein belongs to an uncharacterized family of proteins with an overall structure resembling the LON protease N‐terminus. Co‐immunoprecipitation assays allowed us to identify the Liberibacter chaperonin GroEL as the main LotP‐interacting protein. The specific interaction between LotP and GroEL was reconstructed and confirmed using a two‐hybrid system in Escherichia coli. Furthermore, it was demonstrated that LotP has a native molecular weight of 44 kDa, corresponding to a dimer in solution with ATPase activity in vitro. In Liberibacter crescens, LotP is strongly induced in response to conditions with high osmolarity but repressed at high temperatures. Electrophoretic mobility shift assay (EMSA) results suggest that LotP is a member of the LdtR regulon and could play an important role in tolerance to osmotic stress.

Texas Phoenix Palm Decline

Texas Pheonix palm decline is a new disease in Florida, caused by an unculturable bacterium. It is a fatal, systemic disease that kills palms relatively quickly. This revised 6-page fact sheet explains the pathogen and hosts of TPPD, its symptoms, how to diagnose it, and provides disease management practices. Written by Nigel A. Harrison and Monica L. Elliott, and published by the Plant Pathology Department, June 2016. PP243/PP163: Lethal Bronzing Disease (LB) (ufl.edu)

High-Fidelity PCR Improves the Detection of ‘Candidatus Liberibacter solanacearum’ in Potato Tubers

Zebra chip disease (ZC) of potato tubers, caused by the unculturable bacterium ‘Candidatus Liberibacter solanacearum’ (Lso) can be difficult to detect from potato tuber tissue. The polymerase chain reaction is an important tool for the detection of plant pathogens, but there are limits to its sensitivity. High-fidelity PCR incorporates a proofreading enzyme with 3′-5′ exonuclease activity and has been shown to increase detection of pathogens when the titer is low or when large amounts of competitor DNA or other host cellular contents inhibit PCR amplification. In this study, high-fidelity PCR and conventional PCR protocols were performed on tubers presenting ZC symptoms. Asymptomatic tubers were used as control. The high-fidelity PCR increased detection of Lso from symptomatic tubers by 30–40 % over conventional PCR. Slight modifications were made from previous high-fidelity protocols to decrease testing time and simplify the protocol.

Phylogenetic study of methanol oxidizers from chilika-lake sediments using genomic and metagenomic approaches.

Group-wise diversity of sediment methylotrophs of Chilika lake (Lat. 19°28'-19°54'N; Long. 85°06'-85°35'E) Odisha, India at various identified sites was studied. Both the culturable and unculturable (metagenome) methylotrophs were investigated in the lake sediments employing both mxaF and 16S rRNA genes as markers. ARDRA profiling, 16S rRNA gene sequencing, PAGE profiling of HaeIII, EcoRI restricted mxaF gene and the mxaF gene sequences using culture-dependent approach revealed the relatedness of α-proteobacteria and Methylobacterium, Hyphomicrobium and Ancyclobacter sp. The total viable counts of the culturable aerobic methylotrophs were relatively higher in sediments near the sea mouth (S3; Panaspada), also demonstrated relatively high salinity (0.1M NaCl) tolerance. Metagenomic DNA from the sediments, amplified using GC clamp mxaF primers and resolved through DGGE, revealed the diversity within the unculturable methylotrophic bacterium Methylobacterium organophilum, Ancyclobacter aquaticus, Burkholderiales and Hyphomicrobium sp. Culture-independent analyses revealed that up to 90% of the methylotrophs were unculturable. The study enhances the general understandings of the metagenomic methylotrophs from such a special ecological niche.

Influence of the pathogen Candidatus Liberibacter solanacearum on tomato host plant volatiles and psyllid vector settlement.

Candidatus Liberibacter solanacearum (CLso) is an unculturable bacterium vectored by the tomato potato psyllid (TPP) Bactericera cockerelli and has been associated with Zebra chip disease in potato and with other economically relevant symptoms observed in solanaceous crops. By altering their host and vector's biological system, pathogens are able to induce changes that benefit them by increasing their transmission rate. Understanding these changes can enable better targeting of mechanisms to control pathogen outbreaks. Here, we explored how the CLso infectious status affects the volatile organic compounds (VOCs) of the tomato plant, and whether the CLso infectious status of TPP influences host plant settlement. These chemical and behavioral changes can ultimately affect the rate of encounter between the host and the vector. Results from headspace volatile collection of tomato plants showed that CLso infected tomato plants emitted a qualitatively and quantitatively different blend of VOCs compared to sham-infected plants. By a factorial experiment, we showed that CLso negative (CLso-) TPP preferred to settle 70 % more often on infected tomato plants, while CLso positive (CLso+) TPP were found 68 % more often on sham-infected tomato plants. These results provide new evidence in favor of both host and vector manipulation by CLso.

Induction of innate immune responses by flagellin from the intracellular bacterium, 'Candidatus Liberibacter solanacearum'.

Background‘Candidatus Liberibacter solanacearum’ (Lso) is a phloem-limited alphaproteobacterium associated with the devastating zebra chip disease of potato (Solanum tuberosum). Like other members of Liberibacter, Lso-ZC1 encodes a flagellin domain-containing protein (FlaLso) with a conserved 22 amino-acid peptide (flg22Lso). To understand the innate immune responses triggered by this unculturable intracellular bacterium, we studied the pathogen-associated molecular patterns (PAMPs) that triggered immunity in Nicotiana benthamiana, using the flg22Lso peptide and the full length flaLso gene.ResultsOur results showed that the expression of flaLso via Agrobacterium-mediated transient expression induced a slow necrotic cell death in the inoculated leaves of N. benthamiana, which was coupled with a burst of reactive oxygen species (ROS) production. Moreover, the expression of several representative genes involved in innate immunity was transiently up-regulated by the flg22Lso in N. benthamiana. The FlaLso, however, induced stronger up-regulation of these representative genes compared to the flg22Lso, especially that of flagellin receptor FLAGELLIN SENSING2 (FLS2) and respiratory burst oxidase (RbohB) in N. benthamiana. Although neither cell death nor ROS were induced by the synthetic flg22Lso, a weak callose deposition was observed in infiltrated leaves of tobacco, tomato, and potato plants.ConclusionThe flagellin of Lso and its functional domain, flg22Lso share characteristics of pathogen-associated molecular patterns, and trigger unique innate immune responses in N. benthamiana. Slow and weak activation of the innate immune response in host plants by the flagellin of Lso may reflect the nature of its intracellular life cycle. Our findings provide new insights into the role of the Lso flagellin in the development of potato zebra chip disease and potential application in breeding for resistance.

Bacterial diversity of aMasi, a South African fermented milk product, determined by clone library and denaturing gradient gel electrophoresis analysis

In the present study, we investigated the bacterial diversity of aMasi, a traditional South African fermented milk product, by 16S rRNA clone library and Denaturing Gradient Gel Electrophoresis (DGGE) analysis. Two hundred and eighty two clones from clone library were isolated and identified from aMasi, prepared from the milk of four cows from one herd in the EkuPindiseni Community, North West of Hluhluwe-iMfolozi Park in KwaZulu-Natal Province. The majority of the identified sequences corresponded to lactic acid bacteria (LAB), with the genus Lactococcus as major representative. The speciesLactococcus lactis accounted for 179 of the identified clones. In addition, several species of Lactobacillus, Leuconostoc and Enterococcus were detected. Furthermore, several clones belonging to Acinetobacter, Aeromonas and genera within the Enterobacteriaceaewere detected. It is important to note that human pathogens such as Klebsiella pneumoniae were identified in aMasi in the present study. Conversely, zoonotic bacteria such as Brucella abortus and Mycobacterium bovis were not detected in aMasi, although, they are present in the cattle population in the study area. Thirty (30) clones were identified as uncultured bacterial clones. Nine DGGE bands were successfully sequenced, of which four were classified as L. lactis with other bands belonging to lactobacilli, Clostridium acidurici, Enterobacter sp., Acinetobacter baumannii and an un-culturable bacterium. Even though there was some discrepancy between the two culture independent methods used to study the bacteriological community in aMasi, a general conclusion can be drawn,L. lactis may be considered as the dominant bacterium within a diverse bacterial community in this locally-produced dairy product. Key words: South Africa, aMasi, fermented milk, microbial diversity, clone library, denaturing gradient gel electrophoresis (DGGE).

‘Candidatus Liberibacter solanacearum’

<i>‘Candidatus</i> Liberibacter solanacearum’