scholarly journals Metagenomic Analysis Reveals the Mechanism for the Observed Increase in Antibacterial Activity of Penicillin against Uncultured Bacteria Candidatus Liberibacter asiaticus Relative to Oxytetracycline in Planta

Antibiotics ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 874
Author(s):  
Chuanyu Yang ◽  
Hanqing Hu ◽  
Yihong Wu ◽  
Xiongjie Lin ◽  
Goucheng Fan ◽  
...  
Keyword(s):  
Antibacterial Activity ◽  
Microbial Communities ◽  
Relative Abundance ◽  
Bacterial Species ◽  
Structure And Function ◽  
Candidatus Liberibacter Asiaticus ◽  
Uncultured Bacteria ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
And Function

Citrus huanglongbing (HLB) is a devastating disease for the citrus industry. The previous studies demonstrated that oxytetracycline and penicillin are effective antibiotics against Candidatus Liberibacter asiaticus (CLas). However, since CLas is uncultured, the mechanisms of action of antibiotics against CLas are still unclear. It was recently reported that the endophytic microbial communities are associated with the progression of citrus HLB after oxytetracycline and penicillin treatment. Therefore, we hypothesize that penicillin has greater antibacterial activity against CLas than oxytetracycline, which may be associated with the alteration of the structure and function of endophytic microbial communities in HLB-affected citrus in response to these antibiotics. To test this hypothesis, the microbiome of HLB-affected citrus leaves treated with these two antibiotics was analyzed using a metagenomic method. Our results indicate that the microbial structure and function in HLB-affected citrus were altered by these two antibiotics. The relative abundance of beneficial bacterial species, including Streptomyces avermitilis and Bradyrhizobium, was higher in penicillin-treated plants compared to those treated with oxytetracycline, and the relative abundance of the bacterial species (such as Propionibacterium acnes and Synechocystis sp PCC 6803) associated with CLas survival was lower for penicillin-treated plants compared to oxytetracycline-treated plants. These results indicate that penicillin has greater antibacterial activity against CLas. Based on the metagenomic analysis, this study elucidated the mechanism for the observed increase in antibacterial activity of penicillin against CLas. The data presented here are not only invaluable for developing eco-friendly and effective biocontrol strategies to combat citrus HLB, but also provide a method for revealing mechanism of antimicrobial against uncultured bacteria in host.

scholarly journals Inoculation Period and Citrus Host Effect Establishment of New Infections of ‘Candidatus Liberibacter asiaticus’ Transmitted via Vegetative Grafting

Plant Disease ◽  
2020 ◽  
Vol 104 (7) ◽  
pp. 1894-1899
Author(s):  
Mark E. Hilf ◽  
Weiqi Luo
Keyword(s):  
Survival Analysis ◽  
Bacterial Species ◽  
Systemic Infection ◽  
Candidatus Liberibacter Asiaticus ◽  
Susceptibility To Infection ◽  
Successful Infection ◽  
Significant Difference ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Pathogen Dna

Vegetative grafting is a common method of transmitting and propagating ‘Candidatus Liberibacter asiaticus’, the bacterial species accepted as the causal agent of the citrus disease huanglongbing (HLB). Generally, infected tissue that is grafted to a receptor tree remains in place indefinitely to ensure transmission. In this study, individual HLB-symptomatic leaves were grafted as ‘Ca. L. asiaticus’ inoculum sources to receptor trees of six citrus types and removed after an inoculation period (IP) of 21, 51, or 81 days. The goal was to assess the effect of varying IPs on transmission of bacteria to the receptor tree and on the successful establishment of a new infection. Survival analysis of data from three trials showed a significantly reduced proportion of infected trees with an IP of 21 days compared with IPs of 51 and 81 days but that there was no significant difference in the proportion of infected trees between IPs of 51 and 81 days. In addition, the time to first detection of pathogen DNA in an infected tree was delayed significantly for the 21-day IP when compared with the 51- and 81-day IPs. Survival analysis showed that the probability of infection of sweet orange trees was significantly higher than for trees of five other citrus types throughout the experiment. There was no significant difference between the infection probabilities of these latter five citrus types. The data from this study show that successful infection by grafting is dependent upon the time of exposure to the inoculum, that shorter IPs increase the time needed to establish a systemic infection, and that citrus types vary in their overall susceptibility to infection by ‘Ca. L. asiaticus’.

scholarly journals Structure and function of the bacterial and fungal gut flora of Neotropical butterflies

2017 ◽  
Author(s):  
Alison Ravenscraft ◽  
Michelle Berry ◽  
Tobin Hammer ◽  
Kabir Peay ◽  
Carol Boggs
Keyword(s):  
Amino Acids ◽  
Microbial Communities ◽  
Bacterial Species ◽  
Microbial Community Composition ◽  
Structure And Function ◽  
Feeding Guilds ◽  
Gut Flora ◽  
Microbial Composition ◽  
Host Diet ◽  
And Function

AbstractThe relationship between animals and their gut flora is simultaneously one of the most common and most complex symbioses on Earth. Despite its ubiquity, our understanding of this invisible but often critical relationship is still in its infancy. We employed adult Neotropical butterflies as a study system to ask three questions: First, how does gut microbial community composition vary across host individuals, species and dietary guilds? Second, how do gut flora compare to food microbial communities? Finally, are gut flora functionally adapted to the chemical makeup of host foods? To answer these questions we captured nearly 300 Costa Rican butterflies representing over 50 species, six families and two feeding guilds: frugivores and nectivores. We characterized the bacteria and fungi in guts, wild fruits and wild nectars via amplicon sequencing and assessed the catabolic abilities of the gut flora via culture-based assays.Gut communities were distinct from food communities, suggesting that the gut environment acts as a strong filter on potential colonists. Nevertheless, gut flora varied widely among individuals and species. On average, a pair of butterflies shared 21% of their bacterial species and 6% of their fungi. Host species explained 25-30% of total variation in microbial communities while host diet explained 4%. However, diet was still relevant at the individual microbe level—half of the most abundant microbial species differed in abundance between frugivores and nectivores. Diet was also related to the functional profile of gut flora: compared to frugivores, nectivores’ gut flora exhibited increased catabolism of sugars and sugar alcohols and decreased catabolism of amino acids, carboxylic acids and dicarboxylic acids. Since fermented juice contains more amino acids and less sugar than nectar, it appears that host diet filters the gut flora by favoring microbes that digest compounds abundant in foods.By quantifying the degree to which gut communities vary among host individuals, species and dietary guilds and evaluating how gut microbial composition and catabolic potential are related to host diet, this study deepens our understanding of the structure and function of one of the most complex and ubiquitous symbioses in the animal kingdom.

scholarly journals Metabolic dissimilarity determines the establishment of cross-feeding interactions in bacteria

2020 ◽  
Author(s):  
Samir Giri ◽  
Leonardo Oña ◽  
Silvio Waschina ◽  
Shraddha Shitut ◽  
Ghada Yousif ◽  
...  
Keyword(s):  
Microbial Community ◽  
Microbial Communities ◽  
Community Assembly ◽  
Bacterial Species ◽  
Exchange Interactions ◽  
Structure And Function ◽  
Phylogenetic Distance ◽  
And Function ◽  
Microbial Community Assembly ◽  
Feeding Interactions

AbstractThe exchange of metabolites among different bacterial genotypes is key for determining the structure and function of microbial communities. However, the factors that govern the establishment of these cross-feeding interactions remain poorly understood. While kin selection theory predicts that individuals should direct benefits preferentially to close relatives, the potential benefits resulting from a metabolic exchange may be larger for more distantly related species. Here we distinguish between these two possibilities by performing pairwise cocultivation experiments between auxotrophic recipients and 25 species of potential amino acid donors. Auxotrophic recipients were able to grow in the vast majority of pairs tested (78%), suggesting that metabolic cross-feeding interactions are readily established. Strikingly, both the phylogenetic distance between donor and recipient as well as the dissimilarity of their metabolic networks was positively associated with the growth of auxotrophic recipients. Finally, this result was corroborated in an in-silico analysis of a co-growth of species from a gut microbial community. Together, these findings suggest metabolic cross-feeding interactions are more likely to establish between strains that are metabolically more dissimilar. Thus, our work identifies a new rule of microbial community assembly, which can help predict, understand, and manipulate natural and synthetic microbial systems.SignificanceMetabolic cross-feeding is critical for determining the structure and function of natural microbial communities. However, the rules that determine the establishment of these interactions remain poorly understood. Here we systematically analyze the propensity of different bacterial species to engage in unidirectional cross-feeding interactions. Our results reveal that synergistic growth was prevalent in the vast majority of cases analyzed. Moreover, both phylogenetic and metabolic dissimilarity between donors and recipients favored a successful establishment of metabolite exchange interactions. This work identifies a new rule of microbial community assembly that can help predict, understand, and manipulate microbial communities for diverse applications.

scholarly journals First Report of Candidatus Liberibacter asiaticus Associated with Citrus Huanglongbing in California

Plant Disease ◽  
2013 ◽  
Vol 97 (2) ◽  
pp. 283-283 ◽  
Author(s):  
L. B. Kumagai ◽  
C. S. LeVesque ◽  
C. L. Blomquist ◽  
K. Madishetty ◽  
Y. Guo ◽  
...  
Keyword(s):  
Los Angeles ◽  
Real Time ◽  
16S Rdna ◽  
Real Time Pcr ◽  
Bacterial Species ◽  
The United States ◽  
Candidatus Liberibacter Asiaticus ◽  
Citrus Diseases ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus

Huanglongbing (HLB), also known as citrus greening, is one of the most destructive citrus diseases worldwide and is seen as a major threat to the multimillion dollar citrus industry in California. The vector of the two bacterial species associated with this disease, Candidatus Liberibacter asiaticus and Ca. L. americanus, is the Asian citrus psyllid (ACP), Diaphorina citri (4). ACP was detected in California in August of 2008 and has since been detected in nine counties in southern California. As part of a long term survey and testing program for the ACP carrying the HLB associated bacteria, groups of ACP nymphs and adults were submitted to the Jerry Dimitman Citrus Research Board/Citrus Pest and Disease Prevention Program Laboratory in Riverside, CA. In March 2012, DNA extracted using the Qiagen MagAttract 96 DNA plant kit (QIAGEN Inc., 27220 Turnberry Lane, Suite 200, Valencia, CA 91355) from a group of three ACP adults tested positive for Ca. L. asiaticus with the real-time PCR assay developed by Li et al. (4). ACP adults were collected from a residential citrus tree located in the Hacienda Heights area of Los Angeles County, California. The approximately 1.8 meter tall lemon tree had 23 graft unions, primarily of lemon (Citrus × meyeri) and pomelo (Citrus maxima) varieties. The tree was unthrifty, with yellow shoots and chlorotic leaves. Symptoms on the lemon and pomelo leaves included asymmetrical blotchy mottling, yellowing, and corking of the leaf veins, with the blotchy mottle more prominent in the pomelo leaves. Pomelo leaves appeared crinkled along the thickened veins. Lemon leaves had yellow veins and a few had islands of green tissue completely surrounded by yellow tissue. The entire tree was removed, cut into sections, bagged, and transported to the CDFA Plant Pest Diagnostics Lab for analysis. Two hundred milligrams of petiole and midrib tissue from leaves apical to each graft union was collected, and DNA from each sample was extracted using the Qiagen DNeasy plant mini kit. DNA extracted from both lemon and pomelo leaves tested positive for Ca. L. asiaticus using real-time PCR (4). A 1,160-bp fragment of the 16S ribosomal RNA gene was amplified from the insect and plant DNA extracts using conventional PCR with primers Ol1 and OI2c (2). A 703-bp fragment of the β-operon gene was amplified from the insect and plant extracts with primers A2 and J5 (1). The 16S rDNA fragments from the insect and plant respectively (GenBank Accession Nos. JX430434 and JX455745) and the β-operon fragments (JX430435 and JX455746) showed 100% identity with the corresponding regions of Ca. L. asiaticus (CP001677) strain psy 62. Our 16S rDNA sequence showed 98% identity with Ca. L. africanus (EU921620), 97% identity with Ca. L. solanacearum (HM246509), and 96% with Ca. L. americanus (FJ036892). In response to the detection of HLB, a 241 km2 quarantine area around the detection site was established. Surveys for ACP and symptomatic host plants within the HLB quarantine area are ongoing. To date, there have been no additional positive detections. In the United States, HLB was first detected in Florida in 2005 (4) and in Texas in January of 2012 (3). To our knowledge, this is the first confirmed report of Ca. L. asiaticus associated with HLB in California. References: (1) A. Hocquellet et al. Mol. Cell. Probes 13:373, 1999. (2) S. Jagoueix et al. Mol. Cell. Probes 10:43, 1996. (3) M. Kunta et al. Phytopathology 102:S4.66, 2012. (4) W. Li et al. J. Microbiol. Methods 66:104, 2006.

scholarly journals Two ‘Candidatus Liberibacter asiaticus’ Strains Recently Found in California Harbor Different Prophages

2017 ◽  
Vol 107 (6) ◽  
pp. 662-668 ◽  
Author(s):  
Z. Zheng ◽  
F. Wu ◽  
L. B. Kumagai ◽  
M. Polek ◽  
X. Deng ◽  
...  
Keyword(s):  
Los Angeles ◽  
Geographic Region ◽  
Mapping Technique ◽  
Polymorphism Analysis ◽  
Candidatus Liberibacter Asiaticus ◽  
Next Generation Sequencing Technology ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus

‘Candidatus Liberibacter asiaticus’ (CLas), an α-proteobacterium, is associated with citrus Huanglongbing (HLB; yellow shoot disease). In California, two cases of CLas have been detected in Los Angeles County, one in Hacienda Heights in 2012 and the other in San Gabriel in 2015. Although all infected trees were destroyed in compliance with a state mandate, citrus industry stakeholder concerns about HLB in California are high. Little is known about the biology of CLas, particularly the California strains, hindering effective HLB management efforts. In this study, next-generation sequencing technology (Illumina MiSeq) was employed to characterize the California CLas strains. Data sets containing >4 billion (Giga) bp of sequence were generated from each CLas sample. Two prophages (P-HHCA1-2 and P-SGCA5-1) were identified by the MiSeq read mapping technique referenced to two known Florida CLas prophage sequences, SC1 and SC2. P-HHCA1-2 was an SC2-like or Type 2 prophage of 38,989 bp in size. P-SGCA5-1 was an SC1-like or Type 1 prophage of 37,487 bp in size. Phylogenetic analysis revealed that P-HHCA1-2 was part of an Asiatic lineage within the Type 2 prophage group. Similarly, P-SGCA5-1 was part of an Asiatic lineage within Type 1 prophage group. The Asiatic relatedness of both P-HHCA1-2 and P-SGCA5-1 was further presented by single nucleotide polymorphism analysis at terL (encoding prophage terminase) that has been established for CLas strain differentiation. The presence of different prophages suggests that the two California CLas strains could have been introduced from different sources. An alternative explanation is that there was a mixed CLas population containing the two types of prophages, and limited sampling in a geographic region may not accurately depict the true CLas diversity. More accurate pathway analysis may be achieved by including more strains collected from the regions.

scholarly journals Susceptibility of Sixteen Citrus Genotypes to ‘Candidatus Liberibacter asiaticus’

Plant Disease ◽  
2016 ◽  
Vol 100 (6) ◽  
pp. 1080-1086 ◽  
Author(s):  
Greg McCollum ◽  
Mark Hilf ◽  
Mike Irey ◽  
Weiqi Luo ◽  
Tim Gottwald
Keyword(s):  
The United States ◽  
Insect Vector ◽  
Candidatus Liberibacter Asiaticus ◽  
Citrus Germplasm ◽  
Citrus Production ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Florida Citrus ◽  
Citrus Macrophylla ◽  
Free Ranging

Huanglongbing (HLB) disease is the most serious threat to citrus production worldwide and, in the last decade, has devastated the Florida citrus industry. In the United States, HLB is associated with the phloem-limited α-proteobacterium ‘Candidatus Liberibacter asiaticus’ and its insect vector, the Asian citrus psyllid (ACP; Diaphorina citri). Significant effort is being put forth to develop novel citrus germplasm that has a lower propensity to succumb to HLB than do currently available varieties. Effective methods of screening citrus germplasm for susceptibility to HLB are essential. In this study, we exposed small, grafted trees of 16 citrus types to free-ranging ACP vectors and ‘Ca. L. asiaticus’ inoculum in the greenhouse. During 45 weeks of exposure to ACP, the cumulative incidence of ‘Ca. L. asiaticus’ infection was 70%. Trees of Citrus macrophylla and C. medica were most susceptible to ‘Ca. L. asiaticus’, with 100% infection by the end of the test period in three trials, while the complex genetic hybrids ‘US 1-4-59’ and ‘Fallglo’ consistently were least susceptible, with approximately 30% infection. Results obtained in this greenhouse experiment showed good agreement with trends observed in the orchard, supporting the validity of our approach for screening citrus germplasm for susceptibility to HLB.

scholarly journals A New Diagnostic System for Detection of ‘Candidatus Liberibacter asiaticus’ Infection in Citrus

Plant Disease ◽  
2013 ◽  
Vol 97 (10) ◽  
pp. 1295-1300 ◽  
Author(s):  
Lianming Lu ◽  
Baoping Cheng ◽  
Jinai Yao ◽  
Aitian Peng ◽  
Danchao Du ◽  
...  
Keyword(s):  
Affinity Chromatography ◽  
Polyclonal Antibodies ◽  
Diagnostic System ◽  
Enzyme Linked Immunosorbent Assay ◽  
Candidatus Liberibacter Asiaticus ◽  
Negative Results ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Polymerase Chain

In this study, two polyclonal antibodies were produced against the Omp protein of ‘Candidatus Liberibacter asiaticus’. First, omp genes were sequenced to exhibit 99.9% identity among 137 isolates collected from different geographical origins. Then, two peptides containing the hydrophobic polypeptide-transport-associated (POTRA) domain and β-barrel domain, respectively, were identified on Omp protein. After that, these two peptides were overexpressed in Escherichia coli and purified by affinity chromatography to immunize the white rabbits. Finally, the antiserum was purified by affinity chromatography. The two Omp antibodies gave positive results (0.454 to 0.633, 1:1,600 dilution) in enzyme-linked immunosorbent assay against ‘Ca. L. asiaticus’-infected samples collected from different geographical origins but revealed negative results against other pathogen-infected, nutrient-deficient and healthy samples. The antibody against the POTRA domain of Omp protein could detect ‘Ca. L. asiaticus’ in 45.7% of the symptomatic samples compared with a 56.2% detection rate with a polymerase chain reaction assay. These new antibodies will provide a very useful supplement to the current approaches to ‘Ca. L. asiaticus’ detection and also provide powerful research tools for tracking distribution of this pathogen in vivo.

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