Scientific Reports

AbstractHuanglongbing (HLB) is a serious disease of Citrus sp. worldwide. In Africa and the Mascarene Islands, a similar disease is known as African citrus greening (ACG) and is associated with the bacterium Candidatus Liberibacter africanus (Laf). In recent years, Candidatus Liberibacter asiaticus (Las) associated with the severe HLB has been reported in Ethiopia. Thus, we aimed to identify the Liberibacter species affecting citrus, the associated vectors in Eastern Africa and their ecological distribution. We assessed the presence of generic Liberibacter in symptomatic leaf samples by quantitative PCR. Subsequently, we sequenced the 50 S ribosomal protein L10 (rplJ) gene region in samples positive for Liberibacters and identified the species by comparison with public sequence data using phylogenetic reconstruction and genetic distances. We detected generic Liberibacter in 26%, 21% and 66% of plants tested from Uganda, Ethiopia and Kenya, respectively. The rplJ sequences revealed the most prevalent Liberibacters in Uganda and Ethiopia were LafCl (22%) and Las (17%), respectively. We detected Las in Kenya for the first time from three sites in the coastal region. Finally, we modelled the potential habitat suitability of Las in Eastern Africa using MaxEnt. The projection showed large areas of suitability for the pathogen in the three countries surveyed. Moreover, the potential distribution in Eastern Africa covered important citrus-producing parts of Ethiopia, Kenya, Uganda and Tanzania, and included regions where the disease has not been reported. These findings will guide in the development of an integrated pest management strategy to ACG/HLB management in Africa.

AbstractBois noir, a disease of the grapevine yellows complex, is associated with ‘CandidatusPhytoplasma solani’ and transmitted to grapevines in open fields by the cixiidsHyalesthes obsoletusandReptalus panzeri. In vine-growing areas where the population density of these vectors is low within the vineyard, the occurrence of bois noir implies the existence of alternative vectors. The aim of this study was to identify alternative vectors through screening of the Auchenorrhyncha community, phytoplasma typing bystampgene sequence analyses, and transmission trials. During field activities, conducted in Northern Italy in a vineyard where the bois noir incidence was extremely high, nine potential alternative insect vectors were identified according to high abundance in the vineyard agro-ecosystem, high infection rate, and harbouring phytoplasma strains characterized bystampgene sequence variants found also in symptomatic grapevines. Transmission trials coupled with molecular analyses showed that at least eight species (Aphrodes makarovi,Dicranotropis hamata,Dictyophara europaea,Euscelis incisus,Euscelidius variegatus,Laodelphax striatella,Philaenus spumarius, andPsammotettix alienus/confinis) are alternative vectors of ‘CandidatusPhytoplasma solani’ to grapevines. These novel findings highlight that bois noir epidemiology in vineyard agro-ecosystems is more complex than previously known, opening up new perspectives in the disease management.

AbstractHuanglongbing (HLB) is a worldwide deadly citrus disease caused by the phloem-limited bacteria ‘Candidatus Liberibacter asiaticus’ (CLas) vectored by Asian citrus psyllids. In order to effectively manage this disease, it is crucial to understand the relationship among the bacterial isolates from different geographical locations. Whole genome sequencing approaches will provide more precise molecular characterization of the diversity among populations. Due to the lack of in vitro culture, obtaining the whole genome sequence of CLas is still a challenge, especially for medium to low titer samples. Hundreds of millions of sequencing reads are needed to get good coverage of CLas from an HLB positive citrus sample. In order to overcome this limitation, we present here a new method, Agilent SureSelect XT HS target enrichment, which can specifically enrich CLas from a metagenomic sample while greatly reducing cost and increasing whole genome coverage of the pathogen. In this study, the CLas genome was successfully sequenced with 99.3% genome coverage and over 72X sequencing coverage from low titer tissue samples (equivalent to 28.52 Cq using Li 16 S qPCR). More importantly, this method also effectively captures regions of diversity in the CLas genome, which provides precise molecular characterization of different strains.

AbstractRickettsiales bacteria in arthropods play a significant role in both public health and arthropod ecology. However, the extensive genetic diversity of Rickettsiales endosymbionts of arthropods is still to be discovered. In 2016, 515 arthropods belonging to 9 species of four classes (Insecta, Chilopoda, Diplopoda and Arachnida) were collected in Serbia. The presence and genetic diversity of Rickettsiales bacteria were evaluated by characterizing the 16S rRNA (rrs), citrate synthase (gltA) and heat shock protein (groEL) genes. The presence of various Rickettsiales bacteria was identified in the majority of tested arthropod species. The results revealed co-circulation of five recognized Rickettsiales species including Rickettsia, Ehrlichia and Wolbachia, as well as four tentative novel species, including one tentative novel genus named Neowolbachia. These results suggest the remarkable genetic diversity of Rickettsiales bacteria in certain arthropod species in this region. Furthermore, the high prevalence of spotted fever group Rickettsia in Ixodes ricinus ticks highlights the potential public health risk of human Rickettsia infection.

AbstractPlant pathogenic bacteria interact with their insect host(s)/vector(s) at the cellular and molecular levels. This interaction may alter the physiology of their insect vector, which may also promote the growth and transmission of the bacterium. Here we studied the effect of “Candidatus Liberibacter asiaticus” (“Ca. L. asiaticus”) on physiochemical conditions within its insect vector, the Asian citrus psyllid (ACP), and whether these changes were beneficial for the pathogen. The local microenvironments inside ACPs were quantified using microelectrodes. The average hemolymph pH was significantly higher in infected ACPs (8.13 ± 0.21) than in “Ca. L. asiaticus”-free ACPs (7.29 ± 0.15). The average hemolymph oxygen tension was higher in “Ca. L. asiaticus”-free ACPs than in infected ACPs (67.13% ± 2.11% vs. 35.61% ± 1.26%). Oxygen tension reduction and pH increase were accompanied by “Ca. L. asiaticus” infection. Thus, oxygen tension of the hemolymph is an indicator of infection status, with pH affected by the severity of the infection.

AbstractThe tomato-potato psyllid (TPP), Bactericera cockerelli, is a vector for the phloem-limited bacterium Candidatus Liberibacter solanacearum (Lso), the causative agent of economically important diseases including tomato vein-greening and potato zebra chip. Here, we screened 11 wild tomato relatives for TPP resistance as potential resources for tomato (Solanum lycopersicum) cultivar development. Six accessions with strong TPP resistance (survival <10%) were identified within S. habrochaites, S. pennelli, S. huaylasense, S. chmielewskii, S. corneliomulleri, and S. galapagense. Two S. pennelli and S. corneliomulleri accessions also showed resistance to Lso. We evaluated recombinant inbred lines (RILs) carrying resistance from S. habrochaites accession LA1777 in the S. lycopersicum background and identified major quantitative trait loci (QTLs) responsible for adult TPP mortality and fecundity in several RILs carrying insertions in different chromosomes, indicating the polygenic nature of these traits. Analysis of a major resistance QTL in RIL LA3952 on chromosome 8 revealed that the presence of Lso is required to increase adult TPP mortality. By contrast, the reduced TPP oviposition trait in LA3952 is independent of Lso. Therefore, resistance traits are available in wild-tomato species, although their complex inheritance and modes of action require further characterisation to optimise their utilisation for tomato improvement.

Abstract We used phenotypic, genomic and phylogenetic information following the taxono-genomics approach to demonstrate that strain Marseille–P3254, isolated from an ileal sample of a 76-year old woman who underwent upper and lower digestive tract endoscopy for esophagitis and colonic polyp, is representative of a novel bacterial genus within the family Erysipelotrichaceae in the phylum Firmicutes. It is an anaerobic Gram-negative bacterium without catalase and oxidase activities. The genome of strain Marseille–P3254 is 2,468,496-bp long with a 40.1% G + C content. This new bacterium is most closely related to Eubacterium dolichum, with which it shares 90.7% 16S rRNA sequence similarity. In addition, genomic comparison using the digital DNA–DNA hybridization and OrthoANI analyses between the novel organism and the E. dolichum type strain revealed identities of 25.2 and 68.91%, respectively. The major fatty acids were C16: 0, C18: 1n9 and C18: 0. Based on these data, we propose the creation of the new genus Merdibacter gen. nov., with strain Marseille-P3254T (=CSUR P3254 = DSM 103534) being the type strain of the new species Merdibacter massiliensis gen. nov., sp. nov.

AbstractThe Liberibacter genus comprises insect endosymbiont bacterial species that cause destructive plant diseases, including Huanglongbing in citrus and zebra chip in potato. To date, pathogenic ‘Candidatus Liberibacter spp.’ (CLs) remain uncultured, therefore the plant-associated Liberibacter crescens (Lcr), only cultured species of the genus, has been used as a biological model for in vitro studies. Biofilm formation by CLs has been observed on the outer midgut surface of insect vectors, but not in planta. However, the role of biofilm formation in the life cycle of these pathogens remains unclear. Here, a model system for studying CLs biofilms was developed using Lcr. By culture media modifications, bovine serum albumin (BSA) was identified as blocking initial cell-surface adhesion. Removal of BSA allowed for the first time observation of Lcr biofilms. After media optimization for biofilm formation, we demonstrated that Lcr attaches to surfaces, and form cell aggregates embedded in a polysaccharide matrix both in batch cultures and under flow conditions in microfluidic chambers. Biofilm structures may represent excellent adaptive advantages for CLs during insect vector colonization helping with host retention, immune system evasion, and transmission. Future studies using the Lcr model established here will help in the understanding of the biology of CLs.

Abstract‘Candidatus Phytoplasma solani’ is the causal agent of Bois noir (BN) in grapevine (Vitis vinifera). It is usually detected in leaves, where typical disease symptoms are seen. However, little information is available on the presence of this phytoplasma in grapevine roots. Here, we investigated ‘Ca. P. solani’ in roots collected from 28 symptomatic, 27 recovered and eight asymptomatic grapevine plants. Protocols based on high-resolution melting (HRM) combined with real-time quantitative PCR (qPCR-HRM) and nested-qPCR-HRM were developed to identify ‘Ca. P. solani’ tuf-type variants with single nucleotide polymorphisms. In all, 21.4% of roots from symptomatic plants were positive to ‘Ca. P. solani’ using qPCR-HRM, and 60.7% with nested-qPCR HRM. Also, 7.4% of roots from recovered plants were positive using qPCR-HRM, which reached 44.4% using nested-qPCR HRM. These analyses identified tuf-type b1 on 88.2% of the positive samples from symptomatic grapevines, and 66.6% from recovered grapevines, with all other samples identified as tuf-type a. This study reports the presence of ‘Ca. P. solani’ in the roots of both symptomatic and recovered grapevines. These qPCR-HRM and nested-qPCR-HRM protocols can be applied to increase the sensitivity of detection of, and to simplify and speed up the screening for, ‘Ca. P. solani’ tuf-types.

AbstractMembers of the order Rickettsiales are often found in association with ciliated protists. An interesting case is the bacterial endosymbiont “Candidatus Megaira”, which is phylogenetically closely related to the pathogen Rickettsia. “Candidatus Megaira” was first described as an intracellular bacterium in several ciliate species. Since then it has been found in association with diverse evolutionary distantly-related hosts, among them other unicellular eukaryotes, and also algae, and metazoa, such as cnidarians. We provide the characterization of several new strains of the type species “Candidatus Megaira polyxenophila”, and the multidisciplinary description of a novel species, “Candidatus Megaira venefica”, presenting peculiar features, which highlight the diversity and variability of these widespread bacterial endosymbionts. Screening of the 16S rRNA gene short amplicon database and phylogenetic analysis of 16S rRNA gene hypervariable regions revealed the presence of further hidden lineages, and provided hints on the possibility that these bacteria may be horizontally transmitted among aquatic protists and metazoa. The phylogenetic reconstruction supports the existence of at least five different separate species-level clades of “Candidatus Megaira”, and we designed a set of specific probes allowing easy recognition of the four major clades of the genus.