Publications

4369

Citrus Huanglongbing (HLB, also known as “citrus greening”), an important disease worldwide, is associated with three species of phloem-limited Candidatus liberibacter, of which Candidatus L. asiaticus (CLas) is the predominant one that has severely affected citrus production. TaqMan real-time polymerase chain reaction (PCR) (TM) has been the standard and very efficient method to diagnose several strains of Candidatus Liberibacter in citrus; however, it detects total bacteria and is unable to differentiate dead from live Liberibacter. The detection of only live bacteria is essential for testing methods of control for this important citrus disease. It is well known that ethidium monoazide and propidium monoazide (PMA) are compounds that supposedly enter only dead or membrane-damaged bacteria, intercalate the DNA strand, and make the DNA unavailable for amplification by PCR. These compounds are widely used when extracting the plant DNA to detect only live bacteria. In this research, we tested primers amplifying products from 79 to 1160 bp in TM and SYBR Green real-time PCR (SG) and PMA as DNA intercalating compound. Specifically, primers amplifying a 500-bp amplicon in SG provided the most reliable live-only detection, whereas those producing a smaller amplicon were unable to distinguish between live and dead. This is the first report of testing primers amplifying various amplicon sizes for the detection of only live CLas cells in citrus.

Citrus huanglongbing (HLB) is present in 10 provinces in China and is associated with ‘Candidatus Liberibacter asiaticus’ (CLas), which is transmitted by the Asian citrus psyllid (Diaphorina citri, ACP). To date, HLB and ACP have expanded to Yibin city of Sichuan Province, posing an imminent threat to the citrus belt of the upper and middle reaches of the Yangtze River, an important late-maturing citrus-producing area in China. To understand the epidemiological route of CLas and ACP in newly invaded regions of Sichuan and thereby better establish an HLB interception zone ranging from Leibo to Yibin, we evaluated the molecular variability of 19 CLas draft genomes from citrus or dodder (Cuscuta campestris). They include three type-specific prophage loci, three variable number tandem repeat loci, a miniature inverted-repeat transposable element, and population diversity of 44 ACP mitochondrial genomes. The results indicated that CLas isolates in the newly invaded area (Pingshan) were more diverse than those in the HLB endemic areas (Leibo and Ningnan). Phylogenetic analysis based on mitochondrial genomes demonstrated that ACPs in Leibo, Pingshan, and Xuzhou (rural areas) represent a new mitochondrial group (MG4), distinguished by the three unique single-nucleotide polymorphisms in cox1, nad4, and cytb. However, the ACPs sampled from the urban areas of Cuiping and Xuzhou belonged to the southeastern China group (MG2-1). Altogether, our study revealed multiple sources of ACP and CLas in the HLB interception zone and proposed their transmission route. This study contributes to the formulation of precise HLB prevention and control strategies in the HLB interception zone in Sichuan and could be useful for HLB management efforts in other regions.

Abstract A new distribution map is provided for Candidatus Phytoplasma cynodontis Marcone et al. Mollicutes: Acholeplasmatales: Acholeplasmataceae. Main host: Bermuda grass ( Cynodon dactylon ). Information is given on the geographical distribution in Africa (Ethiopia, Kenya, Sudan, Tanzania, Uganda), Asia (China, India, Karnataka, Kerala, Madhya Pradesh, Tamil Nadu, Uttar Pradesh, Indonesia, Iran, Iraq, Israel, Malaysia, Myanmar, Pakistan, Saudi Arabia, Singapore, Sri Lanka, Taiwan, Thailand, Turkey, Vietnam), Europe (Albania, Italy, Serbia), North America (Cuba), Oceania (Australia, Northern Territory, Western Australia).

Grapevine Bois noir (BN) is associated with ‘Candidatus Phytoplasma solani’. It has been recorded in vineyards throughout Europe as well as in different countries in Asia, where it now constitutes a threat to Iranian viticulture. BN is strictly dependent on ‘Ca. P. solani’ strains, wild host plants, and insect vectors. The molecular typing of ‘Ca. P. solani’, based on the nonribosomal gene tuf and the two hypervariable markers vmp1 and stamp, is valuable for the reconstruction and clarification of the pathways of BN spread. In this study, an RFLP analysis was performed on the vmp1 gene, and a single-nucleotide polymorphism analysis confirmed new vmp types in ‘Ca. P. solani’. A stamp gene phylogenetic analysis allowed us to distinguish between the new genotype infections in the grapevines and the ‘weeds’ Convolvulus arvensis and Erigeron bonariensis in Iranian vineyards, highlighting the close genetic relatedness of the strains of ‘Ca. P. solani’ found in Iran and Azerbaijan. The most common genotype in the grapevines was tuf b/V24/stamp III, which was associated with C. arvensis. This information contributes toward the identification of further routes of introduction of ‘Ca. P. solani’ in Iran to sustain the control measures for the management of BN.

‘Candidatus Liberibacter solanacearum’ (CaLsol) is an uncultured bacterium, transmitted by psyllids and associated with several diseases in Solanaceae and Apiaceae crops. CaLsol detection in psyllids often requires insect destruction, preventing a subsequent morphological identification. In this work, we have assessed the influence on the detection of CaLsol by PCR in Bactericera trigonica (Hemiptera: Psyllidae), of four specimen preparations (entire body, ground, cut-off head, and punctured abdomen) and seven DNA extraction methods (PBS suspension, squashing on membrane, CTAB, Chelex, TRIsureTM, HotSHOT, and DNeasy®). DNA yield and purity ratios, time consumption, cost, and residues generated were also evaluated. Optimum results were obtained through grinding, but it is suggested that destructive procedures are not essential in order to detect CaLsol. Although CaLsol was detected by qPCR with DNA obtained by the different procedures, HotSHOT was the most sensitive method. In terms of time consumption and cost, squashed on membrane, HotSHOT, and PBS were the fastest, while HotSHOT and PBS were the cheapest. In summary, HotSHOT was accurate, fast, simple, and sufficiently sensitive to detect this bacterium within the vector. Additionally, cross-contamination with CaLsol was assessed in the ethanol solutions where B. trigonica specimens were usually collected and preserved. CaLsol-free psyllids were CaLsol-positive after incubation with CaLsol-positive specimens. This work provides a valuable guide when choosing a method to detect CaLsol in vectors according to the purpose of the study.

AbstractThe phylum “Candidatus Nanohaloarchaeota” is a representative halophilic lineage within DPANN superphylum. They are characterized by their nanosized cells and symbiotic lifestyle with Halobacteria. However, the development of the symbiosis remains unclear for the lack of genomes located at the transition stage. Here, we performed a comparative genomic analysis of “Ca. Nanohaloarchaeota”. We propose a novel family “Candidatus Nanoanaerosalinaceae” represented by five de-replicated metagenome-assembled genomes obtained from hypersaline sediments and the enrichment cultures of soda-saline lakes. Phylogeny analysis reveals that the novel family are placed at the root of the family “Candidatus Nanosalinaceae” including the well-researched taxa. Most members of “Ca. Nanoanaerosalinaceae” contain lower proportion of putative horizontal gene transfers from Halobacteria than “Ca. Nanosalinaceae”, while they maintain moderately acidic proteomes for hypersaline adaptation of “salt-in” strategy, suggesting that “Ca. Nanoanaerosalinaceae” have not established an intimate association with Halobacteria, and may descend from an intermediate stage. Functional prediction discloses that they exhibit divergent potentials in carbohydrate and organic acids metabolism, and environmental responses. Historical events reconstruction illustrates that the involved genes acquired at the putative ancestors possibly drive the evolutionary and symbiotic divergences. Globally, this research on the new family “Ca. Nanoanaerosalinaceae” enriches the taxonomic and functional diversity of “Ca. Nanohaloarchaeota”, and provides insights into the evolutionary process of “Ca. Nanohaloarchaeota” and their Halobacteria-associated symbiosis.ImportanceDPANN superphylum is a group of archaea widely distributing in various habitats. They generally have small cells, and perform a symbiotic lifestyle with other archaea. The archaeal symbiotic interaction is important to understand microbial community. However, the formation and evolution of the symbiosis between the DPANN lineages and other diverse archaea remain unclear. Based on phylogeny, hypersaline adaptation, functional potentials, and historical events of “Ca. Nanohaloarchaeota”, a representative phylum within the DPANN superphylum, we report a novel family descending from an intermediate stage, and we illustrate the evolutionary process of “Ca. Nanohaloarchaeota” and their Halobacteria-associated symbiosis. Furthermore, we find the acquired genes involved in carbohydrate and organic acids metabolism and environmental responses possibly drive the evolutionary and symbiotic divergences. Altogether, this research helps in understanding the evolution of the archaeal symbiosis, and provides a model for the evolution of the other DPANN lineages.

As the causal agent of the grapevine yellows disease Bois noir, ‘Candidatus Phytoplasma solani' has a major economic impact on grapevines. To improve the control of Bois noir, it is critical to understand the very complex epidemiological cycles that involve the multiple “Ca. P. solani” host plants and insect vectors, of which Hyalesthes obsoletus is the most important. In the present study, multiple genotyping of the tuf, secY, stamp, and vmp1 genes was performed. This involved archived grapevine samples that were collected during an official survey of grapevine yellows throughout the wine-growing regions of Slovenia (from 2003 to 2016), plus samples from Austrian grapevines, stinging nettle, field bindweed, and insect samples (collected from 2012 to 2019). The data show that the tuf-b2 type of the tuf gene has been present in eastern Slovenia since at least 2003. The hypotheses that the occurrence of the haplotypes varies due to the geographical position of Slovenia on the Italian–Slovenian Karst divide and that the haplotypes are similar between Slovenian and Austrian Styria were confirmed. The data also show haplotype changes for host plants and H. obsoletus associated with ‘Ca. P. solani,' which might be linked to new epidemiological cycles of this phytoplasma that involve not just new plant sources and new insect vectors, but also climate and land-use changes.

Objetivo. Informe sobre especies de garrapatas que parasitan a los humanos en un área del bioma semiárido de Caatinga, noreste de Brasil, con nota de infección por rickettsias. Materiales y métodos. Todas las garrapatas se identificaron morfológicamente con estereomicroscopio. Algunas de las garrapatas recolectadas se analizaron molecularmente para detectar la presencia de ADN de bacterias del género Rickettsia. Las garrapatas se sometieron individualmente a la extracción de ADN (gen de la citrato sintasa [gltA] y gen de la proteína de la membrana externa [ompA]). Resultados. Se recolectaron un total de 78 garrapatas en humanos, identificadas como adultos de Amblyomma parvum (62 hembras y 16 machos). De estas, 15 mujeres fueron infectadas por ‘Candidatus Rickettsia andeanae’. Conclusiones. El presente estudio confirma una zona más de riesgo humano de picaduras de garrapatas en Brasil, también de la rickettsia no patógena 'Ca. Rickettsia andeanae'

AbstractMembers of the bacterial genusRickettsiawere originally identified as causative agents of vector-borne diseases in mammals. However, manyRickettsiaspecies are arthropod symbionts and close relatives of ‘CandidatusMegaira’, which are symbiotic associates of microeukaryotes. Here, we clarify the evolutionary relationships between these organisms by assembling 26 genomes ofRickettsiaspecies from understudied groups, including the Torix group, and two genomes of ‘Ca. Megaira’ from various insects and microeukaryotes. Our analyses of the new genomes, in comparison with previously described ones, indicate that the accessory genome diversity and broad host range of TorixRickettsiaare comparable to those of all otherRickettsiacombined. Therefore, the Torix clade may play unrecognized roles in invertebrate biology and physiology. We argue this clade should be given its own genus status, for which we propose the name ‘CandidatusTisiphia’.