Publications

4493

Summary ‘ Candidatus Liberibacter asiaticus’ (CLas) is a phloem‐limited non‐culturable α‐proteobacterium associated with citrus Huanglongbing, a highly destructive disease threatening global citrus industry. Research on CLas is challenging due to the current inability to culture CLas in vitro and the low CLas titre in citrus plant. Here, we develop a CLas enrichment system using the holoparasitic dodder plant ( Cuscuta campestris ) as an amenable host to acquire and enrich CLas from CLas‐infected citrus shoots maintained hydroponically. Forty‐eight out of fifty‐five (87%) dodder plants successfully parasitized CLas‐infected citrus shoots with detectable CLas by PCR. Among 48 dodders cultures, 30 showed two‐ to 419‐fold CLas titre increase as compared to the corresponding citrus hosts. The CLas population rapidly increased and reached the highest level in dodder tendrils at 15 days after parasitizing citrus shoot. Genome sequencing and assembly derived from CLas‐enriched dodder DNA samples generated a higher resolution than those obtained for CLas from citrus hosts. No genomic variation was detected in CLas after transmission from citrus to dodder during short‐term parasitism. Dual RNA‐Seq experiments showed similar CLas gene expression profiles in dodder and citrus samples, yet dodder samples generated a higher resolution of CLas transcriptome data. The ability of dodder to support CLas multiplication to high levels, as well as its advantage in CLas genomic and transcriptomic analyses, make it an optimal model for further studies on CLas–host interaction.

The Asian citrus psyllid Diaphorina citri is the transmission vector of Huanglongbing (HLB), a devastating disease of citrus plants. The bacterium “Candidatus Liberibacter asiaticus” (CLas) associated with HLB is transmitted between host plants by D. citri in a circulative manner. Understanding the interaction between CLas and its insect vector is key for protecting citrus cultivation from HLB damage. Here, we used RNA sequencing and liquid chromatography-mass spectrometry (LC-MS) to analyze the transcriptome and metabolome of D. citri interacting with CLas. We identified 662 upregulated and 532 downregulated genes in CLas-infected insects. These genes were enriched in pathways involving carbohydrate metabolism, the insects’ immune system, and metabolism of cofactors and vitamins. We also detected 105 differential metabolites between CLas-infected and non-infected insects, including multiple nucleosides and lipid-related molecules. The integrated analysis revealed nine pathways—including those of the glycine, serine, threonine, and purine metabolism—affected by the differentially expressed genes from both groups. The network for these pathways was subsequently constructed. Our results thus provide insights regarding the cross-talk between the transcriptomic and metabolomic changes in D. citri in response to CLas infection, as well as information on the pathways and genes/metabolites related to the CLas–D. citri interaction.

Ammonia tolerance of AOA is usually much lower than that of the AOB, which makes the AOB rather than AOA a predominant ammonia oxidizer in agricultural soils, contributing to global N 2 O emission. Recently, some AOA species from the genus “ Ca. Nitrosocosmicus” were also found to have high ammonia tolerance.

Abstract Background Hemotropic mycoplasmas (hemoplasmas) infect animals and humans and can lead to clinical syndromes mainly characterized by hemolytic anemia. A novel pathogen, Candidatus Mycoplasma haemohominis, was recently associated with a case of human hemoplasmosis in Europe. Here we report the first detection of this pathogen in an Australian patient exhibiting persistent fever, hemolytic anemia, and pancytopenia over a 10-month period. Methods After exhaustive negative testing for human infectious diseases, whole genome sequencing (WGS) was performed on the patient’s bone marrow aspirate, using an Illumina NextSeq500 platform. Conventional polymerase chain reaction (PCR), followed by Sanger sequencing, was then performed on blood samples using novel Mycoplasma-specific primers targeting the 16S ribosomal RNA gene. In addition, a Mycoplasma-specific fluorescence in situ hybridization (FISH) assay was developed to differentiate Mycoplasma cells from other erythrocyte inclusions (eg, Pappenheimer and Howell-Jolly bodies) which are morphologically similar to bacterial cocci by light microscopy. Results WGS analysis revealed that approximately 0.04% of the total number of unmapped reads to human genome corresponded to Mycoplasma species. A 1-kb Mycoplasma 16S fragment was successfully amplified by conventional PCR, and sequence analyses revealed 100% identity with Candidatus Mycoplasma haemohominis. FISH confirmed that several (approximately 2%) epierythrocytic inclusions initially observed by light microscopy corresponded to Mycoplasma cells. Conclusions This represents the second report of hemolytic anemia associated with hemoplasma infection in a human, and the first report of human hemoplasmosis in Australia. This study highlights the importance of new and emerging diagnostic approaches and need for further investigations on the epidemiology of Candidatus Mycoplasma haemohominis in Australia.

The present study examined the effects of Candidatus Phytoplasma solani infection on antioxidative metabolism in leaves and roots of carrot (Daucus carota L.). Disease symptoms appeared at the end of June in the form of the chlorosis on some of the leaves, which became intensely red one week later, while the previously healthy leaves from the same branch becme chlorotic. A few days later, all leaves from the infected leaf branch were intensely red. Infected plants also had slower growth compared to the healthy ones with fewer leaf branches developed. The roots of infected plants were less developed, seared, or gummy with or without brown-colored root hair. The presence of the pathogen was detected by sequencing the 16S rRNA. National Center for Biotechnology Information (NCBI) BLAST analyses of the obtained sequence revealed 100% identity of tested strain with deposited Ca. Phytoplasma solani strains from various countries and hosts, all belonging to the “stolbur” group (16SrXII-A). Identity of 99.74% was found when the tested Serbian strain (MF503627) was compared with the reference stolbur strain STOL11 (AF248959). The oxidative damage of membranes in carrot cells was accompanied by a decrease in the content of photosynthetic pigments. Furthermore, for the determination of specific scavenging properties of the extracts, in vitro antioxidant assay was performed. In phytoplasma-infected carrot leaves, there was a greater reduction in the level of glutathione content (GSH); however; flavonoids and anthocyanidins seem to be responsible for the accompanied increased antioxidative capacity against hydroxyl radical and hydrogen peroxide.

Aster Yellows phytoplasma (AYp; ‘Candidatus Phytoplasma asteris’) is an obligate bacterial pathogen that is the causative agent of multiple diseases in herbaceous plants. While this phytoplasma has been examined in depth for its disease characteristics, knowledge about the spatial and temporal dynamics of pathogen spread is lacking. The phytoplasma is found in plant’s phloem and is vectored by leafhoppers (Cicadellidae: Hemiptera), including the aster leafhopper, Macrosteles quadrilineatus Forbes. The aster leafhopper is a migratory insect pest that overwinters in the southern United States, and historical data suggest these insects migrate from southern overwintering locations to northern latitudes annually, transmitting and driving phytoplasma infection rates as they migrate. A more in-depth understanding of the spatial, temporal and genetic determinants of Aster Yellows disease progress will lead to better integrated pest management strategies for Aster Yellows disease control. Carrot, Daucus carota L., plots were established at two planting densities in central Wisconsin and monitored during the 2018 growing season for Aster Yellows disease progression. Symptomatic carrots were sampled and assayed for the presence of the Aster Yellows phytoplasma. Aster Yellows disease progression was determined to be significantly associated with calendar date, crop density, location within the field, and phytoplasma subgroup.

AbstractCitrus Greening disease (CG) in South Africa (SA) is associated with the fastidious bacterium ‘CandidatusLiberibacter africanus’ (Laf). It has been observed that Laf isolates obtained from different geographic localities in SA differed in the rate of transmission during grafting experiments leading to the hypothesis that genetic variation of Laf may exist in this country. To determine this, 167 Laf isolates obtained from Limpopo, North West, Mpumalanga and the Western Cape were subjected to microsatellite analyses, using four polymorphic markers. From UPGMA and STRUCTURE analysis, it was shown that most sources belong to one of two major genetic groups of Laf and these comprise 25 distinct haplotypes. Four samples included within this study did not group with these two major groups, suggesting a potential third and fourth genetic group of Laf being present, which can be validated by further sampling. Results further indicate that Laf populations in SA are formed by geographic locality. The high genetic diversity observed for Laf within this study is consistent with the hypothesis that Laf originated on the African continent, warranting further genetic analysis of Laf populations from Africa. This is the first study to unveil the genetic diversity of Laf.

Abstract The symbiosis between bacteria and sponges has arguably the longest evolutionary history for any extant metazoan lineage, yet little is known about bacterial evolution or adaptation in this process. An example of often dominant and widespread bacterial symbionts of sponges is a clade of uncultured and uncharacterised Proteobacteria. Here we set out to characterise this group using metagenomics, in-depth phylogenetic analyses, metatranscriptomics, and fluorescence in situ hybridisation microscopy. We obtained five metagenome-assembled-genomes (MAGs) from different sponge species that, together with a previously published MAG (AqS2), comprise two families within a new gammaproteobacterial order that we named Ca. Tethybacterales. Members of this order share a heterotrophic lifestyle but vary in their predicted ability to use various carbon, nitrogen and sulfur sources, including taurine, spermidine and dimethylsulfoniopropionate. The deep branching of the Ca. Tethybacterales within the Gammaproteobacteria and their almost exclusive presence in sponges suggests they have entered a symbiosis with their host relatively early in evolutionary time and have subsequently functionally radiated. This is reflected in quite distinct lifestyles of various species of Ca. Tethybacterales, most notably their diverse morphologies, predicted substrate preferences, and localisation within the sponge tissue. This study provides new insight into the evolution of metazoan–bacteria symbiosis.