Publications

4861

Citrus huanglongbing (HLB) disease associated with the ‘Candidatus Liberibacter asiaticus’ (CLas) bacterium has caused significant financial damage to many citrus industries. Large-scale pathogen surveys are routinely conducted in California to detect CLas early in the disease cycle by lab-based qPCR assays. We have developed an improved reference gene for the sensitive detection of CLas from plants in diagnostic duplex qPCR and analytical digital droplet PCR (ddPCR) assays. The mitochondrial cytochrome oxidase gene (COX), widely used as a reference, is not ideal because its high copy number can inhibit amplification of small quantities of target genes. In ddPCRs, oversaturation of droplets complicates data normalization and quantification. The variable copy numbers of COX gene in metabolically active young tissue, greenhouse plants, and citrus relatives suggest the need for a non-variable, nuclear, low copy, universal reference gene for analysis of HLB hosts. The single-copy nuclear gene, malate dehydrogenase (MDH), developed here as a reference gene, is amenable to data normalization, suitable for duplex qPCR and ddPCR assays. The sequence of MDH fragment selected is conserved in most HLB hosts in the taxonomic group Aurantioideae. This study emphasizes the need to develop standard guidelines for reference genes in DNA-based PCR assays.

Abstract Background Candidatus (Ca.) Neoehrlichia (N.) mikurensis is an emerging tick-borne pathogen of humans that is closely related to Ehrlichia and Anaplasma species. This strict intracellular bacterium escapes detection by routine microbiologic diagnostic methods such as blood culture, leading to considerable under-diagnosis of the infectious disease it causes, neoehrlichiosis. Methods Here, we describe the vascular and thromboembolic events afflicting a series of 40 patients diagnosed with neoehrlichiosis in Sweden during a 10-year period (2009–2019). Results The majority of the patients (60%) developed vascular events ranging from repeated thrombophlebitis, deep vein thrombosis, pulmonary embolism, transitory ischemic attacks, to arteritis. Younger age was a risk factor for vascular complications. In contrast, there was no difference in the incidence of vascular events between immunosuppressed and immunocompetent patients. However, there were qualitative differences, such that deep vein thrombosis exclusively afflicted the immunosuppressed patients, whereas arteritis was restricted to the immunocompetent persons. We also present the case histories of two patients who developed vasculitis mimicking polyarteritis nodosa and giant cell arteritis. Both were cured by doxycycline treatment. Conclusions Ca. N. mikurensis infection should be considered in patients living in tick-endemic areas of Europe and northern Asia who present with atypical vascular and/or thromboembolic events. Early diagnosis and antibiotics targeting this emerging infectious agent can eradicate the infection and prevent the development of new vascular events.

Abstract Background Hemotropic mycoplasmas, previously classified in the genus Eperythrozoon, have been reported as causing human infections in Brazil, China, Japan, and Spain. Methods In 2017, we detected DNA from Candidatus Mycoplasma haemohominis in the blood of a Melanesian patient from New Caledonia presenting with febrile splenomegaly, weight loss, life-threatening autoimmune hemolytic anemia, and hemophagocytosis. The full genome of the bacterium was sequenced from a blood isolate. Subsequently, we retrospectively (2011–2017) and prospectively (2018–2019) tested patients who had been hospitalized with a similar clinico-biological picture. In addition, as these patients had been in contact with frugivorous bats (authorized under conditions for hunting and eating in New Caledonia), we investigated the role of these animals and their biting flies by testing them for hemotropic mycoplasmas. Results There were 15 patients found to be infected by this hemotropic mycoplasma. Among them, 4 (27%) died following splenectomy performed either for spontaneous spleen rupture or to cure refractory autoimmune hemolytic anemia. The bacterium was cultivated from the patient’s blood. The full genome of the Neocaledonian Candidatus M. haemohominis strain differed from that of a recently identified Japanese strain. Of 40 tested Pteropus bats, 40% were positive; 100% of collected bat flies Cyclopodia horsfieldi (Nycteribiidae, Diptera) were positive. Human, bat, and dipteran strains were highly similar. Conclusions The bacterium being widely distributed in bats, Candidatus M. haemohominis, should be regarded as a potential cause of severe infections in humans.

Candidatus Liberibacter solanacearum (CaLso) is associated with diseases in tomato crops and transmitted by the tomato psyllid Bactericera cockerelli. A polymeric water-dispersible nanobactericide (PNB) was evaluated against CaLso as a different alternative. PNB is a well-defined polycationic diblock copolymer designed to permeate into the vascular system of plants. Its assessment under greenhouse conditions was carried out with tomato plants previously infected with CaLso. Using a concentration as low as 1.0 mg L−1, a small but significant reduction in the bacterial load was observed by real-time qPCR. Thus, to achieve an ecologically friendly dosage and set an optimum treatment protocol, we performed experiments to determine the effective concentration of PNB to reduce ~65% of the initial bacterial load. In a first bioassay, a 40- or 70-fold increase was used to reach that objective. At this concentration level, other bioassays were explored to determine the effect as a function of time. Surprisingly, a real reduction in the symptoms was observed after three weeks, and there was a significant decrease in the bacterial load level (~98%) compared to the untreated control plants. During this period, flowering and formation of tomato fruits were observed in plants treated with PNB.

Tomato is one of the most economically important crops in Republic of Moldova. However, it is affected by a number of pathogens. One of the wide spread diseases is stolbur caused by the infection agent ‘Candidatus Phytoplasma solani’. Accurate diagnostics of the disease at an early stage is essential for successful control of the disease. In this work, we describe detection and quantification of ‘Candida-tus Phytoplasma solani’ in tomato by real-time PCR, as well as suitability of the method for assessing resistance of different tomato varieties to Phytoplasma infection.

The status Candidatus was introduced to bacterial taxonomy in the 1990s to accommodate uncultured taxa defined by analyses of DNA sequences. Here I review the strengths, weaknesses, opportunities and threats (SWOT) associated with the status Candidatus in the light of a quarter century of use, twinned with recent developments in bacterial taxonomy and sequence-based taxonomic discovery. Despite ambiguities as to its scope, philosophical objections to its use and practical problems in implementation, the status Candidatus has now been applied to over 1000 taxa and has been widely adopted by journals and databases. Although lacking priority under the International Code for Nomenclature of Prokaryotes, many Candidatus names have already achieved de facto standing in the academic literature and in databases via description of a taxon in a peer-reviewed publication, alongside deposition of a genome sequence and there is a clear path to valid publication of such names on culture. Continued and increased use of Candidatus names provides an alternative to the potential upheaval that might accompany creation of a new additional code of nomenclature and provides a ready solution to the urgent challenge of naming many thousands of newly discovered but uncultured species.

The phloem-limited ‘Candidatus Liberibacter asiaticus’ (Las) causes huanglongbing, a destructive citrus disease. Graft-inoculated potted plants were used to assess Las speed of movement in phloem in the greenhouse, and the impacts of temperature on plant colonization in growth-chamber experiments. For assessment of Las speed, plants were inoculated at the main stem and assessed over time by quantitative PCR (qPCR) or symptoms at various distances from the inoculum. For colonization, the plants were inoculated in one of two opposite top branches, maintained at from 8 to 20°C, from 18 to 30°C, or from 24 to 38°C daily range, and assessed by qPCR of samples taken from noninoculated shoots. For all experiments, frequencies of Las-positive sites were submitted to analysis of variance and binomial generalized linear model and logistic regression analyses. Probabilities of detecting Las in greenhouse plants were functions of time and distance from the inoculation site, which resulted in 2.9 and 3.8 cm day−1 average speed of movement. In growth chambers, the temperature impacted plant colonization by Las, new shoot emission, and symptom expression. After a 7-month exposure time, Las was absent in all new shoots in the cooler environment (average three per plant), and present in 70% at the milder environment (six shoots, severe symptoms) and 25% in the warmer environment (eight shoots, no visible symptoms). Temperature of 25.7°C was the optimum condition for plant colonization. This explains the higher impact and incidence of huanglongbing disease during the winter months or regions of milder climates in Brazil.