Publications

4372

Objectives The objective of this study was to evaluate wild-caught mosquitoes for evidence of hemotropic Mycoplasma species DNA and to determine whether the feline hemoplasmas, Mycoplasma haemofelis (Mhf) and ‘ Candidatus Mycoplasma haemominutum’ (Mhm), can be transmitted by Aedes aegypti mosquitoes in a laboratory setting. Methods Wild-caught mosquito pools (50 mosquitoes per pool, 84 pools) utilized in routine public health department disease surveillance programs were tested for hemotropic Mycoplasma species DNA using PCR with primers designed to amplify all known hemoplasmas. Additionally, mosquitoes were trapped in the vicinity of known feral cat colonies, pooled (50 mosquitoes per pool) and tested (84 pools). Purpose-bred cats housed in a research facility were infected with Mhf or Mhm and then colonized laboratory A aegypti were fed upon the bacteremic cats. After a 7 day incubation period, mosquitoes previously fed on infected cats were allowed to feed again on naive cats, which were monitored for bacteremia for 10 weeks. Results Mycoplasma wenyonii DNA was confirmed in one wild-caught mosquito pool by DNA sequencing. While 7% of cats tested in feral colonies were hemoplasma positive, none of the mosquitoes trapped near colonies were positive. Hemoplasma DNA was amplified from A aegypti by PCR immediately after the infectious blood meal, but DNA was not detected at 7 and 14 days after feeding. Although evidence for uptake of organisms existed, hemoplasma DNA was not amplified from the experimentally infested cats in the 10 week observation period. Conclusions and relevance While wild-caught mosquitoes contained hemoplasma DNA and laboratory reared A aegypti mosquitoes take up hemoplasmas during the blood meal, there was no evidence of biologic transmission in this model.

Huanglongbing, a destructive disease of citrus, is caused by the fastidious bacterium ‘Candidatus Liberibacter asiaticus’ and transmitted by Asian citrus psyllid, Diaphorina citri. The impact of ‘Ca. L. asiaticus’ infection or D. citri infestation on Valencia sweet orange (Citrus sinensis) leaf metabolites was investigated using gas chromatography mass spectrometry, followed by gene expression analysis for 37 genes involved in jasmonic acid (JA), salicylic acid (SA), and proline-glutamine pathways. The total amino acid abundance increased after ‘Ca. L. asiaticus’ infection, while the total fatty acids increased dramatically after infestation with D. citri, compared with control plants. Seven amino acids (glycine, l-isoleucine, l-phenylalanine, l-proline, l-serine, l-threonine, and l-tryptophan) and five organic acids (benzoic acid, citric acid, fumaric acid, SA, and succinic acid) increased in ‘Ca. L. asiaticus’-infected plants. On the other hand, the abundance of trans-JA and its precursor α-linolenic increased in D. citri-infested plants. Surprisingly, the double attack of both D. citri infestation and ‘Ca. L. asiaticus’ infection moderated the metabolic changes in all chemical classes studied. In addition, the gene expression analysis supported these results. Based on these findings, we suggest that, although amino acids such as phenylalanine are involved in citrus defense against ‘Ca. L. asiaticus’ infection through the activation of an SA-mediated pathway, fatty acids, especially α-linolenic acid, are involved in defense against D. citri infestation via the induction of a JA-mediated pathway.

Summary Cotylorhiza tuberculata is an important scyphozoan jellyfish producing population blooms in the Mediterranean probably due to pelagic ecosystem's decay. Its gastric cavity can serve as a simple model of microbial–animal digestive associations, yet poorly characterized. Using state‐of‐the‐art metagenomic population binning and catalyzed reporter deposition fluorescence in situ hybridization (CARD‐FISH), we show that only four novel clonal phylotypes were consistently associated with multiple jellyfish adults. Two affiliated close to Spiroplasma and Mycoplasma genera, one to chlamydial ‘ Candidatus Syngnamydia’, and one to bacteroidetal Tenacibaculum , and were at least one order of magnitude more abundant than any other bacteria detected. Metabolic modelling predicted an aerobic heterotrophic lifestyle for the chlamydia, which were found intracellularly in Onychodromopsis ‐like ciliates. The Spiroplasma ‐like organism was predicted to be an anaerobic fermenter associated to some jellyfish cells, whereas the Tenacibaculum ‐like as free‐living aerobic heterotroph, densely colonizing the mesogleal axis inside the gastric filaments. The association between the jellyfish and its reduced microbiome was close and temporally stable, and possibly related to food digestion and protection from pathogens. Based on the genomic and microscopic data, we propose three candidate taxa: ‘ Candidatus Syngnamydia medusae’, ‘ Candidatus Medusoplasma mediterranei’ and ‘ Candidatus Tenacibaculum medusae’.

AbstractGrapevine (Vitis vinifera) is one of the most important fruits in Iran where the provinces of Qazvin, Lorestan and Markazi are main producers. During 2013–2015, vineyards located in these provinces were surveyed to verify the presence of phytoplasma. The sample collection was based on symptomatology including decline, leaf yellowing and shortening of internodes. Total DNA was extracted from symptomatic and symptomless grapevine samples and used in nested‐polymerase chain reaction (PCR) assays with phytoplasma ribosomal primers (P1/Tint followed by R16F2n/R2, R16mF1/mR1, R16(I)F1/R1 or 6R758f/16R1232r). Nested‐PCR products were obtained only for symptomatic samples while samples from symptomless plants yielded no PCR products. Restriction fragment length polymorphism (RFLP) analyses with Tru1I, TaqI and Tsp509I and direct sequencing of amplicons followed by phylogenetic analyses indicated the presence of ‘Candidatus Phytoplasma fraxini’, ‘Ca. P. aurantifolia’, ‘Ca. P. solani’ and ‘Ca. P. phoenicium’‐related strains. In Marzaki province, there ‘Ca. P. aurantifolia’ strains were mainly detected, while in the other two provinces, all the four ‘Candidatus species’ were identified with the prevalence of ‘Ca. P. solani’‐related strains. In both provinces in one case, mixed phytoplasma infection was also detected by RFLP analyses. The presence of different phytoplasmas in positive samples indicates great phytosanitary significance due to grapevine economic importance for country. Grapevine phytoplasma infection represents a threat for other crops suggesting grapevine as alternative host species for the phytoplasmas already reported in Iran, while the ‘Ca. P. fraxini’ is for the first time identified in Iran.

The phloem‐sucking psyllid Cacopsylla picta plays an important role in transmitting the bacterium ‘ Candidatus Phytoplasma mali’, the agent associated with apple proliferation disease. The psyllid can ingest ‘ Ca . Phytoplasma mali’ from infected apple trees and spread the bacterium by subsequently feeding on uninfected trees. Until now, this has been the most important method of ‘ Ca . Phytoplasma mali’ transmission. The aim of this study was to investigate whether infected C. picta are able to transmit ‘ Ca . Phytoplasma mali’ directly to their progeny. This method of transmission would allow the bacteria to bypass a time‐consuming reproductive cycle in the host plant. Furthermore, this would cause a high number of infected F 1 individuals in the vector population. To address this question, eggs, nymphs and adults derived from infected overwintering adults of C. picta were reared on non‐infected apple saplings and subsequently tested for the presence of ‘ Ca . Phytoplasma mali’. In this study it was shown for the first time that infected C. picta individuals transmit ‘ Ca . Phytoplasma mali’ to their eggs, nymphs and F 1 adults, thus providing the basis for a more detailed understanding of ‘ Ca . Phytoplasma mali’ transmission by C. picta .

‘Candidatus Liberibacter asiaticus’, the bacterium associated with citrus Huanglongbing (HLB), was reported from Uganda and tentatively from Tanzania, posing a threat to citriculture in Africa. Two surveys of citrus expressing typical HLB symptoms were conducted in Uganda, Kenya, and Tanzania to verify reports of ‘Ca. L. asiaticus’ and to assess the overall threat of HLB to eastern and southern African citrus production. Samples were analyzed for the presence of ‘Candidatus Liberibacter’ species by real-time PCR and partial sequencing of three housekeeping genes, 16S rDNA, rplJ, and omp. ‘Ca. L. africanus’, the bacterium historically associated with HLB symptoms in Africa, was detected in several samples. However, samples positive in real-time PCR for ‘Ca. L. asiaticus’ were shown not to contain ‘Ca. L. asiaticus’ by sequencing. Sequences obtained from these samples were analogous to ‘Ca. L. africanus subsp. clausenae’, identified from an indigenous Rutaceae species in South Africa, and not to ‘Ca. L. asiaticus’. Results indicate a nontarget amplification of the real-time assay and suggest that previous reports of ‘Ca. L. asiaticus’ from Uganda and Tanzania may be mis-identifications of ‘Ca. L. africanus subsp. clausenae’. This subspecies was additionally detected in individual Diaphorina citri and Trioza erytreae specimens recovered from collection sites. This is the first report of ‘Ca. L. africanus subsp. clausenae’ infecting citrus and being associated with HLB symptoms in this host.