Identification and quantification of ‘Candidatus Phytoplasma pyri’ in declining trees of a Swiss cider pear orchard after incision treatment at the stem base

Candidatus Orchards of 30 to >100 years old fruit trees used for cider production are endangered by an accumulation of abiotic and biotic stress factors. Among biotic stress factors, diseases such as pear decline (PD) caused by the bacterial pathogen ' Phytoplasma ' contributes to a weakening and reduced life time of affected trees. Since direct treatment of this disease is not possible, approaches have gained attention, which might lead to an increased resilience against this pathogen, such as incisions of the cambium at the graft union at the stem base. Six 35 years old pear trees () of a Swiss cider production orchard, all affected by mild decline were chosen for this study. Four out of them were treated with 2-4 incisions per tree in February 2016. Symptoms were visually assessed during summer and autumn 2016 and 2017, respectively, and ' P ' measured in branch samples with a newly developed duplex TaqMan qPCR assay. No effect could be determined within these two subsequent growing seasons after treatment. Both, visual assessment of symptoms and qPCR measurement of the pathogen in branches did not show any difference between treated and untreated trees. The sequencing of two marker genes of the pathogen detected in this orchard confirmed its identity as ' ' and revealed that it belongs to the major genotype present in Europe

A Novel Betaproteobacterial Agent of Gill Epitheliocystis in Seawater Farmed Atlantic Salmon (Salmo salar)

Epitheliocystis, a disease characterised by cytoplasmic bacterial inclusions (cysts) in the gill and less commonly skin epithelial cells, has been reported in many marine and freshwater fish species and may be associated with mortality. Previously, molecular and ultrastructural analyses have exclusively associated members of the Chlamydiae with such inclusions. Here we investigated a population of farmed Atlantic salmon from the west coast of Norway displaying gill epitheliocystis. Although ‘Candidatus Piscichlamydia salmonis’, previously reported to be present in such cysts, was detected by PCR in most of the gill samples analysed, this bacterium was found to be a rare member of the gill microbiota, and not associated with the observed cysts as demonstrated by fluorescence in situ hybridization assays. The application of a broad range 16 S rRNA targeted PCR assay instead identified a novel betaproteobacterium as an abundant member of the gill microbiota. Fluorescence in situ hybridization demonstrated that this bacterium, tentatively classified as ‘Candidatus Branchiomonas cysticola’, was the cyst-forming agent in these samples. While histology and ultrastructure of ‘Ca. B. cysticola’ cysts revealed forms similar to the reticulate and intermediate bodies described in earlier reports from salmon in seawater, no elementary bodies typical of the chlamydial developmental cycle were observed. In conclusion, this study identified a novel agent of epitheliocystis in sea-farmed Atlantic salmon and demonstrated that these cysts can be caused by bacteria phylogenetically distinct from the Chlamydiae

The End of a 60-year Riddle: Identification and Genomic Characterization of an Iridovirus, the Causative Agent of White Fat Cell Disease in Zooplankton

The planktonic freshwater crustacean of the genus Daphnia are a model system for biomedical research and, in particular, invertebrate-parasite interactions. Up until now, no virus has been characterized for this system. Here we report the discovery of an iridovirus as the causative agent of White Fat Cell Disease (WFCD) in Daphnia. WFCD is a highly virulent disease of Daphnia that can easily be cultured under laboratory conditions. Although it has been studied from sites across Eurasia for more than 60 years, its causative agent had not been described, nor had an iridovirus been connected to WFCD before now. Here we find that an iridovirusthe Daphnia iridescent virus 1 (DIV-1)is the causative agent of WFCD. DIV-1 has a genome sequence of about 288 kbp, with 39% G+C content and encodes 367 predicted open reading frames. DIV-1 clusters together with other invertebrate iridoviruses but has by far the largest genome among all sequenced iridoviruses. Comparative genomics reveal that DIV-1 has apparently recently lost a substantial number of unique genes but has also gained genes by horizontal gene transfer from its crustacean host. DIV-1 represents the first invertebrate iridovirus that encodes proteins to purportedly cap RNA, and it contains unique genes for a DnaJ-like protein, a membrane glycoprotein and protein of the immunoglobulin superfamily, which may mediate host-pathogen interactions and pathogenicity. Our findings end a 60-year search for the causative agent of WFCD and add to our knowledge of iridovirus genomics and invertebrate-virus interactions

Dynamic Acquisition and Loss of Dual-Obligate Symbionts in the Plant-Sap-Feeding Adelgidae (Hemiptera: Sternorrhyncha: Aphidoidea)

Sap-sucking insects typically engage in obligate relationships with symbiotic bacteria that play nutritional roles in synthesizing nutrients unavailable or in scarce supply from the plant-sap diets of their hosts. Adelgids are sap-sucking insects with complex life cycles that involve alternation between conifer tree species. While all adelgid species feed on spruce during the sexual phase of their life cycle, each adelgid species belongs to a major lineage that feeds on a distinct genus of conifers as their alternate host. Previous work on adelgid symbionts had discovered pairs of symbionts within each host species, and unusual diversity across the insect family, but left several open questions regarding the status of bacterial associates. Here, we explored the consistency of symbionts within and across adelgid lineages, and sought evidence for facultative vs.obligate symbiont status. Representative species were surveyed for symbionts using 16Sribosomal DNA gene sequencing, confirming that different symbiont pairs were consistently present within each major adelgid lineage. Several approaches were used to establish whether symbionts exhibited characteristics of long-term, obligate mutualists. Patterns of symbiont presence across adelgid species and diversification with host insects suggested obligate relationships. Fluorescent in situ hybridization and electron microscopy localized symbionts to bacteriocyte cells within the bacteriome of each species (with one previously known exception), and detection of symbionts in eggs indicated their vertical transmission. Common characteristics of long-term obligate symbionts, such as nucleotide compositional bias and pleomorphic symbiont cell shape were also observed. Superimposing microbial symbionts on the adelgid phylogeny revealed a dynamic pattern of symbiont gains and losses over a relatively short period of time compared to other symbionts associated with sap-sucking insects, with each adelgid species possessing an older, “senior” symbiont and a younger “junior” symbiont. A hypothesis relating adelgid life cycles to relaxed constraints on symbionts is proposed, with the degradation of senior symbionts and repeated acquisition of more junior symbionts creating opportunities for repeated colonization of new alternate-conifer hosts by adelgids

The role of macro-aggregation in regulating enzymatic depolymerization of soil organic nitrogen

Extracellular enzymatic depolymerization of polymeric organic nitrogen (PON) is a rate-limiting step in N mineralization. However, enzymatic accessibility to PON might be regulated by physical occlusion of the PON resulting from the architectural packing of soil minerals during aggregate formation. To examine the extent to which enzymatic accessibility to PON is regulated by soil aggregation, we put forward a new approach involving the comparison of relationships between potential N depolymerase activity (protease and β-glucosaminidase; as an estimate of the potential to produce depolymerized products) and net N mineralization (as a bioassay for actual low molecular weight dissolved ON production) in aggregated and corresponding disaggregated soil. Soils were sampled from grassland (GL) and arable land (AL), separated by dry sieving into fractions (4.75-2, 2-0.25 and 0.25-0.063 mm) and fractions mixed (4:4:1 by mass, respectively) to obtain constructed aggregated soils. Corresponding disaggregated soils were prepared using a mortar and pestle. This procedure mainly disrupted the 4.75-2 mm (large macro-aggregate) fraction. Disaggregation significantly promoted (p<0.05) net N mineralization rates by 1.3 times and 1.5 times in GL and AL soil, respectively. When net N mineralization - potential N depolymerase relationships for GL were examined, a greater slope parameter for disaggregated compared to aggregated soil (p=0.001; ANCOVA) quantified the extent to which this promoted N mineralization could be attributed to disruption of macroaggregate-increased enzymatic accessibility to PON. For AL, which had low protease and β-glucosaminidase activity, promoted N mineralization rate could not be attributed to increased protease + β-glucosaminidase accessibility to PON reflecting a possible role for other N depolymerases and/or osmolyte/lysate effects. By proposing how differences between mineralization-depolymerase relationships for soils differing in aggregation status might, with assumptions, be interpreted to identify the role of physical occlusion in protection of PON, we give new insight on the regulation of enzymatic depolymerization by physical protection through macro-aggregation for soils from contrasting land use