Association of kidney disease measures with risk of renal function worsening in patients with type 1 diabetes

Background: Albuminuria has been classically considered a marker of kidney damage progression in diabetic patients and it is routinely assessed to monitor kidney function. However, the role of a mild GFR reduction on the development of stage 653 CKD has been less explored in type 1 diabetes mellitus (T1DM) patients. Aim of the present study was to evaluate the prognostic role of kidney disease measures, namely albuminuria and reduced GFR, on the development of stage 653 CKD in a large cohort of patients affected by T1DM. Methods: A total of 4284 patients affected by T1DM followed-up at 76 diabetes centers participating to the Italian Association of Clinical Diabetologists (Associazione Medici Diabetologi, AMD) initiative constitutes the study population. Urinary albumin excretion (ACR) and estimated GFR (eGFR) were retrieved and analyzed. The incidence of stage 653 CKD (eGFR < 60 mL/min/1.73 m2) or eGFR reduction > 30% from baseline was evaluated. Results: The mean estimated GFR was 98 \ub1 17 mL/min/1.73m2 and the proportion of patients with albuminuria was 15.3% (n = 654) at baseline. About 8% (n = 337) of patients developed one of the two renal endpoints during the 4-year follow-up period. Age, albuminuria (micro or macro) and baseline eGFR < 90 ml/min/m2 were independent risk factors for stage 653 CKD and renal function worsening. When compared to patients with eGFR > 90 ml/min/1.73m2 and normoalbuminuria, those with albuminuria at baseline had a 1.69 greater risk of reaching stage 3 CKD, while patients with mild eGFR reduction (i.e. eGFR between 90 and 60 mL/min/1.73 m2) show a 3.81 greater risk that rose to 8.24 for those patients with albuminuria and mild eGFR reduction at baseline. Conclusions: Albuminuria and eGFR reduction represent independent risk factors for incident stage 653 CKD in T1DM patients. The simultaneous occurrence of reduced eGFR and albuminuria have a synergistic effect on renal function worsening

Developing a method for phytoplasma identification in cactus pear samples from California.

Cactus pear plants showing proliferation and stunting of cladodes in Californian cultivations were tested in order to define a molecular methodology for reliable phytoplasma detection. After several unsuccessful trials a simple extraction method was developed to reduce the mucilage content in nucleic acid preparations that was seriously affecting pathogen detection. Nested PCR on 16S ribosomal gene and RFLP analyses together with sequencing of obtained amplicons allow to verify the presence in symptomatic plants of 16SrV-A and 16SrI-B phytoplasmas respectively related to \u2018Candidatus Phytoplasma ulmi\u2019 and \u2018Ca. P. asteris\u2019

Molecular identification of \u201cBois Noir\u201d phytoplasmas in grapevine in Bulgaria.

Field inspection in 4 vineyards located near Plovdiv (Bulgaria) allows observation of yellows symptom presence. PCR/RFLP analyses on 16S ribosomal gene identified phytoplasmas belonging to 16SrXII-A ribosomal subgroup in both symptomatic grapevines and bindweed growing in infected vineyard. Grapevine mother plant and young plants were also infected suggesting that, even if this is the first report of this disease in Bulgaria, the environment is epidemically affected

Phytoplasma detection in corn with reddening in Italy

During the second half of August 2009 in corn fields located in Northern Italy scattered plants showing reddening symptoms were observed, mainly located at the edge of the fields. Symptoms were clearly visible on the main leaf midribs, and/or on the stalks, and eventually affect the whole plant. Symptomatic plants had smaller size than healthy ones, and corn cobs were sometime malformed and of very little size. In some of the symptomatic plants the cobs produced were of regular size and contains poor shrivelled grains as reported for reddening disease of corn in Serbia (Duduk & Bertaccini, Plant Disease, 90, 1313-1319. 2006). Ten samples of symptomatic, and 4 of asymptomatic corn plants were collected in two different locations and nested PCR assays were carried out on total nucleic acids from 1 g of main leaf midrib and phloem stalk tissues chloroform/phenol extracted. Direct PCR assays with phytoplasma universal primer pair P1/P7 followed by nested PCR with 16S758F/16S1242R (Gibb et al., Phytopathology, 85, 169-174. 1995) primers allowed amplification of 500 bp amplicons from all samples from symptomatic plants, no bands were obtained from asymptomatic samples. Identification of detected phytoplasmas done using RFLP analyses with TruI, Tsp509I and MboII restriction enzymes allow preliminary identification of phytoplasmas belonging to 16SrI (aster yellows), 16SrIII (X disease) and 16SrXII (stolbur) groups, in same cases in mixed infection. Further molecular characterization of these phytoplasmas is in progress together with epidemiological studies to verify the presence of phytoplasma sources, and of possible insect vectors in the two environments. Presence of stolbur phytoplasmas in corn samples with reddening symptoms is confirming the finding in Serbia (Duduk & Bertaccini, above), however this is the first report in Europe of 16SrI group phytoplasmas, and the first report of 16SrIII in corn. The diverse phytoplasmas are associated with indistinguishable symptoms in plants as already worldwide reported in this and in other plant species for phytoplasma infection

Multi-gene analysis for differentiation of aster yellows phytoplasmas infecting carrots in Serbia. Annals of Applied Biology, 154(2): 219-229.

During a survey of large carrot fields in Serbia, plants showing leaf reddening and/or yellowing, adventitious shoot production and reduction in taproot size and quality were observed in a low percentage of plants. To verify phytoplasma association with the described symptoms and to carry out pathogen differentiation, PCR assays followed by restriction fragment length polymorphism (RFLP) analyses and/or sequencing of phytoplasma 16Sr DNA and ribosomal protein genes l22 and s3, tuf, putative aa kinase plus ribosomal recycling factor genes and DNA helicase gene were carried out. Phytoplasmas belonging to 16SrI-A and 16SrI-B ribosomal subgroups and to rpI-A and rpI-B ribosomal protein subgroups, respectively, were identified by RFLP analyses in 13 of 15 symptomatic plants tested. No amplification was obtained with non-symptomatic carrot samples. The identification was confirmed by sequence analyses of the phytoplasma genes studied. In two carrot samples, presence of interoperon sequence heterogeneity was detected and phytoplasma strains were identified as belonging to 16SrI group but were not assigned to any 16S rRNA or ribosomal protein subgroup. This research allowed the first molecular identification of phytoplasmas infecting carrot in Serbia using several molecular markers, and it indicates that under field conditions in non-epidemic outbreaks a certain amount of genetic mutation may occur in conserved genes of these prokaryotes

Virescence of tenweeks stock associated to phytoplasma infection in Sicily

In April 2007, a severe disease occurred in Sicily (Italy) in a glasshouse cultivation of tenweeks stock belonging to the cultivar White-Beach. Plants were stunted and rosetted, and the flowers were of small size and characterized by virescence symptoms. Phytoplasma presence and identity was detected by applying PCR/RFLP techniques and sequencing of 16S ribosomal gene. Phytoplasmas were identified as belonging to ribosomal subgroup 16SrII-A, never reported before in Italy and showed 99% of homology with ‘Candidatus Phytoplasma aurantifolia’ and related phytoplasmas. This is the first report of a phytoplasma disease of tenweeks stock. Considering that this Brassicaceae ornamental species is widely grown in Italy, it could play an important role in spreading these phytoplasmas, new for Italy

A new phytoplasma associated with witches\u2019 -broom of Cassia italica in Oman.

In several Oman locations plants of Cassia italica exhibit witches\u2019 broom symptoms. Samples were collected from four locations, and examined for phytoplasma presence. PCR amplification using ribosomal phytoplasma primers followed by RFLP analyses indicates that the phytoplasmas present in these samples were undistinguishable from each other, but differed from those reported in the literature. RFLP and phylogenetic analysis of 16S rRNA gene plus spacer sequence confirmed that the closest phytoplasma relatives were members of the pigeon pea witches\u2019 broom phytoplasma ribosomal group (16SrIX), sharing a 93-97% sequence similarity. Nested PCR experiments using primers amplifying the gene coding for ribosomal protein S3 provided amplification from phytoplasmas detected in C. italica, after sequencing 600 bp in this gene the higher homology showed was 78% with phytoplasmas related to \u2018Candidatus Phytoplasma phoenicium\u2019 and 77% with phytoplasmas belonging to ribosomal group 16SrIX

\u2018Candidatus Phytoplasma omanense\u2019, a phytoplasma associated with witches\u2019 broom of Cassia italica (Mill.) Lam. in Oman.

Samples from plants of Cassia italica exhibiting typical witches\u2019-broom symptoms (Cassia witches\u2019-broom; CWB) were examined for the presence of plant pathogenic phytoplasmas by PCR amplification using universal phytoplasma primers. All affected plants yielded positive results. RFLP analyses of rRNA gene products indicated that the phytoplasmas detected were different from those described previously. Phylogenetic analysis of 16S rRNA gene sequences confirmed that CWB represents a distinct lineage and shares a common ancestor with \u2018Candidatus Phytoplasma phoenicium\u2019. Molecular comparison revealed that the 16S rRNA gene sequences of the four CWB strains (IM-1, IM-2, IM-3 and IM-4) identified in symptomatic C. italica samples were nearly identical (99.6\u2013100% similarity). The closest relatives were members of the pigeon pea witches\u2019-broom phytoplasma ribosomal group (16SrIX; 95\u201397% sequence similarity). On the basis of unique 16S rRNA gene sequences and biological properties, the phytoplasma associated with witches\u2019-broom of C. italica in Oman represents a coherent but discrete novel phytoplasma, \u2018Candidatus Phytoplasma omanense\u2019, with GenBank/DDBJ/EMBL accession number EF666051 representing the reference strain

Molecular identification of \u2018Candidatus Phytoplasma asteris\u2019 inducing histological anomalies in Silene nicaeensis.

Numerous plants of Silene nicaeensis having symptoms resembling those associated with the presence of phytoplasmas were observed in an extensive coastal area in the south of Italy. Microscopic observation showed histological abnormalities in the organization of tissues in symptomatic plants, and molecular tests, including PCR/RFLP analyses and nucleic acid sequencing, revealed the presence of phytoplasmas belonging to the aster yellows group (\u2018Candidatus Phytoplasma asteris\u2019). This is the first report of phytoplasma infection in S. nicaeensis, a wild species that colonizes the Calabrian coast