Detection and identification of phytoplasmas in two vineyards located in a restricted geographic area in Italy

A survey to verify the presence of grapevine yellows was carried out in a restricted area of the Marche region (central east Italy) in a small geographic area. In two locations six grapevine varieties were examined and phytoplasmas belonging to four ribosomal groups were detected by PCR/RFLP analyses. The surveyed varieties were Passerina, Pecorino, Merlot, Sangiovese, Fedit and Montepulciano. The different phytoplasmas detected were present in a scattered manner in all the varieties, but only Passerina, Pecorino and Sangiovese where shown to be infected by “bois noir” (16SrXII-A) phytoplasmas. Considering the landscape shape and the age of the vineyards the source of BN infection and of the other phytoplasmas is very likely in the surrounding environment or vineyards

Isolation of diverse phytoplasmas from symptomatic grapevine samples

Phytoplasmas from different ribosomal groups were isolated from grapevine samples in which “flavescence dorée” or “bois noir” phytoplasmas had been identified. The results indicate that the developed medium is not phytoplasma specific and supports the growth of phytoplasmas which cannot be detected by other molecular methods and are very likely present in a very low titre in the endobiome of the plants. The applied method discriminates the presence of bacterial contamination already in the liquid phase, and the colony purification technique allows excluding the contamination

Molecular variability and host distribution of ‘candidatus phytoplasma solani’ strains from different geographic origins

The knowledge of phytoplasma genetic variability is a tool to study their epidemiology and to implement an effective monitoring and management of their associated diseases. ‘Candidatus Phytoplasma solani’ is associated with “bois noir” disease in grapevines, and yellowing and decline symptoms in many plant species, causing serious damages during the epidemic outbreaks. The epidemiology of the diseases associated with this phytoplasma is complex and related to numerous factors, such as interactions of the host plant and insect vectors and spreading through infected plant propagation material. The genetic variability of ‘Ca. P. solani’ strains in different host species and in different geographic areas during the last two decades was studied by RFLP analyses coupled with sequencing on vmp1, stamp, and tuf genes. A total of 119 strains were examined, 25 molecular variants were identified, and the variability of the studied genes was linked to both geographic distribution and year of infection. The crucial question in ‘Ca. P. solani’ epidemiology is to trace back the epidemic cycle of the infections. This study presents some relevant features about differential strain distribution useful for disease monitoring and forecasting, illustrating and comparing the phytoplasma molecular variants identified in various regions, host species, and time periods

Survey for ‘Candidatus Liberibacter’ and ‘Candidatus Phytoplasma’ in Citrus in Chile

The considerable economic losses in citrus associated with ‘Candidatus Liberibacter’ and ‘Candidatus Phytoplasma’ presence have alerted all producing regions of the world. In Chile, none of these bacteria have been reported in citrus species. During the years 2017 and 2019, 258 samples presenting symptoms similar to those associated with the presence of these bacteria were examined. No detection of ‘Ca. Liberibacter’ associated with “huanglongbing” disease was obtained in the tested samples; therefore, this quarantine pest is maintained as absent in Chile. However, 14 plants resulted positive for phytoplasmas enclosed in subgroups 16SrV-A (12 plants) and 16SrXIII-F (2 plants). Although they have been found in other plant species, this is the first report of these phy-toplasmas in citrus worldwide

Induction of resistance and enhancing agronomic performance in grapevines under greenhouse and in open fields by applications of plasma activated water

The exposure of water to a cold atmospheric pressure plasma (CAP) enables the production of plasma activated water (PAW), having high content of reactive species, whose applications were tested on grapevine plants, both in greenhouse and in vineyard conditions. Two different CAPs were used for PAW production, evaluating their effectiveness as a possible mean to control plant diseases. Grapevines infected with yellows associated with the presence of phytoplasmas were treated evaluating qualitative and quantitative yield parameters, phytoplasma presence, and gene expression. The results show the capability of PAW to enhance plant defence mechanisms and, as demonstrated in the field trials, confirmed its ability to improve the health status of the treated plants. Quantitative (q)RT-PCR analyses allowed to determine the transcription level of genes involved in the plant defence response (phenylalanine ammonia-lyase, pal) and in the plant phytoalexin metabolism of PAW-treated materials. The number of symptomatic grapevine plants in vineyards was significantly reduced by the treatments. Transcriptional and post-transcriptional molecular analyses highlighted the PAW ability to enhance the expression of genes encoding the main enzymes involved in the phytoalexin biosynthetic pathway (flavonoids and stilbenes). The PAW ability to enhance some of the plant defence mechanisms also improving the health status of the treated plants was therefore experimentally demonstrated. After three years of trials the overall results demonstrated the possible use of PAW to reduce the disease severity, induce plant resistance both in open field and greenhouse, improving plant healthy status and grapevine yield production

Transcriptional profiling of phytoplasma infected plants treated with plasma activated water (PAW).

Background. Phytoplasmas are insect-transmitted plant pathogenic prokaryotes, associated with severe diseases in agronomic important crops. Management of these diseases has mainly focused on insect vector chemical control and on infected plant rouging. There is therefore a strong need for effective and friendly control strategies for phytoplasma-associated diseases and the possibility to use plasma activated water (PAW) as sustainable and effective method to them was therefore evaluated. PAW is produced by treating distilled water with atmospheric pressure plasmas, inducing the production of reactive oxygen and nitrogen species (RONS) and pH reduction. PAW has good potential for bacterial decontamination, degradation of organic compounds and was shown to positively affect plant growth. Methods. Sterile deionized water (SDW) was exposed to a nanosecond pulsed dielectric barrier discharge, operating in ambient air for 10 min treatment with a peak voltage of 19 kV and a pulse repetition frequency of 1 kHz, which induced production of nitrates, nitrites and peroxides, and a pH decrease. Phytoplasma infected and healthy periwinkles micropropagated shoots were exposed to PAW for about 25 minutes and gene expression studies were then performed. The theses used were: shoots treated with PAW, Fosetyl aluminum (as positive control) and SDW (as negative control), with an exposition of about 25 minutes. Nine shoots for each thesis were then collected at 6 different times after treatment and stored at -80\ub0C. Quantitative RT-PCR analyses were carried out to determine the expression level of genes involved in the plant defense response. Parallel experiments were carried out treating grapevine plants in vineyards previously tested for the phytoplasma presence. Treatments were performed for three years injecting into the plant vascular tissues 10-20 ml of PAW or sterile distilled water (as control) on each selected plant for a total of 60 plants (40 with phytoplasmas and 20 without phytoplasmas). Results. Overexpression of selected genes involved in the phytoalexin metabolism was detected in the periwinkles micropropagated shoots treated with PAW in comparison with the shoots treated with Fosetyl-Al and distilled water. In the field trials, in a relevant number of cases, the PAW-treated symptomatic plants showed reduction of symptoms, while the SDW-treated and untreated plants did not show symptom reduction. No phytotoxicity was observed in the PAW treated grapevine and periwinkle plants. Conclusion. The results obtained showed the capability of PAW to enhance plant defence mechanisms and, as demonstrated in the field trials, confirmed its ability to improve the health status of the treated plant

The cost of inflammatory bowel disease management matches with clinical course: A single outpatient centre analysis

Inflammatory bowel diseases (IBD) have a large economic burden on health systems. Our single-centre observational retrospective study aimed to assess an economic evaluation in two IBD outpatient cohorts (biological and conventional therapy) in relation to disease activity within a three-year follow-up. Four hundred and seventeen consecutive IBD patients referred to our tertiary gastroenterology unit (Bari-Puglia-Southern Italy) on January 2014–December 2016 were included. For each group (conventional/biological), we assessed direct/indirect costs and clinical/endoscopic activity within the first year and along the three-year follow-up. Statistical analyses: Wilcoxon signed-rank test (continuous variables), chi-square and Fisher’s test (categorical variables), Spearman ranks (single outcome) and ANOVA (detection time, clinical/endoscopic scores) were used. Continuous variables were expressed as mean ± standard deviation and range and/or median, interquartile range and range; categorical variables were expressed as proportions with 95% confidence interval. Direct and indirect cost items of 2014 and 2014–2016 were higher in patients treated with biological than conventional therapy. Subjects on biological therapy were younger and showed clinical and endoscopic moderate-to-severe disease activity. After three years, they reached a significant improvement from baseline. Conversely, disease activity was mild when conventional treatment had a beneficial effect. In conclusion, overall IBD management cost matches with clinical course and needs long-term evaluation in critical patients

The USe of pac1 Gene from Schizosaccaromyces pombe to Protect Potato from Potato virus Y (PVY) and Potato X (PVX) Infections

The effect that pac1, a yeast-derived ds-RNA specific RNase gene, has on Potato virus X (PVX) and on Potato virus Y (PVY) infections in potato was investigated. Tuber disks of cv. Desirée were transformed with LB4404 Agrobacterium tumefaciens carrying a modified PBI121 binary vector in which the transgene replaced GUS. Transformed plantlets were tested for possible acquired resistance by inoculation with purified virus suspensions at different concentrations and were ELISA-tested for virus content 40 days later. Plants of two clones had a significantly lower PVX content (66 to 75% that of the control, respectively), when inoculated with a 1 µg ml-1 suspension of the virus. One of these clones showed also a reduced PVY accumulation (41% that of the control), when inoculated with a 10 µg ml-1 suspension of this virus. Results demonstrated the practical utility of the pac1 gene for countering potato virus infections

Identification of ‘Candidatus Phytoplasma’ species in “huanglongbing” infected citrus orchards in the Caribbean

“Huanglongbing” (HLB) is one of the most devastating diseases of citrus orchards worldwide. Samples from 183 citrus plants of different cultivars and rootstock/cultivar combinations, showing HLB symptoms in three Caribbean countries (Cuba, Jamaica, and Guadeloupe-France), were collected to verify the possible co-infection of ‘Candidatus Phytoplasma’ and ‘Candidatus Liberibacter’ species. The 64% of the samples resulted positive to the ‘Ca. L. asiaticus’ and the 27% to diverse ‘Ca. Phytoplasma’-related species, moreover about the 14% of the samples infected with ‘Ca. Liberibacter’ were also found positive to phytoplasmas, indicating the presence of mixed infection especially in the orchards located in Cuba. Moreover, in one of the samples from Jamaica mixed phytoplasma infection was detected. Moreover the detection of only phytoplasmas in 11 symptomatic citrus samples collected from Cuba and Guadeloupe without ‘Ca. Liberibacter’ detection, confirmed that the symptomatology cannot be the sole criterium to discriminate between the presence of the two pathogens, and molecular detection is necessary to identify single or mixed infections. Diaphorina citri insects collected from Cuba and Guadeloupe resulted infected with ‘Ca. L. asiaticus’ confirming its active role in the dissemination of the pathogen. Only one insect of the Cicadidae family, collected in Guadeloupe, was found positive for phytoplasma presence. Considering that the phytoplasmas belonging to some ‘Candidatus species’ were detected in the three countries in different citrus varieties, a relevant role as phytoplasma reservoir can be attribute to citrus orchards