Detection of grapevine yellows symptoms in Vitis vinifera L. with artificial intelligence

Grapevine yellows (GY) are a significant threat to grapes due to the severe symptoms and lack of treatments. Conventional diagnosis of the phytoplasmas associated to GYs relies on symptom identification, due to sensitivity limits of diagnostic tools (e.g. real time PCR) in asymptomatic vines, where the low concentration of the pathogen or its erratic distribution can lead to a high rate of false-negatives. GY's primary symptoms are leaf discoloration and irregular wood ripening, which can be easily confused for symptoms of other diseases making recognition a difficult task. Herein, we present a novel system, utilizing convolutional neural networks, for end-to-end detection of GY in red grape vine (cv. Sangiovese), using color images of leaf clippings. The diagnostic test detailed in this work does not require the user to be an expert at identifying GY. Data augmentation strategies make the system robust to alignment errors during data capture. When applied to the task of recognizing GY from digital images of leaf clippings—amongst many other diseases and a healthy control—the system has a sensitivity of 98.96% and a specificity of 99.40%. Deep learning has 35.97% and 9.88% better predictive value (PPV) when recognizing GY from sight, than a baseline system without deep learning and trained humans respectively. We evaluate six neural network architectures: AlexNet, GoogLeNet, Inception v3, ResNet-50, ResNet-101 and SqueezeNet. We find ResNet-50 to be the best compromise of accuracy and training cost. The trained neural networks, code to reproduce the experiments, and data of leaf clipping images are available on the internet. This work will advance the frontier of GY detection by improving detection speed, enabling a more effective response to the disease

The monitoring program of grapevine phytoplasmas in Tuscany (Italy): Results of a four year survey

Quantitative PCR protocols for phytoplasma detection were used to monitor grapevine yellows (GY) in 373 vineyards located in nine Tuscan districts. Among more than 70,000 plants visually monitored, 1.867 plants were sampled and “flavescence dorée” phytoplasmas (FD) were detected in 122 plants and mainly identified as trains belonging to 16SrV-C subgroup. The “bois noir” (BN) phytoplasma was found in 734 samples, with prevalence of tufB type-b strains. The 2013–2015 monitoring program was strongly influenced by the first survey (2012) in which FD was found consistently in the North West (15 samples), whereas only a few cases were observed in the East territory (2 samples). Both areas were thoroughly monitored in the following years: few foci were found in the East (2 in 2014, 1 in 2015), while several infected areas were found in the North West (6, 10 and 22 foci in 2013, 2014 and 2015, respectively). Definitely, the novel FD foci detected in the survey (17, 6, 12 and 23 in each year of survey) and the widespread of BN, suggest a dangerous distribution of GY in Tuscan

Optimization of organotypic cultures of mouse spleen for staining and functional assays

By preserving cell viability and three-dimensional localization, organotypic culture stands out among the newest frontiers of cell culture. It has been successfully employed for the study of diseases among which neoplasias, where tumoral cells take advantage of the surrounding stroma to promote their own proliferation and survival. Organotypic culture acquires major importance in the context of the immune system, whose cells cross-talk in a complex and dynamic fashion to elicit productive responses. However, organotypic culture has been as yet poorly developed for and applied to primary and secondary lymphoid organs. Here we describe in detail the development of a protocol suitable for the efficient cutting of mouse spleen, which overcomes technical difficulties related to the peculiar organ texture, and for optimized organotypic culture of spleen slices. Moreover, we used microscopy, immunofluorescence, flow cytometry and qRT-PCR to demonstrate that the majority of cells residing in spleen slices remain alive and maintain their original location in the organ architecture for several days after cutting. The development of this protocol represents a significant technical improvement in the study of the lymphoid microenvironment in both physiological and pathological conditions involving the immune system

Characterization of a Bacillus cereus strain associated with a large feed-related outbreak of severe infection in pigs

Aims: Bacillus cereus is often responsible for foodborne diseases and both local and systemic infections in humans. Cases of infection in other mammals are rather rare. In this study, we report a B. cereus feed-related outbreak that caused the death of 6234 pigs in Italy. Methods and Results: Massive doses of a Gram-positive, spore-forming bacterium were recovered from the animal feed, faeces of survived pigs and intestinal content of dead ones. The B. cereus MM1 strain was identified by MALDI-TOF MS and typified by RAPD-PCR. The isolate was tested for the production of PC-PLC, proteases, hemolysins and biofilm, for motility, as well as for the presence of genes encoding tissue-degrading enzymes and toxins. Antimicrobial resistance and pathogenicity in Galleria mellonella larvae were also investigated. Our results show that the isolated B. cereus strain is swimming-proficient, produces PC-PLC, proteases, hemolysins, biofilm and carries many virulence genes. The strain shows high pathogenicity in G. mellonella larvae. Conclusions: The isolated B. cereus strain demonstrates an aggressive profile of pathogenicity and virulence, being able to produce a wide range of determinants potentially hazardous to pigs' health. Significance and Impact of Study: This study highlights the proficiency of B. cereus to behave as a devastating pathogen in swine if ingested at high doses and underlines that more stringent quality controls are needed for livestock feeds and supplements

Use of Saccharomyces boulardii CNCM I-745 as therapeutic strategy for prevention of nonsteroidal anti-inflammatory drug-induced intestinal injury

Background and Purpose: Nonsteroidal anti-inflammatory drugs (NSAIDs) can be associated with severe adverse digestive effects. This study examined the protective effects of the probiotic Saccharomyces boulardii CNCM I-745 in a rat model of diclofenac-induced enteropathy. Experimental Approach: Enteropathy was induced in 40-week-old male rats by intragastric diclofenac (4 mg·kg−1 BID for 14 days). S. boulardii CNCM I-745 (3 g·kg−1 BID by oral gavage) was administered starting 14 days before (preventive protocol) or along with (curative protocol) diclofenac administration. Ileal damage, inflammation, barrier integrity, gut microbiota composition and toll-like receptors (TLRs)–nuclear factor κB (NF-κB) pathway were evaluated. Key Results: Diclofenac elicited intestinal damage, along with increments of myeloperoxidase, malondialdehyde, tumour necrosis factor and interleukin-1β, overexpression of TLR2/4, myeloid differentiation primary response 88 (Myd88) and NF-κB p65, increased faecal calprotectin and butyrate levels, and decreased blood haemoglobin levels, occludin and butyrate transporter monocarboxylate transporter 1 (MCT1) expression. In addition, diclofenac provoked a shift of bacterial taxa in both faecal and ileal samples. Treatment with S. boulardii CNCM I-745, in both preventive and curative protocols, counteracted the majority of these deleterious changes. Only preventive administration of the probiotic counteracted NSAID-induced decreased expression of MCT1 and increase in faecal butyrate levels. Occludin expression, after probiotic treatment, did not significantly change. Conclusions and Implications: Treatment with S. boulardii CNCM I-745 prevents diclofenac-induced enteropathy through anti-inflammatory and antioxidant activities. Such effects are likely to be related to increased tissue butyrate bioavailability, through an improvement of butyrate uptake by the enteric mucosa

Delivery Mode Shapes the Composition of the Lower Airways Microbiota in Newborns

Radical alterations in the human microbiota composition are well-known to be associated with many pathological conditions. If these aberrations are established at the time of birth, the risk of developing correlated pathologies throughout life is significantly increased. For this reason, all newborns should begin their lives with a proper microbiota in each body district. The present study aimed at demonstrating a correlation between the mode of delivery and the development of a well-balanced microbiota in the lower airways of newborns. 44 pregnant women were enrolled in this study. Microbiological comparative analysis was carried out on tracheobronchial secretions of babies born through vaginal delivery (VD) or caesarean section (CS). All samples showed the presence of bacterial DNA, regardless of the mode of delivery. No viable cultivable bacteria were isolated from the CS samples. On the contrary, VD allowed colonization of the lower airways by alive cultivable bacteria. The identification of bacterial species revealed that Lactobacillus spp. and Bacteroides vulgatus were the most common microorganisms in the lower airways of vaginally-delivered newborns. Data obtained from quantitative PCRs showed a significantly higher total bacterial load, as well as Firmicutes and Lactobacillus spp. amount, in VD samples than CS ones, while no statistically significant difference was found in Torque Teno Virus (TTV) load between samples. Taken together, our findings confirm the hypothesis that passage through the maternal vaginal canal determines more beneficial colonization of the lower airways in newborns

The sustainability of old grapevine mother plants in relation to new mandatory diagnostic tests for virus control

In 2011, the methods to perform phytosanitary tests to check for viruses in grapevine nurseries were reassessed and new regulations were defined (DM 13 December 2011). The mandatory tests require serological assays for the diagnosis of five viruses in grapevine mother plants transplanted in Tuscany in 2001 or before. The aim of the present paper is to report the impact of certification programs applied before 2001 in Tuscany and the sustainability of older mother plants with relation to the new mandatory diagnostic tests. Among the cultivars, virus infection was reported in 19.2% of pool samples, whereas 2.4% of rootstock pool samples showed a compromised health status. GLRaV-3 is the most frequently found virus (10.4% and 1.3% of cultivar and rootstock pools, respectively), and it is also included in the most frequent multiple infections. Multiple infections represent about 25% of infected cultivar pools and almost 50% of infected rootstock pools

Signaling Cross-Talk between Salicylic and Gentisic Acid in the ‘Candidatus Phytoplasma Solani’ Interaction with Sangiovese Vines

“Bois noir” disease associated with ‘Candidatus Phytoplasma solani’ seriously compromises the production and survival of grapevines (Vitis vinifera L.) in Europe. Understanding the plant response to phytoplasmas should help to improve disease control strategies. Using a combined metabolomic and transcriptomic analysis, this work, therefore, investigated the phytoplasma–grapevine interaction in red cultivar Sangiovese in a vineyard over four seasonal growth stages (from late spring to late summer), comparing leaves from healthy and infected grapevines (symptomatic and symptomless). We found an accumulation of both conjugate and free salicylic acids (SAs) in the leaves of ‘Ca. P. solani’-positive plants from early stages of infection, when plants are still asymptomatic. A strong accumulation of gentisic acid (GA) associated with symptoms progression was found for the first time. A detailed analysis of phenylpropanoids revealed a significant accumulation of hydroxycinnamic acids, flavonols, flavan 3-ols, and anthocyanin cyanidin 3-O-glucoside, which are extensively studied due to their involvement in the plant response to various pathogens. Metabolomic data corroborated by gene expression analysis indicated that phenylpropanoid biosynthetic and salicylic acid-responsive genes were upregulated in ‘Ca. P. solani-positive plants compared to -negative ones during the observed period

The monitoring program of grapevine phytoplasmas in Tuscany (Italy): results of a four year survey

Quantitative PCR protocols for phytoplasma detection were used tomonitor grapevine yellows (GY) in 373 vineyards located in nine Tuscan districts. Among more than 70,000 plants visually monitored, 1.867 plants were sampled and “flavescence dorée” phytoplasmas (FD) were detected in 122 plants and mainly identified as trains belonging to 16SrV-C subgroup. The “bois noir” (BN) phytoplasma was found in 734 samples, with prevalence of tufB type-b strains. The 2013–2015 monitoring program was strongly influenced by the first survey (2012) in which FD was found consistently in the North West (15 samples), whereas only a few cases were observed in the East territory (2 samples). Both areas were thoroughly monitored in the following years: few foci were found in the East (2 in 2014, 1 in 2015), while several infected areas were found in the North West (6, 10 and 22 foci in 2013, 2014 and 2015, respectively). Definitely, the novel FD foci detected in the survey (17, 6, 12 and 23 in each year of survey) and the widespread of BN, suggest a dangerous distribution of GY in Tuscany

Robot-assisted pelvic floor reconstructive surgery: an international Delphi study of expert users.

Robotic surgery has gained popularity for the reconstruction of pelvic floor defects. Nonetheless, there is no evidence that robot-assisted reconstructive surgery is either appropriate or superior to standard laparoscopy for the performance of pelvic floor reconstructive procedures or that it is sustainable. The aim of this project was to address the proper role of robotic pelvic floor reconstructive procedures using expert opinion. We set up an international, multidisciplinary group of 26 experts to participate in a Delphi process on robotics as applied to pelvic floor reconstructive surgery. The group comprised urogynecologists, urologists, and colorectal surgeons with long-term experience in the performance of pelvic floor reconstructive procedures and with the use of the robot, who were identified primarily based on peer-reviewed publications. Two rounds of the Delphi process were conducted. The first included 63 statements pertaining to surgeons' characteristics, general questions, indications, surgical technique, and future-oriented questions. A second round including 20 statements was used to reassess those statements where borderline agreement was obtained during the first round. The final step consisted of a face-to-face meeting with all participants to present and discuss the results of the analysis. The 26 experts agreed that robotics is a suitable indication for pelvic floor reconstructive surgery because of the significant technical advantages that it confers relative to standard laparoscopy. Experts considered these advantages particularly important for the execution of complex reconstructive procedures, although the benefits can be found also during less challenging cases. The experts considered the robot safe and effective for pelvic floor reconstruction and generally thought that the additional costs are offset by the increased surgical efficacy. Robotics is a suitable choice for pelvic reconstruction, but this Delphi initiative calls for more research to objectively assess the specific settings where robotic surgery would provide the most benefit