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

Microbiological quality and resistance to an artificial gut environment of two probiotic formulations

The quality control of probiotic products is the focus of numerous organizations worldwide. Several studies have highlighted the poor microbiological quality of many commercial probiotic formulations in terms of the identity of the contained microorganisms, viability, and purity, thus precluding the expected health benefits and representing a potential health risk for consumers. In this paper, we analyzed the contents of two probiotic formulations, one composed of an encapsulated mixture of lactobacilli and bifidobacteria, and one by a lyophilized yeast. The microorganisms contained in the products were quantified and identified using up-to-date methodologies, such as MALDI-TOF MS and metagenomic analysis. Moreover, as acid and bile tolerance is included among the criteria used to select probiotic microorganisms, in vitro tests were performed to evaluate the behavior of the formulations in conditions mimicking the harsh gastric environment and the intestinal fluids. Our results indicate the high quality of the formulations in terms of the enumeration and identification of the contained organisms, as well as the absence of contaminants. Moreover, both products tolerated the acidic conditions well, with encapsulation providing further protection for the microorganisms. A good tolerance to the simulated artificial intestinal conditions was also evidenced for both preparations

Effects of extracellular K+ on grapevine membrane potential as influenced by the antiviral mycophenolic acid. An electrophysiological study

Mycophenolic acid (MPA) is an effective antiviral drug in plants, and its action in modulating the activity of KATP channels is already known in animals. In the present work, an electrophysiological study was carried out to investigate MPA effects on plant K+ channels, through the measurement of trans-plasma membrane potential in samples of Vitis vinifera cv. Sangiovese treated with extracellular K+. Tests confirmed that the administration of MPA (in preincubated samples or in those maintained under chemical treatment) can reduce the membrane depolarization induced by K+. However, MPA-induced alteration in membrane potential was sensitive to the KATP channel opener diazoxide, as well to treatments with guanosine. This result confirms the effectiveness of MPA in influencing KATP channel activity as well as inhibiting activity of the inward-rectifier potassium ion channel which could be mediated by guanosine depletion induced by MPA

Molecular typing of bois noir phytoplasma strains in the chianti classico area (Tuscany, Central Italy) and their association with symptom severity in vitis vinifera 'sangiovese'

Bois noir (BN) is the most widespread disease of the grapevine yellows complex in the Euro-Mediterranean area. BN is caused by 'Candidatus Phytoplasma solani' (BNP), transmitted from herbaceous plants to grapevine by polyphagous insect vectors. In this study, genetic diversity among BNP strains and their prevalence and possible association with grapevine symptom severity were investigated in a Sangiovese clone organic vineyard in the Chianti Classico area (Tuscany). Field surveys over 2 years revealed a range of symptom severity on grapevine and an increase of BN incidence. ATaqMan allelic discrimination assay detected only tufB type b among BNP strains, suggesting the prevalence of the bindweed-related ecology. Nucleotide sequence analyses of vmp1 and stamp genes identified 12 vmp1 and 16 stamp sequence variants, showing an overall positive selection for such genes. The prevalent genotype was Vm43/St10, reported for the first time in this study and closely related to strains identified only in the French Eastern Pyrenees. BNP strains identified in the examined vineyard and mostly grouped in separate bindweed-related phylogenetic clusters showed statistically significant differences in their distribution in grapevines exhibiting distinct symptom severity. These results suggest the possible occurrence of a range of virulence within BNP strain populations in the Chianti Classico area

Proposal of A New Bois Noir Epidemiological Pattern Related to ‘Candidatus Phytoplasma Solani’ Strains Characterized by A Possible Moderate Virulence in Tuscany

Bois noir (BN), associated with 'Candidatus Phytoplasma solani' (CaPsol), is the most widespread disease of the grapevine yellows complex worldwide. In this work, BN epidemiology was investigated in a case study vineyard where an unusual CaPsol strain, previously detected only in other host plants, was found to be prevalent in grapevine. Experimental activities included: symptom observation; sampling of symptomatic vines, Auchenorrhyncha specimens, and weeds; molecular detection and typing of CaPsol strains; statistical analyses for determining possible relationships between CaPsol relative concentration, strain type, and symptom severity. Among insects, Reptalus quinquecostatus was the most abundant and was found to be highly infected by CaPsol, while Hyalesthes obsoletus, the main CaPsol vector, was not caught. Moreover, R. quinquecostatus harbored CaPsol strains carrying uniquely the stamp sequence variant St10, also identified as prevalent in vines and in the majority of weeds, and all the secY variants identified in the vineyard. Statistical analyses revealed that CaPsol strains carrying the St10 variant are not associated with severe symptoms, suggesting their possible moderate virulence. Based on such evidence, a new BN epidemiological pattern related to these CaPsol strains and involving grapevine, R. quinquecostatus, and/or weeds is proposed. Furthermore, the possible presence of other players (vectors and weeds) involved in CaPsol transmission to grapevines was highlighted

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

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

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

Propagative material of grapevine: RFID technology for supporting traceability of "basic" and "certified" material along the wine production chain

Four main categories of propagative material in the European Union (EU) have been indentified for grapevine: the primary source, pre-basic, basic and certified material. Each type of material has to be periodically assayed for pathogens and each material stage is intrinsically characterized by increasing risks. Radiofrequency (RFID) can be considered an efficient and durable traceability system to provide retrieval of propagated material or check producer identity. RFID tagging of grapevines of different categories along the production line could establish a durable link between stakeholder and products. To evaluate this approach, histological observations and growth parameters of basic or certified RFID- tagged grapevine were performed, as well as requirement analysis for management of sanitary checks and for traceability of the wine production chain. Basic material can be safety tagged with RFID tags to establish mother plant vineyards; derived certified material can also safely be tagged. No detrimental performance in terms of vegetative growth and bud production were reported for mother plant vineyards from the first year of production life. Requirement analysis made it possible to individuate traceability objectives, materials and stakeholders involved, as well as the RFID-tagging steps and methods to collect sanitary and production data that are useful for traceability purposes