Tomato brown rugose fruit virus (ToBRFV) represents an emerging viral threat to the productivity of tomato and pepper protected cultivation worldwide. This virus has got the status of quarantine ...organism in the European Union (EU) countries. In particular, tomato and pepper seeds will need to be free of ToBRFV before entering the EU and before coming on the market. Thus, lab tests are needed. Here, we develop and validate a one-step reverse transcription LAMP platform for the detection of ToBRFV in tomato and pepper leaves, by real-time assay reverse transcription loop-mediated isothermal amplification (RT-LAMP) and visual screening (visual RT-LAMP). Moreover, these methods can also be applied successfully for ToBRFV detection in tomato and pepper seeds. The diagnostic specificity and sensitivity of both RT-LAMP and visual RT-LAMP are both 100%, with a detection limit of nearly 2.25 fg/μl, showing the same sensitivity as RT-qPCR Sybr Green, but 100 times more sensitive than end-point RT-PCR diagnostic methods. In artificially contaminated seeds, the proposed LAMP assays detected ToBRFV in 100% of contaminated seed lots, for up to 0.025-0.033% contamination rates in tomato and pepper, respectively. Our results demonstrate that the proposed LAMP assays are simple, inexpensive, and sensitive enough for the detection of ToBRFV, especially in seed health testing. Hence, these methods have great potential application in the routine detection of ToBRFV, both in seeds and plants, reducing the risk of epidemics.
The health status of the native grapevine Vitis vinifera subsp. sylvestris (Gmeli) Hegi in natural areas in Europe has received little attention. A survey was carried out on wild grapevines in ...Tuscany (Italy), where isolates of the Grapevine rupestris stem pitting virus (GRSPaV), Grapevine leafroll-associated virus 1 and 3 (GLRaV-1 and GLRaV-3) and Grapevine virus A (GVA) were detected. The complete coat protein (CP) region of these isolates was sequenced to investigate the relationship of the viral variants from Tuscan wild grapevines with isolates from different geographical origins. According to the phylogenetic analyses, GLRaV-1 and GLRaV-3 isolates from Tuscan wild grapevines clustered with isolates from cultivated grapevines with nucleotide sequence identities ranging from 66% to 87% and from 72.5% to 99% respectively, without any correlation between the distribution and geographical origin. Conversely, GRSPaV and GVA isolates clustered together with other Italian isolates from V. vinifera with nucleotide sequence identities ranging from 71.14% to 96.12% and from 73.5% to 92%, respectively. Our analysis of the whole amino acid sequences revealed a high conservation level for the studied proteins explained by a selective pressure on this genomic region, probably due to functional constraints imposed on CP, such as specific interactions with cellular receptors in the insect vectors necessary for successful transmission. In addition, analyses of genetic recombination suggest no significant point mutations that might play a significant role in genetic diversification. The dN/dS ratio also estimated a low number of non-silent mutations, highlighting the purifying selective pressure. The widespread distribution of the Rugose wood complex (GRSPaV and GVA associated disease) in comparison with the Grapevine Leafroll associated viruses (GLRaV-1 and -3) could explain the major geographical correlation found for the viral variants detected in Tuscany.
The present study focused on trees of Tuscany autochthonous plum cv. Coscia di Monaca in order to evaluate the presence of viruses and elucidate the physiological and biochemical responses to virus ...infections under real field conditions. Among the several investigated viruses, plums tested positive only to plum pox virus (PPV) and plum bark necrosis steam pitting associated virus (PBNSPaV), occurring as both singular and co-infections. This is the first report of PBNSPaV in a Tuscany orchard. Furthermore, the present study not only confirmed the detrimental effects of PPV on the carbon dioxide assimilation rate due to both stomatal limitations and mesophyll impairments, but also showed that although PBNSPaV did not induce such photosynthetic impairments when occurring as singular infection, it enhanced this damaging effect when present as a co-infection with PPV, as confirmed by a severe decrease in the chlorophyll content. Infection-specific responses in terms of accessory pigments (i.e., carotenoids and xanthophylls), as well as sugars and organic acids, were also reported, these being likely related to photoprotective mechanisms and osmotic regulations under virus-induced oxidative stress. Overall, the results here presented represent an important step to fill knowledge gaps about the interaction of plant viruses and autochthonous Prunus cultivars.
"Bois noir" disease associated with '
Phytoplasma solani' seriously compromises the production and survival of grapevines (
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 '
. 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-
-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 '
. P. solani-positive plants compared to -negative ones during the observed period.
Bois noir is a disease associated with the presence of phytoplasma '
Phytoplasma solani' belonging to the Stolbur group (subgroup 16SrXII-A), which has a heavy economic impact on grapevines. This ...study focused on the changes induced by phytoplasma in terms of the profile and amount of secondary metabolites synthesized in the phenylpropanoid pathway in leaves of
L. red-berried cultivar Sangiovese. Metabolic alterations were assessed according to the disease progression through measurements of soluble sugars, chlorophyll, and phenolic compounds produced by plant hosts, in response to disease on symptomatic and asymptomatic Bois noir-positive plants. Significant differences were revealed in the amount of soluble sugars, chlorophyll, and accumulation/reduction of some compounds synthesized in the phenylpropanoid pathway of Bois noir-positive and negative grapevine leaves. Our results showed a marked increase in phenolic and flavonoid production and a parallel decrease in lignin content in Bois noir-positive compared to negative leaves. Interestingly, some parameters (chlorophyll
, soluble sugars, total phenolic or flavonoids content, proanthocyanidins, quercetin) differed between Bois noir-positive and negative leaves regardless of symptoms, indicating measurable biochemical changes in asymptomatic leaves. Our grapevine cultivar Sangiovese results highlighted an extensive modulation of the phenylpropanoid biosynthetic pathway as a defense mechanism activated by the host plant in response to Bois noir disease.
Grapevine “bois noir”, related to the presence of ‘Candidatus Phytoplasma solani’ (‘Ca. P. solani’), represents a serious threat in several vine-growing areas worldwide. In surveys conducted over two ...years, mild and/or moderate symptoms and lower pathogen titer were mainly associated with ‘Ca. P. solani’ strains harboring a secY gene sequence variant (secY52), whereas severe symptoms and higher titer were mainly observed in grapevines infected by phytoplasma strains carrying any one of another four variants. A comparison of amino acid sequences of the protein SecY of ‘Ca. P. solani’ strains revealed the presence of conservative and semi-conservative substitutions. The deduced three-dimensional (3D) protein analysis unveiled that one semi-conservative substitution identified in the sequence variant secY52 is responsible for a structural disordered region that probably confers a flexibility for binding to distinct molecular complexes. In fact, the other analyzed variants show an organized structure and the 3D in silico prediction allowed the identification of β-sheets. Thus, differences in symptom severity and pathogen concentration observed in grapevines infected by ‘Ca. P. solani’ strains carrying distinct secY gene sequence variants suggest a possible relationship between SecY protein structure and phytoplasma strain virulence.
Bois noir (BN), associated with '
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,
was the most abundant and was found to be highly infected by CaPsol, while
, the main CaPsol vector, was not caught. Moreover,
harbored CaPsol strains carrying uniquely the
sequence variant St10, also identified as prevalent in vines and in the majority of weeds, and all the
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,
, and/or weeds is proposed. Furthermore, the possible presence of other players (vectors and weeds) involved in CaPsol transmission to grapevines was highlighted.
Sharka, caused by plum pox virus (PPV), is by far the most harmful disease of stone fruits (genus
Prunus
). Although Italy is a major producer of stone fruits, and sharka has spread in most Italian ...regions since its first identification in the 1970s, information on the diffusion of the disease in Italy results still scattered and incomplete. To address this knowledge gap, the present review aimed to firstly provide a historical overview of sharka in Italy, summarizing outcomes on the disease achieved since the first identification of its causal agent PPV. Data retrieved from a variety of sources (i.e., published literature, official reports and personal communications) showed that (i) sharka spread across almost the whole Italian peninsula as only Liguria, Umbria and Aosta Valley regions officially result disease-free; (ii) the regions with higher rates of infected districts are not always those more dedicated to the production of stone fruits; (iii) only the three major PPV strains causing sharka, i.e., PPV-Dideron (D), PPV-Marcus (M), and PPV-Recombinant (Rec), have been reported in Italy; and (iv) only apricot, peach and plum (i.e., widespread and economically relevant species) are hosts of PPV, with different scenarios among regions. A focus on Tuscany (Central Italy) showed that, (i) sharka has been reported since the 1990s, but extensive PPV monitoring campaigns were run from 2013 to 2021, especially in nurseries; (ii) higher number of symptomatic and PPV positive samples were recorded in farms, mostly in the Arezzo and Florence districts; and (iii) only PPV-M and PPV-Rec strains seem still present. We believe that the present overview will benefit and encourage both researchers for future investigations on sharka and farmers that have to manage the disease.
Wild herbaceous dicotyledonous plants (dicots) showing symptoms ascribable to phytoplasma disorders were found to be widely distributed in organic vineyards in central Tuscany (Italy) affected by ...bois noir, a grapevine yellows disease caused by "Candidates Phytoplasma solarii". In 2010 symptomatic dicots were tentatively identified to species level and the incidence of symptoms estimated in two selected vineyards in the province of Florence. Incidence ranged from 2 to 77%, and was not related to the relative abundance of hosts since very common species as well as relatively rare ones were consistently found to be symptomatic. PCR indexing and 16S rRNA sequence analyses indicated that two phytoplasmas co-existed in the vineyards: "Ca. P. solarii", infecting the root systems of 17 taxa, and a phytoplasma closely related to "Ca. P. phoenicium", infecting 11 taxa, and occasionally co-infecting the same plant. Regardless of the high frequency of both pathogens in the vineyards, only "Ca. P. solarii" could be detected in the grapevines. Population screening by means of tuf sequence analyses revealed the presence of only the tuf-b "Ca. P. solani" type both in dicot hosts and grapevine. This supports current notions of bois noir epidemiology, indicating that some infected dicots act as sources of "Ca. P. solani" inoculum whereas others are dead-end hosts. When the same specimens were screened by sequence analysis of the vmpl gene, evidence was found that different phytoplasma genotypes may be predominant in grapevines and dicots.
•A Grapevine yellows (GY) disease detection system was developed.•It can distinguish GY disease from other diseases with similar symptoms.•Six neural network architectures were utilized and ...evaluated.•This automatic tool is crucial to avoid missing GY-positive plants.•It offers an end-to-end detection of GY.
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.
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