A virus with highly flexuous filamentous particles c. 800 nm long, showing distinct transverse striations was isolated with high frequency (60%) by inoculation of Nicotiana occidentalis with sap from grapevine accessions indexing positive for corky bark. The virus, for which the name grapevine virus B (GVB) is proposed, has an ssRNA genome with mol. wt. of c. 2.5 x 10(6) Da (c. 7600 nt) and coat protein subunits with mol. wt. of c 23,000 Da. GVB has a very restricted herbaceous host range and was experimentally transmitted by the mealybug Pseudococcus ficus. The physicochemical and ultrastructural properties of GVB resemble those of closteroviruses. However, it is serologically unrelated to other grapevine closteroviruses including grapevine virus A, with which it shares some biological and physicochemical properties.

Four stable hybridoma cell lines secreting monoclonal antibodies to grapevine closterovirus A (GVA) were obtained by fusing spleen cells of immunized BALB/c mice with mouse myeloma cell line Sp2/0-Ag 14. In ELISA all MAbs reacted with virus in leaf extracts from Nicotiana benthamiana, glass-house-, field-, or in vitro-grown grapevines, or with cortical shavings from mature grape canes. In IEM tests, only one of the MAbs (PA3.F5) decorated virus particles on the entire surface. This MAb was likely induced by a surface antigenic determinant, whereas the other three MAbs (PA3.D 11, PA3.C 6, and PA3.B 9) were originated by cryptotopes. Coupling polyclonal antibodies for coating protein A-sensitized plates, and monoclonal antibody conjugates for antigen detection, gave highly efficient and reproducible results for identification of GVA in field-grown grapevines.

Pathogens ultra-sensitive detection is vital for early diagnosis and provision of restraining actions and/or treatments. Among plant pathogens, Xylella fastidiosa is among the most threatening as it can infect hundreds of plant species worldwide with consequences on agriculture and the environment. An electrolyte-gated transistor is here demonstrated to detect X. fastidiosa at a limit-of-quantification (LOQ) of 2 ± 1 bacteria in 0.1 mL (20 colony-forming-unit per mL). The assay is carried out with a millimeter-wide gate functionalized with Xylella-capturing antibodies directly in saps recovered from naturally infected plants. The proposed platform is benchmarked against the quantitave polymerase chain reaction (qPCR) gold standard, whose LOQ turns out to be at least one order of magnitude higher. Furthermore, the assay selectivity is proven against the Paraburkholderia phytofirmans bacterium (negative-control experiment). The proposed label-free, fast (30 min), and precise (false-negatives, false-positives below 1%) electronic assay, lays the ground for an ultra-high performing immunometric point-of-care platform potentially enabling large-scale screening of asymptomatic plants.

No abstract

Xylella fastidiosa is among the most dangerous plant bacteria worldwide causing a variety of diseases, with huge economic impact on agriculture and environment. A surveillance tool, ensuring the highest possible sensitivity enabling the early detection of X. fastidiosa outbreaks, would be of paramount importance. So far, a variety of plant pathogen biomarkers are studied by means of surface plasmon resonance (SPR). Herein, multiparameter SPR (MP‐SPR) is used for the first time to develop a reliable and label‐free detection method for X. fastidiosa. The real‐time monitoring of the bioaffinity reactions is provided as well. Selectivity is guaranteed by biofunctionalizing the gold transducing interface with polyclonal antibodies for X. fastidiosa and it is assessed by means of a negative control experiment involving the nonbinding Paraburkholderia phytofirmans bacterium strain PsJN. Limit of detection of 105 CFU mL−1 is achieved by transducing the direct interaction between the bacterium and its affinity antibody. Moreover, the binding affinity between polyclonal antibodies and X. fastidiosa bacteria is also evaluated, returning an affinity constant of 3.5 × 107 m−1, comparable with those given in the literature for bacteria detection against affinity antibodies.