No abstract

Early diagnosis of seedborne fungal pathogens is particularly important, as often, infected seeds appear symptomless; seed diagnosis can avoid uncontrolled propagation of pathogens through long‐distance exchange of such material. This will prevent economic losses and unnecessary use of fungicides, so reducing costs and the introduction of toxic substances into the environment. Traditional techniques for detection of seedborne fungi are based on incubation and grow‐out methods. Although these are frequently used because of their simplicity of application, they are time‐consuming, require mycological skills, and are sometimes not sensitive enough to low levels of seed infection. Recently, new identification techniques, based on DNA analysis, have been applied and are very efficient due to high sensitivity and specificity. The most common technique is conventional PCR, while other recent techniques include nested PCR, to obviate low levels of target pathogens, multiplex PCR, to detect several pathogens simultaneously, real‐time PCR, to quantify fungi on seeds, and magnetic‐capture hybridization PCR. The main drawbacks of molecular methods are the inability to distinguish between vital and non‐vital inocula, and the difficulty in obtaining quality DNA template, due to PCR inhibitors in seeds. To reduce inhibitory effects, several modified PCR protocols, such as loop‐mediated isothermal amplification, and non‐destructive testing methods have been developed. Loop‐mediated isothermal amplification and next‐generation sequencing have been widely applied in nucleic acid analysis and, given the numerous advantages provided, their application can be substantially extended in the future for detection of fungal pathogens in seeds.

Aim:  Evaluation of the genetic variability of stolbur phytoplasma infecting grapevines, bindweeds and vegetables, collected in different central and southern Italian regions. Materials and Results:  Phytoplasma isolates belonging to stolbur subgroup 16SrXII‐A were subjected to molecular characterization by polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP), to investigate two different nonribosomal genes: tuf and vmp1. In grapevines, 32% of samples were infected by tuf‐a type and 68% by tuf‐b type, with different relative incidences in the regions surveyed. All herbaceous samples (bindweeds, tomato, tobacco, pepper, celery) were infected by tuf‐b. The gene vmp1 showed higher polymorphism in grapevines (nine profiles) than herbaceous plants (six) by RFLP analysis, in agreement with nucleotide sequences’ analysis and virtual digestions. Conclusions:  The phylogenetic analysis of vmp1 gene sequences supports the RFLP data and demonstrates the accuracy of RFLP for preliminary assessments of genetic diversity of stolbur phytoplasmas and for screening different vmp types. Significance and Impact of the Study:  Stolbur represents a serious phytosanitary problem in the areas under investigation, owing to heavy economic losses in infected grapevines and vegetables. Molecular information about the complex genotyping of the vmp1 gene provides useful data towards a better understanding of stolbur epidemiology. Moreover, this study clarifies some different vmp1 genotype classifications of stolbur, providing molecular data in comparison with previous investigations.

Bois noir (BN) is one of the main phytoplasma diseases of grapevine (Vitis vinifera). It is widespread, and can cause severe losses in European vineyards. The infective agent colonizes phloem elements and induces visible symptoms of leaf yellowing or reddening after a relatively long incubation period. As the most sensitive cultivars to BN, Chardonnay plants were grouped as healthy or symptomatic in spring, based on the records from the previous year. Leaf gas exchange and chlorophyll a fluorescence were measured weekly from July to September in healthy plants, and in symptomatic and asymptomatic leaves from symptomatic plants. The midday relative water content (mRWC) was measured once per month. The detection of phytoplasma DNA by nested-polymerase chain reaction revealed BN infection in symptomatic leaf samples at the end of September. A significant decrease in pigment content and maximum quantum efficiency of photosystem II (Fv/Fm) of these symptomatic leaves was detected from July to September, although in the asymptomatic leaves of the symptomatic plants the net photosynthesis (Pn) decrease was not significant. In the leaves from the healthy plants, Pn and transpiration were relatively stable. Of note, in July, an initially healthy plant showed a strong Pn reduction that was followed by visible leaf yellowing symptoms only in August. The phytoplasma infection also stimulated significant reductions in mRWC of the symptomatic leaves, with a final large decrease in yield.

Grapevine Bois noir (BN) is a phytoplasma disease that is widespread in most viticultural regions of the world, and it can result in heavy reductions to yields and grape juice quality. At present, there is no effective strategy to reduce the incidence of BN-infected grapevines. However, phytoplasma-infected plants can recover through spontaneous or induced symptom remission. Five elicitors (chitosan, two glutathione-plus-oligosaccharine formulations, benzothiadiazole, and phosetyl-Al) were applied weekly to the canopy of BN-infected 'Chardonnay' grapevines from early May to late July. The best and most constant recovery inductions were obtained with benzothiadiazole and the two glutathione-plus-oligosaccharine formulations. The plants that recovered naturally or following the elicitors showed qualitative and quantitative parameters of production no different from healthy plants. In another vineyard, diseased plants showed reduced shoot length and production compared with healthy plants, and there were no negative effects on these parameters for grapevines sprayed with a glutathione-plus-oligosaccharine formulation. The application of resistance inducers promoted the recovery of BN-infected grapevines with no adverse effects on the plants. Therefore, grapevine can be used as a model species to test this innovative strategy to contain phytoplasma diseases.