Department of Environmental and Crop Sciences

Table grapes, strawberries and sweet cherries are perishable fruits and can be affected by different diseases, both in the field and even more during the postharvest storage. The main decay are gray mold, which infect all such fruit, and Rhizopus rot, which attacks mainly strawberry, at times sweet cherries, and rarely table grapes. Moreover, sweet cherries can suffer heavy losses mainly by brown rot, and to a lesser extent, by blue mold (at times this can infect table grapes and strawberries too), Alternaria rot, and Cladosporium rot. Table grapes, strawberries and sweet cherries are often cold stored soon after harvest, to retain quality, delay senescence, and reduce decay development. In several countries the use of synthetic fungicides after harvest is not allowed or there is a very short list of approved active ingredients. Therefore, together with consumer demand for food free of pesticide residues, the use of alternative means to control postharvest decay of fruit has gained increasing interest. Among these, chitosan has been identified as having the properties of an ideal coating for fruit. Preharvest and postharvest chitosan treatments of table grapes, strawberries and sweet cherries reduce their decay in the field and during storage, with the best performance at the highest tested concentration (usually 1%). Chitosan-based commercial products are available commercially, and they have shown the same effectiveness as the biopolymer dissolved in an acid solution. Chitosan has a double mechanism of action: it reduces the growth of decay causing fungi, and it induces resistance responses in host tissues. With this double effectiveness, chitosan can be considered as the first compound of a new class of plant protection products.

The effectiveness of chitosan treatment of table grapes, alone or in combination with UV-C, to control postharvest gray mold, caused by Botrytis cinerea, was determined in California, USA. The influence of these treatments on catechin and resveratrol contents and chitinase activity in grape berry skins was also assessed. Clusters of cvs. Thompson Seedless, Autumn Black, and Emperor were sprayed in the vineyard with 1% chitosan, then harvested daily for five days. Promptly after harvest, they were inoculated with B. cinerea. Decay incidence and disease severity were significantly reduced by chitosan, which was most effective on berries harvested 1 or 2 days after treatment. In another experiment, grapes were sprayed in the vineyard with chitosan, harvested 2 days later, irradiated 5 min with UV-C (0.36 J/cm2), and inoculated with B. cinerea two days later. Combined chitosan and UV-C treatments applied to cv. Autumn Black or selection B36-55 were synergistic in the reduction of gray mold incidence and severity compared to either treatment alone. Preharvest chitosan treatment did not increase concentration of catechin or resveratrol or activity of chitinase in berry skin. Conversely, UV-C irradiation, alone or combined with chitosan treatment, induced catechin in cv. Autumn Black berries and trans-resveratrol in both cv. Autumn Black and selection B36-55.

The effectiveness of pre and postharvest treatments with chitosan (0.1, 0.5, and 1.0%) to control Botrytis cinerea on table grapes was investigated. In postharvest treatments, small bunches dipped in chitosan solutions and inoculated with the pathogen showed a reduction of incidence, severity, and nesting of grey mould, in comparison with the control. Single berries artificially wounded, treated with the polymer and inoculated with B. cinerea showed a reduced percentage of infected berries and lesion diameter. The higher the chitosan concentration, the greater the decay reduction. All preharvest treatments significantly reduced the incidence of grey mould, as compared to the control. Table grapes treated with 1.0% chitosan showed a significant increase of phenylalanine ammonia-lyase (PAL) activity. Consequently, besides a direct activity against B. cinerea, chitosan produces other effects contributing to reduce decay.

Ulmus L. and Zelkova Spach are two representative genera of the Ulmaceae family that are found in Europe. Elm (Ulmus spp.) contains about 45 species that show a range of susceptibilities to Elm Yellows (EY), the most widespread and serious phytoplasma disease for these species. Japanese elm (Zelkova spp.) is known for its resistance to a wide range of diseases and pests, although Zelkova Yellows (ZY) have been recently reported on Z. serrata.
Since June 2006, we have carried out surveys to identify the causal agent of the decline of Ulmus spp. and Zelkova serrata in the Marche Region. Twenty-five leaf samples were collected from pre-bonsai and bonsai elms (U. parvifolia and Ulmus sp.), and Z. serrata showing typical EY symptoms or symptomless, and they were analysed by nested-PCR and RFLP analysis based on phytoplasma 16S rRNA gene.
These molecular analyses confirmed a single infection of ‘Candidatus Phytoplasma ulmi’ (16SrV-A) in all of the symptomatic leaf samples. Sequence analysis revealed 99% nucleotide homology with the EY reference strain EY-627 (Genbank accession no. AY197658). This appears to be the first report of ‘Ca. Phytoplasma ulmi’ associated with EY in Ulmus spp. and Z. serrata that have been trained as bonsai.

The Japanese elms are temperate broad-leaved tree species in the genus Zelkova. This genus includes species that are nowadays distributed in East Asia (three species), Western Asia (one species) and the Mediterranean (two species), while it is absent from North America (Denk and Grimm, 2005). Even though a member of the elm family, the Japanese Elm had no disease or pest problems of significance, including Dutch elm disease. Since June 2006, however, several plants of Zelkova serrata grown in Ancona, Marche region (central eastern Italy), have shown symptoms of chlorosis which involve the whole plant or some of the branches, foliar reddening on one or more branches, attenuation of apical dominance and proliferation of lateral shoots, witches’ broom, reduced growth and stunting of the plant.

Leaf samples from symptomatic and symptomless plants were collected and the DNA was extracted using the Plant DNeasy mini kit (Qiagen, Hilden, Germany). A molecular diagnosis was carried out to detect phytoplasma by PCR with universal primers P1/P7 (Seemüller et al., 1998) followed by a nested PCR with specific group primers 16Sr(V)F1/R1 (Lee et al., 1994). All of the five symptomatic samples yielded a PCR product of the expected size (1100 bp), both during 2006 and 2007. This PCR product was purified from the 1% agarose gels using Spin Column Wizard SV Gel and the PCR Clean–Up System (Promega), and eluted with sterile ultrapure water, according to the manufacturer’s instructions. Five-µl aliquots of the PCR product were digested with 3 U of restriction endonuclease BfaI (New England BioLabs, Beverly, MA, USA), over night at 37°C. The restriction pattern was characterized by two bands of 650 and 450 bp, identical to those in the reference strain of ‘Candidatus Phytoplasma ulmi’ (EY, kindly provided by C. Marzachì, IVV-CNR, Torino, Italy) (Lee et al., 2004). The pathogen was not found in samples from symptomless plants. Those plants were inoculated by grafting with samples from infected symptomatic plants, and disease symptoms appeared in the entire plant. Leaf samples from artificially inoculated plants tested positive for the presence of the phytoplasma. The sequenced isolate from Z. serrata showed more than 99% identity with ‘Ca. Phytoplasma ulmi’ strain EY1T (GenBank Acc. No. AY197655) (Lee et al., 2004).

Several natural infections of ‘Ca. Phytoplasma ulmi’ have been described on different species of the genus Ulmus (Lee et al., 2004), although it has not been reported on Zelkova spp. This thus appears to be the first report of ‘Ca. Phytoplasma ulmi’ infection in symptomatic Z. serrata, in Italy and worldwide.

Coltsfoot (Tussilago farfara L.) is a perennial herb with a creeping rhizome and long runners. It is an invasive weed that prefers heavy clay soils throughout Europe, North Africa, Asia and the USA. Bois noir (BN), caused by phytoplasma belonging to the stolbur group (16SrXII-A), is spread in the main Italian viticultural areas and it is common in vineyards of the Abruzzi region (Romanazzi et al., 2007). BN is transmitted to grapevines by its vector, Hyalesthes obsoletus, that can feed on BN-infected wild plants.

In 2007, during a survey carried out in a cv Chardonnay vineyard with a 30% of BN-infected plants (Romanazzi et al., 2007), at Atri (TE) in the Abruzzi region, spontaneous T. farfara plants were collected. Most of these showed typical symptoms ascribed to phytoplasma infection, such as foliar reddening and rolling (Fig. 1). The DNA was extracted from leaf samples of symptomatic and symptomless T. farfara plants using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). The samples were amplified with the P1/P7 universal primers and nested-PCR was carried out with group-specific primers: R16(I)F1/R1, R16(III)F1/R2 and R16(V)F1/R1. Five-µl aliquots of the R16(I)F1/R1 amplicon were digested with 3 U restriction endonuclease MseI (New England BioLabs, Beverly, MA, USA), overnight at 37°C. Further molecular characterization was carried out by amplifying the tuf gene in nested-PCR and digesting the amplicon with 3 U HpaII (New England BioLabs), overnight at 37 °C (Langer and Maixner, 2004).

All symptomatic samples of T. farfara were infected by stolbur, showing an identical restriction pattern to that in the reference strain (STOL, kindly provided by C. Marzachì, IVV-CNR, Turin, Italy). No phytoplasma of groups 16SrI, 16SrIII and 16SrV were detected. The pathogen was not found in samples from symptomless plants, which developed disease symptoms on the entire plant when inoculated by grafting with samples from infected symptomatic ones. Leaf samples from these artificially inoculated plants tested positive for the presence of the phytoplasma. The molecular characterization of the stolbur isolates revealed tuf type II infections (Fig. 2).

Several infection of phytoplasma have been detected on different yellows diseased weeds in Italy (Credi et al., 2006; Borgo et al., 2008), although stolbur infection has not previously reported for T. farfara. The information on weeds that can be sources of BN phytoplasma in the vineyard is basilar for the disease management on grapevine. This is thus the first report of stolbur infection in symptomatic T. farfara in Italy, and worldwide.

Esca is one of the most important grapevine trunk diseases, and it can induce severe declines. In the past, the disease occurred mostly with mature vines, but today it is also present in young vineyards. The aim of this study was to investigate the incidence of esca in young (less than 7 years old) and mature (more than 11 years old) vineyards with cvs. Montepulciano, Sangiovese, Verdicchio and Passerina located in the main viticultural areas of the Marche Region, central-eastern Italy. The average incidence of diseased plants was higher in mature (32.58%) than young (5.21%) vineyards, and Verdicchio and Passerina appeared to be the most sensitive among the cultivars considered, followed by Sangiovese and Montepulciano. The analysis of the spatial spread of esca carried out in two mature vineyards with cv. Verdicchio and a young vineyard with cv. Sangiovese showed a fluctuation in the numbers of infected plants over the three years of observation. The fungi associated with symptomatic plants were detected by classical and, in part, molecular tools. Isolation on agar media yielded colonies of Phaeomoniella chlamydospora (Pch), Botryosphaeria spp. (Bot), Fomitiporia mediterranea (Fomed) and, sporadically, Phaeoacremonium aleophilum (Pal). In samples from young plants, Bot and Pch were recurrent, while Pch and Fomed were found with mature vines and old rootstocks. Molecular detection with specific primer pairs for Pch, Pal and B. dothidea confirmed the data obtained using classical tools, and in some cases it was more sensitive. This study thus provides a further contribution as to the presence of different causal agents in the expression of esca symptoms, and it confirms the importance of molecular tools for the sensitive detection of associated pathogens, which can also be present in propagative materials.

Gray mold is caused by Botrytis cinerea Pers.:Fr., a cosmopolitan and polyphagous fungus that is responsible for significant losses on several fruit crops, including kiwifruit (Actinidia deliciosa), both in the field and during storage (2). The hardy kiwifruit, A. arguta, is a kiwifruit with a smooth skin grown in several countries (e.g Japan, China, Korea, Russia, USA) (1,3) and regions of Italy, including Apulia (south eastern), Basilicata (southern), Marche (central eastern), Tuscany (central western), Piedmont, Friuli Venezia Giulia and Trentino Alto Adige (northern) (4). A. arguta produces fruit with edible thin skin in large clusters. The oblong-to-oval shaped fruit bear tips with a persistent style, and weight about 5 to 15 g (1,3,4). Several fruits of cv. Ananasnaya selection “Anna red” harvested from September to October in 2007 and 2008 in the Apulia and Marche regions, showed browning and depressions in the surface, and a decay of the flesh. The disease affected about 5% of 420 examined fruits; symptoms mainly developed in the equatorial zone, eventually expanding through the entire fruit. Severely infected fruit showed deformation, followed by drying. In the field or after storage in an environment with high RH, fungal mycelia with sporulation appeared on the surface of the symptomatic areas of the fruit. Nineteen symptomatic fruit were surface disinfested by immersion in a 0.5% sodium hypochlorite solution for 2 min, then rinsed in sterile distilled water. Portions of the flesh were plated onto Petri dishes containing potato dextrose agar (PDA) supplemented with 250 mg/l each ampicillin and streptomycin sulphate. Colonies that originated from symptomatic portions of the fruit were transferred to new PDA plates, and identified as B. cinerea by the morphological features of conidiophores and conidia. Conidia were produced on dichotomously branched conidiophores, which had globose
basal cells from mycelia. The conidia were hyaline or pigmented, ellipsoid-obovoid, globoid, and without septa (2). Conidia were collected from an isolate (accession n. CBS 125087) of B. cinerea, recovered from diseased A. arguta fruit grown in Monopoli (BA), Apulia, in September 2007, and maintained in pure culture on PDA.  A spore suspension was created by flooding plates with a small volume of sterile distilled water plus surfactant (0.05% Triton X-100). The suspension was filtered through four layers of cheesecloth and diluted with sterile water to an absorbance of 0.25 at 425 nm as determined by a spectrophotometer. This suspension contained about 1.0 × 106 conidia/ml and was diluted with sterile water to 1 × 104 spores/ml. Twenty μl droplets of spore suspension were deposited within the equitorial zone on each of ten non-wounded fruits of cv. Ananasnaya selection “Anna red”. All of these fruit developed the typical gray mold symptoms after 4 to 5 days of incubation at 20±2°C and 95 to 98% RH. Reisolation from the decayed tissues on PDA produced pure colonies of B. cinerea. To our knowledge, this is the first report of B. cinerea infection on A. arguta in Italy.

Phytoplasmoses are severe diseases of grapevine and other crops, for which no effective means of control are available. However, plants infected by phytoplasma can undergo spontaneous symptom remission (recovery), a long-known phenomenon. A strategy to reduce the number of symptomatic plants could thus arise from the stimulation of plant defence systems to induce recovery. The aim of the present study was to evaluate the effectiveness of field treatments with resistance inducers to promote recovery in Bois noir (BN) infected grapevines. Five commercial products (Chito Plant, Aliette, Kendal, Bion, and Olivis) described as plant defence promoters were sprayed on the canopy of BN-infected grapevines of cv. Chardonnay. Treatments consisted of  weekly sprays in spring-summer 2007 (seven applications) and 2008 (thirteen applications). All treatments increased the number of recovered plants with respect to the control. The best results were obtained with Kendal, Olivis and Bion, while Aliette and Chito Plant performed better in the first than in the second year. Molecular analysis of leaf vein extracts from recovered plants failed to detect the phytoplasma. An induction of host defence is likely to be responsible for the effectiveness of these resistance promoters in the control of BN.

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 PCR/RFLP, to investigate two different non-ribosomal 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, due 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 for comparison with previous investigations.